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RNA-BINDING OF LC3 TO THE AU-RICH ELEMENT OF FIBRONECTIN -A: A STRUCTURAL AND FUNCTIONAL STUDY Agatha Lau A thesis submitted in conformity with the requirements for the Degree of Master of Science in the Graduate Department of Labonatory Medicine and Pathobiology, University of Toronto Q Copyright by Agatha Lau, 1999

OF LC3 THE AU-RICH ELEMENT OF FIBRONECTIN · RNA-BINDING OF LC3 TO THE AU-RICH ELEMENT OF FIBRONECTIN mRNA: A STRUCTURAL AND FUNCTIONAL STUDY Master of Science, 1999 Agatha Lau Department

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Page 1: OF LC3 THE AU-RICH ELEMENT OF FIBRONECTIN · RNA-BINDING OF LC3 TO THE AU-RICH ELEMENT OF FIBRONECTIN mRNA: A STRUCTURAL AND FUNCTIONAL STUDY Master of Science, 1999 Agatha Lau Department

RNA-BINDING OF LC3 TO THE AU-RICH ELEMENT OF FIBRONECTIN

-A: A STRUCTURAL AND FUNCTIONAL STUDY

Agatha Lau

A thesis submitted in conformity with the requirements for the Degree of Master of

Science in the Graduate Department of Labonatory Medicine and Pathobiology,

University of Toronto

Q Copyright by Agatha Lau, 1999

Page 2: OF LC3 THE AU-RICH ELEMENT OF FIBRONECTIN · RNA-BINDING OF LC3 TO THE AU-RICH ELEMENT OF FIBRONECTIN mRNA: A STRUCTURAL AND FUNCTIONAL STUDY Master of Science, 1999 Agatha Lau Department

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The author retains ownership of the L'auteur consewe la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation,

Page 3: OF LC3 THE AU-RICH ELEMENT OF FIBRONECTIN · RNA-BINDING OF LC3 TO THE AU-RICH ELEMENT OF FIBRONECTIN mRNA: A STRUCTURAL AND FUNCTIONAL STUDY Master of Science, 1999 Agatha Lau Department

RNA-BINDING OF LC3 TO THE AU-RICH ELEMENT OF FIBRONECTIN

mRNA: A STRUCTURAL AND FUNCTIONAL STUDY

Master of Science, 1999

Agatha Lau

Department of Laboratory Medicine and Pathobiology, University of Toronto

ABSTRACT

During neointima formation in the Fetal ductus arteriosus (DA) and in diseased coronary arteries,

migration of smooth muscle cells (SMC) is modulated by upregulation of fibronectin (FN). This

involves increased translation of FN mRNA when microtubule-associated protein 1 light chain 3

(LC3) binds an AU-rich element (ARE) in the 3' untranslated region (32JTR) of FN rnRNA. We

now investigate how LC3 binds to the ARE by generating LC3 peptides and mutant LC3, followed

by northwestern (NW) immunoblotting and electrophoretic mobility shift assay (EMSA). The

positive charge of 3 arginine residues (the arginine-nch motif)(ARM) appears critical for LC3-ARE

binding. The significance of this motif in regulating FN mRNA translation is dernonstrated by

stably-transfecting wild-type (WT) vs mutant LC3 into LC3-nul1 HT1080 human fibrosarcoma

cells and observing increased fibronectin with wild-type LC3 only, associated with an elongated,

slower growing phenotype.

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First of d l , 1 would like to express my deepest gratitude to my supervisor, Dr. Marlene Rabinovitch, for her tireless support and encouragement in these past two years. Her enthusiasm and talent in science, as weli as her determination and perseverance, have inspired me enormously in shaping my future path in iife and career. It has been an invaluable experience to pursue science under her supervision.

1 would also like to thank the members of my advisory cornmittee for contributing their precious time and scholarly advice to this work Dr Philip Marsden, Dr Fred Keeley, Dr Mario Moscarello and Dr Emil Pai.

1 am indebted to the members in Dr Rabinovitch's laboratory and the Division of

Cardiovascular Research for their help, consideration and humor. Special and most sincere thanics to Dr Bin Zhou, who is always so eager and patient to help and taught me al1 those

speciai 'tricks' in doing experiments. Also, 1 sincerely thank Dr Hassan Zaidi and Dr Haisong Ju, the most comprehensive and user-fnendiy RNA and Cellular Biology

'Dic tionaries' ; Dr Catherine Mason, the Northem 'superstar'; Claire Coulber and Alecia Lennard, my cells' most caring 'baby-sitters' while 1 was away; Joan Jowlabar, our wonderful secretary, who helped me with ail those letters and slides; and Dr Pascale Dufourcq, who kindly granted me the pnvilege to stay in her apartment while she was away, saving me from commuting during al1 the hazards of snow storms and TTC strike!

It was also my fortune to have met, worked and become fkiends with al1 these excellent people: Dr Peter Jones, Andrea Burry, Stacy O'Blenes, Caroline Fallery, Sandra Demaries,

Kyle Cowan and Arian Khandari (a very special one h m ET!)

Lastly, 1 would like to dedicate this thesis to my beloved parents, Francis and Cecilia Lau, for their unconditional and unlimited support and care, as well as endurance and understanding in the past 25 years.

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TABLE OF CONTENTS

ABSTRACT

ACKNOWLEDGEMENTS

TABLE OF CONTENTS

LIST OF FIGURES

LIST OF ABBREVIATIONS

INTRODUCTION

Overview

1. Fibronectin and Intima1 Cushion in the Developing Ductus Arteriosus

Fibronectin and Srnooth Muscle Cell Migration

Regulation of Fibronectin Synrhesis

Light Chain 3 (LC3) Upregulates Fibronectin mRNA Translation

Microtubule Involvement in LC3-mediated Enhunced FN M A Translation

Phosphorylation of LC3 Enhmrces LC3-mediated FN Translational Upregulatron

Cell Motiliv is Relared to Other LC3-reguiafed Genes Bedes Fibronectin

II. Fibronectin in Post-Cardiac Transplant Coronary Arteriopathy

Neointinml Formation in Posr-Cardiac Tramplant Coronary Aneriopathy

Fibronectin and Migration of Inflammatory Cells und Smooth Muscle Cells

Coronary Artery Smooth Muscle Cells

iii

viii

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III.

IV.

Fibronectin Reverses the Transformed Phenotype of HTlOSO, a - -

Human Fibrosarcoma Ceïï Line

Dom-regulatr8n of Fibronectin in Oncogenic Trmisformuhon

Fibronectin Suppresses the Transfomd Phenotype of HTIOBO Cells

Role of EGR-2 in FN Upregulation and Tumor Suppression

Stable Transfection of LC3 in HT1û80 Cells Upregulates FN Syntheis

Role of Protein-RNA Interactions in mRNA Translation

Role of 3' Untranslated Region ( 3 ' m ) AU-Rich Element (ARE) in mR Translation

ARE-binding Proteins mtd Their Functiom

RNA-binding Motvs

HYPOTHESES and OBJECTIVES

MATERIALS AND METHODS

Ce11 Culture

Expression of Recombinant LC3

Partial Proteolysis of LC3 by Endoproteinases

5' end-radiolabeling of RhrA Probe

Zn vitro Transmption of RNA Probe

North western (NW) Blot Analysis

Site-directed Mutagenesis

Gel Mobility Shrfi Assays

Stable TrCUZSfection

Western Immunoblot Analysis

Cell Growth Curves

Indirect Immunofluorescence

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FN Biosynthesis

RNA Isolation and Northern Blot AnalysiS

RESULTS

LC3 Binds the ARE of FN mRNA at the IOkD N-terminal Region

LC3 Binds Specificaily to ARE of FN mRNA

Arginine-Rich Motif (ARM) is Cntical in LC3 Binding to ARE of FN rnRNA

LC3 Buidhg to ARE via ARM Codbned in Stably-transfected HTl080 Ceils

Effect of WT and Mutant LC3 on FN Synthesis

LC3 Regulates Ceii Growth and Morphology via ARM-ARE Binding

Subcellular Locahatîon of WT and Mutant LC3 in Stable- transfectants

DISCUSSION

FUTURE STUDES

APPENDIX I

APPENDIX II

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LIST OF FIGURES

Schematic summary of FN regdation by IL-1p. TNF-a and EDP as described in the text-

Figure 1

Figure 2

Figure 3

Figure 4

Schematic ribbon drawing of the hnRNP C RBD (residues 2-94).

Amino acid sequence of rat LC3.

Northwestern (NW) blot anaiyses show LC3-ARE binding localizes to a 10 kD N-tenninal region on LC3.

Figure 5 Arnino acid sequence of rat LC3 showing the 2 candidate RNA binding sites.

Fi,oure 6

Figure 7

Figure 8

Sumrnary diagram of NW biot analysis of LC3-ARE binding.

RNA binding of LC3 protein and lOkD peptide is preferential for the ARE.

LC3 binds ARE at the arginine-rich motif (ARM) via a charge-charge interaction.

Figure 9 Gel mobility shifi assay on HTlO8O cytosolic extracts with stably transfected WT and mutant LC3 confirms the significance of the ARM in AREcbinding.

Figure 10 Tmmunofluorescence labeling of ceil swface FN deposit in WT and mutant LC3 transfected HT1080 celis.

Figure I l FN expression in specific clones of WT and mutant LC3 stably-transfected HT1080 cells.

Figure 12 Steady state levels of FN mRNA in WT and mutant LC3 stably-transfected HT1080 ceiis.

TransIational efficiency of FN mRNA in WT and mutant LC3 stably- transfected HTlO8O cells.

Figure 13

CeU count of WT and mutant LC3 transfected HTlOSO celis. Table 1

Figure 14

Figure 15

Figure 16

Growth curves of WT and mutant LC3 transfected HTlO8O cells.

Effect of WT and mutant LC3 expression on HTlO8O cell morphology.

Immunofluorescence labeling of tubulin in WT and mutant LC3 transfected HTlO8O ceus.

Figure 17 hunofluorescence labeling of WT and mutant LC3 in distinct subceliular locations in stably-transfected HTlOSO cells.

Figure 18 Quantitative analyses comparing the subceiiuia. distribution of WT and mutant LC3 in stably-transfected HT1080 cells by western immunoblot.

vii

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LIST OF ABBREVIATIONS

A

Ao

D

ARE

ARM

ATP

AUBF

BSA

C

CA

CD

cDNA

CNBr

cpm

CS

CS 1

CS-RBD

DA

DAPI

d m

DNA

Dm

EC

ecNOS

EBP

aàenosine

aorta

apoiipoprotein D

adenosine uridine nch element

arginine-nch motif

adenosine S'-triphosphate

adenosine uridine binding factor

bovine serum aibwnin

cytosine

coronary artery

caihepsin D

complementary deoxyribonucleic acid

cyanogen bromide

count per minute

chondroitin sulphate

connecting segment 1

consensus RNA-binding domain

ductus artenosus

4', 6-diamidino-2-phenylîndole

deoxycytidine S'-triphosphate

deoxyribonucleic acid

dithiothreitol

endothelid ceils

endotheiial constitutive nitric oxide synthase

elastin-binding protein

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ECM

EDP

EDTA

EGF

e h

EMEM

EMSA

EPAN

EPLC

F

FBS

FN

FP

G

GAG

GAPDH

GM-CSF

GST

h

HA

HC1

Hel-N1

hnRNP

H R P

IgG

IL-f p

IL-3

extracellular rnahix

eiastinderived peptide

ethylenediamine te-tic acid

epidermal growth factor

embryonic lethal abnormal vision

Eagle's minimal essential medium

electrophoretic mobility shift assay

endoproteinase Lys-C

pheny lalanine

fetal bovine serum

fibronectin

free probe

guanosine

glycosaminoglycan

gl yceraldeh y de 3-phosphate dehydrogenase

granulocyte macrophage colony-stimulating factor

glutathione S-transferase

hour

hyaluronan acid or hemaglutinin

hy drochlonc acid

human elav-like neuronal protein 1

heteronucleoprotein

horseradis h peroxidase

immunoglobulin G

interleukin-1 $

interleukin-3

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N O S

m-'Y IPTG

K

KCI

kD

KH

k-EL

LC3

LPS

M

MAP

MHC

Mgcl?

min

mRNA

N W

n

NaCl

nNOS

NO

NOS

P

PA

PAGE

PBS

PCR

uiduciile niûic oxide synthase

in terferon-y

isopropy l-PD-thiogalactopyranoside

lysine

potassium chloride

kilodaiton

K homology

kappa-elastin

light chah 3

lipopolysaccharide

methionine

microtubule-associateci protein

major histocompatibilty complex

magnesium chloride

minute

messenger ribonucleic acid

northwestem

number of experiments or samples

s o d i u m chloride

neuronal nitric oxide synthase

nitric oxide

nitric oxide synthase

phosphate

pulrnonary artery

pol yacry lamide gel elecirophoresis

phosphate buffered saline

polymerase chain reaction

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PCTCA

PDGF

Phe

PI

PNK

PVDF

Q R

RBD

RER

RGD

RGG

rl?m

RNA

RNP

RNP-CS

RRM

rRNA

SD

SDS

SEM

SMC

snRNA

TBE

TBS-T

TCA

TGF-B

post-cardiac trausplant coronary aaaiopay

platelet derived growth factor

p hen y lalanine

isoelectric point

polynucleoti& kinase

pol yvinyldifiuoride

glutamine

arginine

RNA binding domain

rough endoplasmic reticulum

arginine-giycine-aspartate

@nine-gl ycine-gi ycine

revolutions per minute

ri bonucleic acid

ri bonucleoprotein

ribonucleoprotein consensus sequence

RNA recognition motif

ribosomal ri bonucleic acid

standard deviation

sodium dodecyl sulphate

standard emr of the mean

smwth muscle cells

small nuclear ribonucleic acid

tris-hm&-EDTA

tris buffered saline-tween 20

trichloroace tic acid

transfonning growth factor-$

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tumor necrosis façt0r-a

transfer ribonucleic acid

3' untranslated region

uridine

uridine S'-triphosphate

ultraviolet

very l ate antigena

wild type

xii

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INTRODUCTION

Overview

Upregulation of fibronectin 0 is associated with the smooth muscle cell (SMC)

migratory phenotype contributhg to intimai cushion formation in the closure of the ductus

arteriosus @A) in development and to the formation of the neointima in the post-cardiac

transplant coronary arteriopaîh y (PCTCA) and other occlusive vascular diseases. Studies

on the mechanism of FN upregulation in vascular diseases led to the discovery that hght

chain 3 (LC3) of microtubule-associated protein LA and 1B binds to the AU-rich element

(ARE) in the 3' untranslated region (3'UTR) of the FN mRNA and upregulates its

translation via ribosomal recniitment. This thesis investigates the peptide sequence in LC3

which binds to the ARE of the FN mRNA, and examines its role in regdating FN mRNA

translation using the HTLOSO human fibrosarcoma ceil line. The transformed phenotype of

HT1080 ceils stably transfected with wild type (WT) and mutant LC3 cDNA is dso

examined.

The following Introduction therefore reviews the role of FN in intimai cushion formation

during the closure of the DA as well as in PCTCA, outlining the effect of FN on SMC

migration, and rnechanisms involved in FN upregulation with emphasis on the roles of

LC3 and microtubules. The data supporting the role of FN in reverting the transformed

phenotype of HT1080 cells is then addressed. This is followed by a review of RNA-

protein interactions which modulate mRNA translation, focusing on the importance of

ARE, the function of other ARE-binding proteins, and common RNA-binding motifs.

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1. Fibronectin and Intimal Cushion in the Developing Ductus Arteriosus

Ductus Arîen'osus

The ductus artenosus (DA) is a large fetal shunt comating the pulrnonary artery with the

aorta and allowing the majonty of the right ventricular cardiac output to bypass the

unexpanded lungs. The ductus closes at birth as the lungs expand and blwd oxygen

tension nses. The onset of breathing increases artenal oxygen tension mggenng the strong

vasoconstriction of the ductus. This process is mediated by a cytochrome-P450-dependent

mechanism (Coceani et al., 1988) which results in the release of a potent vasoconstrictor

endothelin-1, from endotheLial and smooth muscle cells (Coceani and Kelsey, 1992).

However, the functional closure of the DA is highly dependent on the prior formation of

the intimal cushions, a process which is initiated around 100 days of a 145-&y gestation

period in the fetal lamb DA, and is more or less completed by day 138. The formation of

intimal cushions is initiated by the accumulation of extracellular matrix @CM) in the

subendothelium, the region separating the endothelial cells (EC) from the intemal elastic

laminae- Smooth muscle cells (SMC) f'm the rnuscular media of the vesse1 wall migrate

in to the rnatrix-enric hed subendothelid region.

Extracellular Matrix-Ce22 Interactions in IntimaZ Cushion Formation

Studies from our iaboratory have shown that a sequence of cell-matrix interactions plays a

criticaï role in the remodeling process associated with the formation of intimal cushions.

The increased production of glycosaminoglycans (GAGS) specifically hyaluronan acid

(HA) by EC and chondroitin sulfate (CS) by SMC leads to their accumulation in the

subendothelium (Boudreau and Rabinovitch, 199 1). The hydrophilic properties of HA

allow it to bind large arnounts of water which causes expansion of a tissue space and

physically facilitates ce11 movement (Toole et al., 1984). The increased production of CS

causes shedding of the 67 k D elastin-binding proteins h m SMC surfaces, leading to the

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impaired assernbly of elastin fibers (Hinek et al., 1991) and to the increased production of

elastin peptides, al1 of which favor the unrestricted movement of SMC into the

subendothehum. The major elastin peptide producecl was identified as the 52 k D muicated

form of tropoelastin (EGnek and Rabinovitch, 1993). This peptide cannot be insolubilized,

but is stable and a potent chernotactic factor to SMC. This elastin peptide can also directly

influence the migration of SMC by inducing their production of the matrix glycoprotein

fibronectin (FN) (Hinek et al-, 1992).

Fibronectin and Smooth Muscle Ce12 Migration

Fibronectin is a homodimenc glycoprotein composeci of subunits of 220-250 kD linked by

disulfide bonds close to their carboxy-terminal ends. It is present in the plasma or

associated with cells or their ECM. It influences ce11 adhesiun, migration, proliferation,

differentiation, cytoskeletal organization and apoptosis (Hynes, 1990; Hynes and Lander,

l992), depending on its interaction wi th the heteroàimeric transmembrane ECM binding

proteins, integrins. A specific RGD (arginine-glycine-aspartate) sequence located in the

type III repeat of FN is recognized by the aspl and a& integins (Pierschbacher and

RuosIahti, 1984; Rouslahti and Pierschbacher, 1987). Studies on SMC adhesion and

migration using blocking antibodies specific to different integrin complexes showed that

SMC adhesion to FN depends exclusively on hinctioning fil integrins while SMC

migration depends on both the UV pj and a4$, integrin receptors (Clyman et al.. 1992;

Molossi et al., 1995~).

FN has been implicated in SMC migration by modulating SMC from a 'contractile' to a

'synthetic' phenotype. In response to FW, cultured rat aortic SMC exhibit ce11 adhesion

and spreading, loss of myofilaments, cessation of the ability to contract, formation of

extensive rough endoplasmic reticulum (RER) and golgi complexes, increased RNA and

protein synthesis, as well as the ability to replicate DNA, divide and produce ECM

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components in response to platelet-derived growth factor (PDGF) and other mitogens

(Hedin and Thyberg, 1987; Hedin et al., 1988). The minïmaI cell-attachment sequence on

FN, RGDS, was found to be responsible for the FN-mediatecl SMC dedifferentiation by

interacting with the B intefins (Hedin et ai., 1989). This FN-mediated-phenotypic change

also likely contributes to the enhanced SMC migration observed in intimai cushion

formation in the DA. In a parallel study comparîng cultured SMC h m the DA, aorta (Ao)

and pulmonary artery (PA) of lûû-day fetal lambs, DA SMC demonstrated a 2-fold

increase in FN synthesis compared to Ao and PA celis (Boudreau and Rabinovitch, 1991).

This upregulation of FN appears to contribute to the spindle-iike etongated morphology of

the DA SMC, as well as theîr enhanced migration in three-dimension collagen gels, since

RGD peptides and antibodies against M can convert DA SMC to a flattened, stellate

morphology and inhibit their migration to a level similar to Ao SMC (Boudreau et al.,

199 1).

In addition to directly altering SMC phenotype, increased production of FN leads to the

stimulation of migration dong a FN gradient as has been demonstrated in chick embryonic

precardiac ceiis duing heart formation. This directional migration can also be abrogated by

an antibody to FN or RGD peptides which interact with integrins and this results in

inhibition of cardiac development (Linask and Lash, 1998a; Linask and Lash, 1998b).

That FN-mediated change in phenotype and directional migration were critical to the infiux

of SMC into the subendothelium which leads to intima1 cushion formation in the DA, was

demonstrated by Mason et al (Mason et al., 1999a), based upon manipulation of the

regulatory mechanism of FN synthesis in fetal lambs.

Regulation of Fibronectin Synthesis

Upregulation of FN synthesis occurs at both transcriptional and pst-transcriptional levels.

Growth factors and cytokines such as epidermal growth factor (EGF), transfonning

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growth factor-6 (TGF-fl), platelet-derived growth factor (PDGF) and interferon-y (IFN-y)

activate FN gene transcription (Blatti et al., 1988; Chen et al., 1977, Diaz and Jimenez,

1997). Interleukin- l$ (IL- 1 f3) upregulates FN gene transcription in vascular SMC

(Clausel1 and Rabinovitch, 1993; Molossi et al., 1995a) whereas hmor necrosis factor-a

(TNF-a) which cooperatively interafts with IL16 appears to influence FN synthesis at a

post-transcriptional level. This will be discussed in the next section. A number of

cytokines and growth factors have been shown to upregulate FN expression at a post-

transcriptional level by modnlatmg mRNA splicuig or stability or translational efficiency.

For example, TGF-8 increases transcription of FN mRNA in fibroblasts Qgnotz et al.,

1987), alters the splicing pattern of FN mRNA in cuitured normal human fibroblasts (Borsi

et al., 1990) and increases FN mRNA stability in cultured human dermal fibroblasts

(Raghow et al., 1987). In contrast, while IFN-y can upregulate FN gene transcription, its

ability to destabilize the FN mRNA and repress FN mRNA translation by inhibiting the

elongation steps results in an overall downregulation of FN synthesis in cultured human

and murine fibroblasts (Diaz and Jimenez, 1997; Levine et al., 1990).

Boudreau et al. found that the increase in FN synthesis in DA compared to aorta SMC was

not associated with an increase in steady state levels of FNA mRNA or in mRNA stability

or in differences in FN mRNA splicing (Boudreau et al., 1992). suggesting that enhanced

translational efficiency of FN mRNA in DA SMC results in the observed FN upregulation.

Zhou et al. (Zhou et al., 1997) later identified a protein which binds the adenosine-uridine

rich element (ARE) of the 3' untranslateci region (UTR) of the FN mRNA and upregulates

its translation through enhanced ribosome recruitment. This RNA-binding protein was

identified as the light chain 3 (LC3) of the microtubule-associated protein w) 1A and

1B.

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Light Chain 3 (LC3) Upreguhtes Fibronectin mRNA TransZation

Light chah 3 (LC3), a 16.4 kD protein enriched in rat brain and CO-purified with

microtubules, was first identified by J. Hammarback as a subunit of the neuronal

~crotubule-associated proteins (MAPs), MAPlA and MAPlB (Mann and Hammarback,

1994). It was thought to play a role in regulating the microtubule binding activity of

MAPlA and MAPlB. Zhou et al. purified LC3 from the DA and Ao SMC as a

cytoplasmic factor which binds the AU-rich element (ARE) of the 3' UTR of the FN

rnRNA (Zhou et al., 1997). It was expressed at higher levels in the cytosolic extracts from

the DA compared to the Ao SMC, and was associateci with increased ARE-binding activity

and increased FN mRNA translation in the DA SMC. Its role in pst-transcriptionai

upregulation of FN, was further established by demonstrating that overexpression of

recombinant LC3 in Ao SMC, resulted in enhanced FW mRNA translation to Ievels

observed in the DA SMC without altering FN mRNA levels (Zhou et al., 1997). ïhis

LC3-mediated upregulation of FN mRNA translation is also dependent on nitric oxide

(NO), which is induced in the DA compared to the Ao associated with both increased

expression of neuronal NO synthase (nNOS) and endothelid constitutive NOS (ecNOS).

Increased NO results in enhanced phosphorylation and binding of LC3 to the ARE of the

FN mRNA (Mason et al., 1999b). Since LC3 is CO-p&ed with microtubules in vivo and

associated with microtubules assembled from purified tubulin in vitro (Mann and

Hammarback, 1994), microtubules appear also be involved in iranslational regulation of

FN (Zhou et al., 1998).

Microtubule Involvement in LC3-mediafed Enhanced FN mRNA Translation

MicrotubuIes have been implicated in the storage, sorting and translational control of

mRNAs (Singer, 1992; St Johnston, 1995). However, most of the studies about

microtubule-mediated translationai regulation or localization involve mRNAs encoding

cytoskeletal or cytoplasmic proteins, whereas little is known about how microtubules

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regulate translation of mRNAs encoding secreted pmteins such as F N Zhou et al. (Zhou

et al., 1998) inves tigated the role of microtubules in influencing LC3-mediated FN mRNA

translation. Since FN is a secreted ECM glycoprotein synthesized at the rough

endoplasmic reticulum (RER) bound to polysomes and then packaged into secretory

vesicles for later release, it was hypothesized that microtubules may help target FN mRNA

to membrane bound polysomes for translation via LC3. When cultured DA SMC were

treated with colchicine to disrupt microtubules, FN mRNA translation was inhibited,

concomitant with the decreased association of FN mRNA and LC3 protein with the RER

(heavy pdysomes in sucrose &nsity gradient) (Zhou et al., 1998).

Phosphorylation of LC3 Enhances LC3-mediafed FN Translational

Upregulation

Consistent with a dual function as both the microtubule-binding protein and the RNA-

binding protein, LC3 was found to distribute between 2 functionaiiy distinct pools within

the ceII: the unphosphoryfated, translationdy inactive form binds the microtubuIes while

the phosphorylated, translationally active form associates with the FN mRNA and the other

components of the translational machinery such as 60s ribosomal units. Phosphorylation of

LC3 has been demonstrated to shift LC3 fimm the microtubules towards the membrane-

bound polysomes probably by enhancing the LC3-ARE binding activity, associateci with

induction of FN mRNA translation (Mason et al., 1999b).

Cell Motiliry is Related to m e r Genes Besides Fibronectin

Studies in DA SMC and in CA SMC in post-cardiac transplant coronary arteriopathy

(PCTC A) (detailed discussion in next section) show a correlation between FN upregulation,

ce11 motility and migratory phenotype. However, other genes in both DA and CA SMC

may also contribute to this phenotype as a 'consteiiation' regulated at a pst-transcriptional

level by the LC3 switch. Using an LC3 protein affinity column and incubating it with

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RNA harvested from adult rat brain (a nch source of microtubule assocîated proteins), a

bound transcript was identified which encoded the 3'UTR of apolipoprotein D (apo D),

which also contains an ARE-like element (UUAUCTUCUU)(Burry, Andrea, M.Sc Thesis,

Department of Lab Medicine and Pathobiology, 1998). Apo D was increased in DA

compared to Ao SMC, and in migratory compared to non-migratory cells. suggesting that it

might be necessary for the motile SMC phenotype.

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II. Fibronectin in Post-Ciràiac Transplant Coronary Arteriopathy

Neointimal FoIlll(lfi0on in Post-Cardiac Transpiant Coronary Artenoopathy

Post-cardiac transplant coronary arteriopathy (PCI'CA) is a major complication affecting

the long-term survival of cardiac transplant recipients (Uretsky et al., 1992). It is

characterized by occIusive neointimal formation in the donor heart coronary arteries. It

represents an ongoing immune-inflammatory reaction in the vesse1 wali with activation of

endotheliai cells expressing major histocompatibilty complex W C ) II antigens which

s tirnulate 1 y mp hoc yte binding and proli feration, and sustained released of growth factors

and cytokines, causing proliferation and migration of SMC into the subendothelium and

accumulation of ECM, notably EN, contributing to intimal thickening (Solaman et al.,

199 1; Clausel1 et al., 1993). The latter is similar to the intimal cushion formation in DA:

Increased fragmentation of the interna1 elastic laminae is also observed in PCTCA

associated with increased activity of a serine elastase measured in donor coronary arteries

following heterotopic heart transplant in piglets (Oho and Rabhovitch, 1994).

Fibronectin and Migration of Inflmmaiory Cells und Sntooth Muscle Cells

Cytokine-mediated FN upregulation in donor coronary EC and SMC in PCTCA was

responsible for recruiting inflamrnatory cells, through interactions of the CS 1 and RGD

motifs on FN with inflammatory ce11 surface integrins, &pi and asBi respectively. In a

porcine endothelial-smwth muscle celi co-culture system, IL-l&stimulated EC and SMC

FN s ynthesis and induced transendothelial lymphocyte migration, which was blocked by

CS 1 and RGD synthetic peptides and FN antibodies (Molossi et al., 199Sb). Blockade of

the FN binding VLA4 (a&) integrins on lymphocytes with CS1 peptides in rabbits

following heterotopic cardiac transplantation resulted in reduced infiltration of T cells, l a s

accumulation of FN and a >50% decrease in the incidence and severity of donor coronary

artery intimal thickening (Molossi et al., 1995~). These studies provided further evidence

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that increased expression of FN was not only critical in fafiüiatiag migration of SMC in the

developing intima1 cushion of the ductus but also in the pathological formation of the

neointima in vascular disease.

Coronary Artery Smooth Muscle Cells

The mechanism responsible for the upregulation of FN synthesis in donor versus host heart

coronary artery (CA) SMC was specificaily investigated, In piglets after heterotopic

cardiac transplantation, the early development of a cmnary arteriopathy is charactenzed by

increased immunostaining for FN and IL-Ip in the vesse1 wall (Clausell et al., 1993).

Further investigation using cultured donor and host CA SMC demonstrated an increased

steady state level of M mRNA in donor cells, which could be reduced to host ce11 levels

by neutralizing antibodies to IL-1s associated with a decrease in FN synthesis to host cell

levels. This supported ILI$ upregulation of M synthesis by inducing FN gene

transcription (Clausell and Rabinovitch, 1993). On the other hand, TNF-a induces FN

synthesis in CA SMC without increasing M mRNA levels, implying that the regulation

may be at a post-transcriptional level (Molossi et al., 1995a). This might explain the

reciprocd interaction of IL-ID and TNF4 in modulating FN synthesis, as illustrateci in Fig

1.

IL- 1 f3 induces its own gene transcription (DinarelIo, 199 1; Wamer et al., 1987) as well as

the transcription of the TNF-a gene by activating the transcription factor NF-- via a

protein kinase C-dependent pathway (Bethea et ai.. 1992). Similarly, TNF-a induces its

own mRNA translation (Clausel1 et aZ., 1994) dong with the induction of the IL-1$ gene

transcription by the activation of IL4 transcription factor NF-- (Kruppa et al., 1992).

TNF-a also stabilizes IL-1$ mRNA via pmtein kinase C activity (Gomspe et al., 1993). It

is proposed that TNF-a upregulates FN synthesis via the upregulation of IL-@, but also

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Figure 1. Schematic summary of FN regulation by IL-lp, TNF-a and EDP

as described in the text.

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IL-1p gene TNF-a gene

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via another pathway leading to the post-transcriptional upregulation of FN. This

phenornenon was demonstrated by the ability of both TNF-a antibodies and IL-lp

antibodies alone to block FN synthesis induced either by IL-1s or TNF-a (Molossi et al.,

1995a).

IL- 1 may also regulate FN synthesis at a pst-tranmiptional level by inducing elastase

activity and the production of elastin-derived peptides (EDP). EDP, in the form of kappa-

elastin @-EL), augments IL-l$ upregulation of FN synthesis in CA SMC (Cowan et al.,

1999, rnanuscipt submitted). It is postulated that EDP causes a conformation change in

the ce11 surface elastin-binding protein (EBP), facilitating the binding of IL-lp to type 1

surface receptor, a process which can be blocked by IL-@ receptor antagonists.

Al ternativel y, EDP, like TNF-a, increases the efficiency of FN mRNA translation,

following enhanced transcription by IL-1$. When only exogenous EDP is added to CA

SMC, FN spthesis can also be induced without a corresponding increase in mRNA levels

(Co wan et al., 1999, manuscript submitted).

Besides regulating Llf5 expression, TNF-a also upregulates FN synthesis via an NO-

dependent pathway. TNF-a causes an induction of NO production in cultured CA SMC,

resulting in an increased binding of LC3 to the ARE in the 3' UTR of the M mRNA,

thereby increasing its translational efficiency (Mason et al., 1999, manuscript in

preparation). A similar NO-dependent LC3-mediated upregulation of FN mRNA

translation has also be demonstrated in intima1 cushion formation in DA (Mason et al.,

1999b) which has been discussed above.

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III. Fibronectin Reverses the Tradormed Phenotypc of HT1080, a Human

Fibrosarcoma Ceii Line

Do wn-regdation of Fibronectin in Oncogenie Tronsformotr-on

Loss of FN fkom the ce11 surface has been show to be closely associated with malignant

transformation of cells, and has been related to the decxeased cellular adhesion and

increased metastasis of tumor cells (Hynes, 1973; Gahmberg and Hakomori, 1973). The

tumongenic down-regulztion of FN has been exclusively studied in N-tas-transfomd

human fibrosarcorna HT1080 ceils, which express low levels of FN and lack cell surface

FN matrix deposits (Oliver et al., 1983; Dean et al., 1988), in response to the N-ras

oncogene (Brown et al., 1984; Paterson et al., 1987). The effect is post-transcriptional

(Chandler and Bourgeois, 1991), and was attributed to a reduction in nuclear processing or

stability of processed FN mRNA (Chandler et al., 1994), causing a shorter half-life of

about Il hours compared to 70 hours in nonnal fibroblasts. The end result is a 50 fold

decrease in basal levels of FN compared to normal fibroblasts. For this m o n transformed

ce11 lines have been commonly used to study the role of FN in ceil growth, differentiation

and adhesion, as well as the different regulatory mechanisms of FN gene expression.

Recent studies suggested that the adhesion of cells to ECM proteins, particularly FN,

through the integrin famil y of adhesion receptors transduces si p a l s that regulate ce11

proliferation, differentiation and apoptosis (Juliano and Haskill, 1993; Miyamoto et al.,

1995).

Fibronectin Suppresses the Transfomard Phenotgpe of HT1080 Cells

To elucidate the role of FN in modulating malignant ce11 phenotype, Akamatsu et ai.

overexpressed a full-length cDNA encoding plasma-type FW in HTlOSO human

fibrosarcoma cells. This resulted in a more flattened morphology, deposition of a

moderately developed FN rnatrix, reduced ceU motility on the substratum and poor growth

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when these cells were injectai S.C. into nu& mice. Overexprcssion of FN also suppressed

the ability of the tumor cells to proliferate in soft agar, which couid be reversed by RGD

peptides and antibodies against FN (Akamatsu et al., 1996). These resuits indicated that

increased deposition of FN in the pericellular matrix per se can suppress the motility and

growth potential of mmor cells through interaction with RGD-recognizing or aspi

integins. Moreover, the binding of the integrin %pl to substrate-adsorbed FN has been

reported to inhibit DNA synthesis in ~ 1 0 8 0 cells (Wang et al., 1995).

Besides overexpressing recombinant FN by transfection, upregulation of FN synthesis in

HT 1080 cells can also be achieved by inducing endogenous FN production at different

molecular levels. For example, while dexamethasone (a synthetic glucocorticoid)

upregulates FN expression by augmenting the steady state level of unspiiced FN tranmipts

and increasing the mRNA half-iife from -11 to 26 hours, it has no effect on either the

morphology or the growth rate of the cells (Ehretsmann et al., 1995; Dean et al., 1988).

Forskoiin (an activator of adenylate cyclase) and transforming growth factor (TGF-$)

both increase the rate of FN gene transcnption. While TGF-p has no effect on the

morphology and the growth rate of the cells, Forskolin causes cells to become more

elongated with numerous projections and significantly decreases their growth rate (Dean et

al., 1988). These studies, in contrast to Akamatsu's mentioned above, suggested that

upregulation of FN is not exclusively related to the revertant phenotype of HT 1080 cells.

Instead, concomitant regulation of other genes or proteins, might be important in reversing

the transformed phenotype of HT 1080 celis.

Role of EGR-I in FN Upreguiation and Tumor Suppression

EGR-1, a transcription factor, is a member of the immediate early growth response gene

family that shares close homology in its DNA-binding domain with a well-known tumor

suppressor gene, WT-1. Its expression level is significantly reduced in human breast

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tumor ce11 lines (Huang et al., 1997) and in the HT1080 human fibrosarcoma ceil Line

(Huang et al., 1995). Transfection of EGR-1 into HT1080 cells leads to FN upregulation

as well as tumor suppression. Transfected EGR-1 increases FN secretion via 2 pathways:

a direct induction of FN gene transcription by binding to a known positive transcription

activation site in the FN prornoter; and an indirect mechanism associated with the

upregulation of TGF-$ (Liu et al., 1999) which enhances FN gene transcription (Dean et

al., 198 8). EGR- 1 exerts its tumor suppressing ability probably by downregulating Bcl-2

expression, since overexpression of Bcl-2 in EGR-1 msfected cells restores the

transformed phenotype to these fibrosarcoma cells (Huang et al-, 1998). This m e r

confirms a dissociation between FN upregulation and tumor suppression observed in

HT 1080 ceils. That is, increased FN may be necessary but not sufficient to revert the

maiignant HT1080 phenotype.

Stable Transfection of LC3 in LIT1080 cells Upregdàtes FN Synthesii

LC3 increases FN expression in cultured DA SMC by binding to the ARE in the 3'UTR of

the FN mRNA thereby upregulating its translation (Zhou et al., 1997). To elucidate the

detailed mechanism involved, HTlOSO cells, with no detectable LC3 expression and

negligible FN ce11 surface deposits, were stably transfected with an LC3-encoding plasmid

to study the regulation of FN synthesis (Zhou et al., 1999, manuscript submined). LC3

expressed in the HT1080 cells localizes to the perinuclear region, where most of the

translational machinery (polysomes) are localized. It also distributes dong the microtubule

filaments as granular particles, consis tent with its property as a microtubule-associated

protein. Synthesis and ce11 surface deposition of FN is selectively induced by the

expression of LC3, together with a morphologicd change from a rounded to a flattened cell

shape consistent with a 'revertant' phenotype (Paterson, 1987), a re-arrangement of

microtubules, and a slower growth rate (Zhou et al., 1999, manuscript submitted). While

there is no difference in FN mRNA levels in LC3 and vector transfected cells, polysome

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proNe analysis in LC3 transfected ceiis &rnonstrated that FN mRNA is more concentrated

in heavy polysornes where LC3 and ribosomai subunits are co-dïstributed. The finding

that LC3 also binds 40s and 60s ribosomai subunits in vitro (Zhou et al., 1999, manuscn'pt

submined) leads us to propose that LC3 upregulates tranlsation of FN mRNA by ribosome

recruitrnent .

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IV. Role of Protein-RNA Interactions in mRNA Translation

Role of 3' Untrcrnsloted Region (3VTR) AU-Rich Eknrent (ARE) in mRNA

Translation

AU-rich elernents are common regdatory sequences within the 3' untranslated regions of

mRNAs encoding infiammatory mediators or immediate early response genes such as

cytokines, oncogenes, and signaling molecules involved in cell growth and differentiation,

as well as FN. Thes elements modulate both mRNA stability and translational efficiency.

Caput et al. found that the octanucleotide sequence, UUAUUUAU, exists as one or

multiple copies, and is important for these hinctions (Caput et al., 1986). Its role as an

rnRNA destabilizing elernent was first discovered in c-fos (Treisman, 1985) and GM-CSF

(Shaw and Kamen, 1986). Insertion of the 3'WR from these 2 genes into globulln

mRNA resulted in the destabilization of the message. Since then, the presence of the ARE

has been shown to destabilize a large number of labile mRNAs, including c-myc (Jones

and Coles, 1987), IFN-$ (Whittemore and Maniatis, 1990) and IL-3 (Wodnar-Filipowicz

and Moroni, 1990).

Ln addition to their function as destabilizing elements, ARES can also modulate translational

efficiency. To date, most of the studies show that ARES inhibit mRNA translation. In the

case of IFN-p, the presence of the ARES in the 3'UTR of the mRNA greatly suppressed

mRNA translation in both Xenopus oocytes and in the reticulocyte lysate, whereas

removing the ARES from the 3'UTR increased the IM-f3 translation up to 100 fold in

Xenopus oocytes and 10 fold in reticulocyte lysates without affecting mRNA stability

(Kruys et al., 1987; Kmys et al., 1988). ARES from c-fos and GM-CSF have also been

shown to have the similar inhibitory effects on mRNA translation as on mRNA stability

(Kniys et al., 1989).

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There are a number of proposed mechanisms for the inhibitory effects of ARES on mRNA

translation. Kmys et al. demonstrated that less wild-type IFN-p mRNA associated with

polysomes (the translationai machinery) compared to mutant IFN-p mRNA without ARE,

suggesting that the ARE inhibits IFN-$ mRNA translation via decreased recniitment into

polysomes (Kniys et al., 1990). On the other han& Sachs et al showed that increasing

poly A tail length of 1 . - $ mRNA significantly decreased its translational efficiency in

reticulocyte lysates while shortening the poly A tail or removing the ARE fiom the 3'UTR

enhanced translation (Sachs and Davis, 1989; Tanin and Sachs, 1996; Tanin and Sachs,

1995; GraFr et al., 1993). This suggested that ARES might bind the poly A tail and inhibit

its interaction with poly A binding proteins which are required for translational initiation.

Alternatively, the opposite might occur, i.e. binding of specific trans-acting factors on

ARES might facilitate the release of poly A tail, allowing for the binding of poly A binding

proteins and translation initiation.

Thus, AREs are not always associated with mRNA translational inhibition. Han et al.

reported that lipopolysaccharide (LPS) induced TNF-a expression by enhancing mRNA

translation (Han et al., 1990a; Han et al., 1990b; Han et al., 1990c) associated with

complex formation beniveen TNF-a mRNA and the RND-binding protein TIAR (Gueydan

et al., 1999). Zhou et al. also showed that FN upregulation in DA SMC was due to

translational enhancement of the ARE-containing mRNA (Zhou et al., 1997). Instead of

selectively modulating mRNA stability or translational efficiency, in some cases these 2

properties of ARES are actually interdependent. Studies have shown that c-fos and GM-

CSF AREs rely on ongoing translation to exercise their mRNA destabilizing functions,

probably related to the translational-dependent assernbly of a >20s degradation complex

(Savant-Bhonsale and Cleveland, 1992; Aharon and Schneider, 1993; Winstall et al.,

1995). These leads to the hypothesis that ARES can either reduce or increase mRNA

translation, depending on their interaction with tissue-specific cytoplasmic proteins.

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ARE-binding Proteins and Their Functions

Since AREs have been demonstrated to be important in modulatuig mRNA stability and

translational efficiency, numemus cytoplasmic proteins that bind ARE-containing 3'UTRs

have been identified in a variety of cells or tissues. These ARE-binding proteins can be

functionally assigneci to two categories: the fmt group contains proteins such as adenosine-

uridine binding factor (AUBF) (Malter, 1989) and embryonic lethal abnormal vision

(Ela)-like proteins such as Hel-N1 and Hu-R (Levine et al., 1993; Chung et d., 1996; Ma

et al., 1996) whose ARE-binding activities are associated with stabilization of labile

-As as well as enhanced mRNA translation; the second group includes RNA-binding

proteins sucn as AUFl (Zhang et ai., 1993) whose ARE-binding activities correlate with

rapid mRNA decay that may or may not depend on ongoing translation.

AUBF was fmt identified by Malter as an ARE-binding protein in lymphocyte cytoplasmic

extracts (Malter, 1989). Further studies showed its binding to ARE-containing labile

mRNAs including GM-CSF, IL-3, IFN-y, c ~ u s and v-myc. AUBF binds specificaily to

the destabilizing motif AUUUA of mRNA. Mutations within the AUUUA motifs

demonstrate that both nucleotide sequence and s e c o n d q structure are important in

AULTUA.AUBF RNA complex formation (Giliis and Maiter, 199 1). Binding of AUBF to

AUUUA stabilizes the ARE-containing labile mRNAs. In vitro decay assay of GM-CSF

mRNA demonstrated that depletion of AUBF binding activity led to the accelerated decay

of the GM-CSF mRNA and its decrease in half-life fiom 90 to 20 minutes (Rajagopalan

and Malter, 1994).

Another RNA-binding protein, AUF1, was initially identified through its involvement in c-

myc mRNA degradation in a cell-free mRNA decay system. AUFl facilitates the

association of c-myc with polysomes (Brewer and Ross, 1988) and accelerates c-myc

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mRNA turnover in vitro (Brewer, 1991). In vivo studies have also demonstrated that

AUFl targets decay of ARE-containing mRNAs. Upregulation or downregulation of

AUFl has been associated with increased and decreased decay of ARE-containing RNAs,

such as GM-CSF (Pende et al., 1996; Buzby et al., 1996). Recently, Laroia et al has

found that the dec2y of ARE mRNAs is associated with the displacement of elF 4G fro,

AUF1, the ubiquitination of AUFl and the degradation of AUFl by pmteosomes (Lamin et

al., 1999). Therefore, there is strong experimental data supporting AUFl as one of the

ARE-binding factors facîiitating mRNA decay via the 3 W ï R ARE, but whether this factor

can also influence mRNA translation is unproven-

ARE-binding proteins are only one class of RNA-binding proteins that regulate post-

transciptional gene expression. Many other RNA-binding proteins that have been

identified, including those binding to pre-mRNA, pre-ribosomai RNA (rRNA) or s m d

nuclear RNA (snRNA) are involved in capping, pre-mRNA spiicing and polyadenylation.

The characterization of these different RNA-binding proteins has led to the identification of

several RNA-binding motifs and studies of their interactions with RNA.

RNA-binding Motifs

Based on the negative-charge nature and specific conformation of RNA, most of the

protein-RNA interactions involve charge-c harge interactions such as hydrogen bonds and

specific secondary structures such as beta-sheets. The most cornmon RNA-binding motifs

identified include the RNP (ribonucleoprotein) consensus motif (or RBD motif), the

Arginine-rich motif (ARM), the RGG Box and the KH (K homology) motif.

The RNP motif is by far the most widely found and bat-characterized RNA-binding

motifs. It is also referred to as the RNA recognition motif (RRM)(Kenan et al., 1991;

Query et al., 1989), RNP consensus sequence (RNP-CS)(Swanson et al., 1987; Dreyfuss

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et al., 1988), and consensus RNA-binding domain (CS-RBD)(Bandziulis et al., 1989),

and is commonly found in riboaucleoproteins such as heterogeneous nuclear RNP C

(hnRNP C). It consists of a 90-amui0 acid sequence containhg 2 short highly conserved

sequences, RNPl (an octapeptide) and RNP;! (a hexapeptide), interspersed with a number

of other, mostly hydrophobie conserved amino acids (Swanson et al., 1987; Dreyfuss et

al., 1988;BandWulis et al., 1989;Kenan et al., 199 1). The three-dimensional structures of

RNP motifs in U1 snRNP A (U1 A) and hnRNP C have been determined and appear to be

very similar (Fig. 2)(Nagai et al., 1990; Hotlinan et al., 1991; Wittekind et al., 1992). The

$aBBaP secondary structural elements of the RNP motif fomis a four-stranded antiparaliel

sheet. The RNPl and RNP;! are located on the cenaal 83 and p l strands respectively.

The charged and aromatic side chains of these 2 sequences are solvent exposeci, aiiowing

them to make direct contact with bound RNA, pmbably through hydrogen bonds and ring

stacking (Nagai et al., 1990; Gorlach et al., 1992). Although the highl y conserved RNPL

and RNP2 are crucial for RNA binding, the highly variable regions, particularly in the

Ioops and the termini, contain the major determinants of RNA-binding specificity. In U1

70K, U1 A and hnRNP C, the amino acids on the immediate COOH-terminal of their RNP

motifs determine the RNA-binding specificity (Query et al., 1989; Gorlach et al., 1992;

Scherly et al., 1991).

The arginine-rich motif (ARM) is usuaiiy short, about 10-20 arnino acids long. Other than

the preponderance of arginine residues, the different ARMs show little sequence identity

(Lazinski et al., 1989), and the structures are diverse as well, including stem-lmps in W-

proteins', interna1 Ioops in 'Rev' or bulges in Tat' (Malim et al., 1989; Dayton et al.,

1989; Gorlach et al., 1992; Iwai et al., 1992). Despite the unconserved secondary

structure for the ARM, the structun, rather than particular sequence, appears to be the

major binding determinant. Interaction with RNA involves both the phosphonbose

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Figure 2. Schematic ribbon drawing of the hnRNP C RBD (residues 2-94).

The arrows represent the 4 antiparalleI$ strands, and the curled ribbons represent the 2 a

helices. The labels pl-84 indicate the 4 saands of the sheet; the labels al and a2

indicate each a h e h . The RNPI and RNP;- consensus sequences are juxtaposed on the

adjacent central antiparallel strands (fi sîrands 3 and 1, respectively). N and C denote the

arnino and carboxyl tennini of the domain, respectively (Modifieci h m Dreyfuss G., et al.,

Annu Rev Biochem 62:289-321, 1993).

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backbone (Bartel et al., 1991) and the bases (iwai et al., 1992) of the RNA. Compared to

other charged residues, such as lysine, arginine residues have more potential for forrning

hydrogen bonds. The positive charges probably increase nonspecific affinity for RNA.

This initiates a t tachent and facilitates searching for a higher afflnity and more specific

binding site (Calnan et al., 1991; Weeks and Crothers, 1991; Tan et al., 1993).

The RGG box was first identified as an RNA-binding domain in hnRNP U (Kiledjian and

Dreyfuss, 1992). It is about 20- to 25-amino acid long, consists of closely spaced Arg-

Gly-Gly (RGG) repeats interspersed with other, often aromatic, amino acids. The nurnber

of RGG repeats varies between proteins, with six in hnRNP A l and eighteen in yeast

GARI. These repeats are often found in combination with other RNA-binding domains

(Kiledjian and Dreyfuss, 1992). In nucleolin which contains 4 RNP motifs and 1 RGG

box, specific binding to pre-ribosomal RNA requires the 4 RNP motifs and the RGG box

increases the overall RNA affinity by IO-fold (Ghisolfi et al., 1992). This might suggest

that RNA binding of RGG box is relatively nonspecinc but it plays a more important role in

facilitating the RNA binding of other RNA-binding domains. However, in the case of

hnRNP U, the RGG box is the only RNA-binding element and is able to discriminate

between different RNA sequences (Kiledjian and Dreyfuss, 1992). The binding affinity of

RGG box to RNA can be modulated by methylation of arginine residues within the RGG

box. hnRNP A l has been shown to be pst-translationally arginine-methylated in vivo

within the RGG box (Kim et al., 1998). Its binding property to single-stranded nucleic

acid is significantly reduced subsequent to methylation, suggesting that pst-translational

methyl group insertion to the arginine residues reduces protein-RNA interaction, perhaps

due to interference of hydrogen bonding between guanidino-nitrogen arginine and

phosphate RNA.

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The K homology (KH) motif was initially identified in the human hnRNP K protein and

was Iater found in other RNA-binding proteins in other orgaaisms as well (Siomi et al.,

1993a; Siomi et al., 1993b). It is a stretch of about 45 amino acids characterized by a core

sequence VIGXXGXXI flanked by few interspersed conserved residues. By NMR

spectroscopy, the secondary structures of the KH motif were identified to consist of 3

stranded fi-sheets connected by 2 helical regions (Castiglone Morelli et al., 1995). In

hnRNP K, 3 K H motifs are present, each of them contributing to RNA binding. In

neuronal proteins Nov-l and Nov-2, 3 KH domains are also identified (Lewis et al.,

1999). The 2 KH domains in human FMR proteins play an important rote in the funcion of

the protein. Mutations at these domains are associated with fragile X syndrome, the most

common inherited cause of mental retaradation (Musco et al., 1996).

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RATIONALE

Previous studies in our laboratory &monstrateci that LC3 binds the AU-rich element (ARE)

at the 3' untranslated region of the FN mRNA and upregulates its translation through

ribosome r e d t m e n t (Zhou et al., 1997; Zhou et al., 1998). In view of the importance of

RNA-protein interaction in regulating gene expression, this thesis investigates the

significance of this LC3-ARE interaction using both structural and functional appmaches.

LC3 is a basic protein, with a pI value of 9.2 (Mann and Hammarback, 1994). From the

amino acid sequence of the molecde, we note that most of the positively charged residues

are Iocated in the N-terminal haif. Since charge interaction plays a critical role in RNA-

protein binding, the RNA-binding site is therefore more likely located in the N-terminal half

of the molecule, as shown in Fig 3. Art arginine-rich motif (ARM) which might represent a

high affinity binding site is present toward the end of the N-terminal half.

The HTlOSO human fibrosarcorna cell line, which is LC3-nul1 in nature, has k e n used to

study the functional significance of LC3-ARE interaction. Stable transfection of IlTl080

cells with LC3-encoding plasmids increases FN mRNA translation through ribosome

recruitment, the same mechanism as in DA cushion foxmation. Flattened cell morphology

and decreased ce11 growth is also observed. Therefore, stable transfection of HT1080 cells

with wild type and mutant LC3-encoding plasmids can be performed to elucidate the direct

correlation between LC3-ARE binding and FN mRNA translation, as well as the cell

morphology and ce11 growth.

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Figure 3. Amino acid sequence of rat LC3.

Positively charged residues (shown as bold) are concentrateci at the N-tenninal half of the

protein. ARM, representing arginine-nch motif, is a common RNA-binding motif.

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....,.... 1....,....2....,.~.œ3~.œœ,œœoœ4œœœœ,œœoœ5o.o.,.~~. 6 MPSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPVLDKTKFLVPDHVNM

....,....7....,....8...., . . . . 9 . . . . , . . . . 10 ...,.... 11 ...,.... 12 SELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESERDEDGFLYMWASQETFG

ARM

. . o . , . . . O 13 . . . , . . . . 14. TALAVTYMSALKATATGREPCL

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1. LC3 binds to the AU-rich elernent (ARE) in the 3' untranslated region (3'UTR) of the

FN mRNA via RNA-binding motif(s) a . the N - t e e a l .

2. LC3-ARE interaction via the LC-3 motif(s) identified is criticai for the upregulation of

FN synthesis in HT 1080 cells and this property is duectly related to slower ceil growth

and reorganization of microtubules.

OBJECTIVES

1. Determine the amino acid sequence in LC3 which binds to the ARE region of the 3 U ï R

in FN mRNA, using proteolytic cleavage and site-directed mutagenesis.

2. Determine the effect of LC3-ARE binding through this motif on FN synthesis by stably

transfecting wild type (WT) and mutant LC3 into LC3-nuii HTlOSO human fibrosarcorna

ce11 line.

3. Determine whether LC3-mediated FN upregulation contributes to the decreased growth

rate and changes celi shape in HTlO8O celis.

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MATERIALS AND METHODS

CelZ Culture

The HT1080 human fibrosarcoma ceii iine was purchascd from American Type CeU

Culture (ATCC) and cultured with Eagle's minimal essential medium 0 (GIBCO

BRL, Buïlington, ON) containing 10% fetal bovine senun (FBS) (Intergen, mirchse,

NY), 1 96 antibioticslantimycotics (GIBCO) and 0.2% gentamicin (GIBCO)(for cells with

stable transfection only). For ail comparative snidies, cells were passageci at the same tirne

and plated at the sarne density. To assess morphologie differences, the cultures were

photographed with a phase-contrast microscope (Nikon Inc. Garden City, NY).

Expression of Recombinant LC3

pGEX-LC3 vector (produced by Zhou) was synîhesized by cloning the LC3 coding region

(produced by J. Hammarback) into pGEX-2T vector (Pharmacia Biotech.) at a 5' BamHI

site and a 3' EcoRl site in the plasmid The plasmid was transformed into DH5a E.coli

competent cells (GIBCO). Positive clones were confirmed by Mini-preps and restriction

enzyme digestions. The plasrnid-containing E-coli was amplified by growing ovemight in

2xYTA medium at 37"~, followed by induction with isopropyl-fl-D-thiogalactopyranoside

(IPTG) for 4 h. Total protein was extracteci and the glutathione S-transferase (GST)-LC3

fusion protein was purified using glutathione sepharose 4B beads (Sigma Chemical Co.).

The recombinant LC3 was eluted from the beads after ovemight incubation with thrombin

(Sigma or Phannacia) which cleaves at the GST fusion site with the recombinant LC3. The

punfied recombinant LC3 protein was confirmed by SDS-PAGE and western

immunoblotting.

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Generation of LC3 Pepîïdes

1. Carhepsin D/ Endopoteinase Asp-N/ EnaOproteeulare Lys-C Digesrion

Lyophilized recombinant LC3 was suspended in buffers containing endoproteinases at

3 7 " ~ for specific t h e penods {C'hepsin D digestibn (cleave specijically beîween Phe-Phe

linknge) : LC3 was suspended in 50mM ammonium acetate pH3.5 at a ratio of lpgllpl of

buffer con taining Cathepsin D (Sigma C hemical Co.)(enyme:substrate= 1 :5O) for 30 min;

Endoproteinase Asp-N Digestion (cleave specificoly at the N-teminal end of Aspartare

Acid residues): LC3 was suspended in SOmM sodium phosphate pH 8.0 at a ratio of

l ~ g f l p l of buffer containing Endoproteinase Asp-N (Boehnnger Mannheim)

(enzyme:substrate=l : 100) for 3 h; Endoproteinose L y s 4 Digestion (cleave specificaly a?

the C-terminal of Lysine residues); LC3 was suspended in 1% (w/v) ammonium

bicarbonate at a ratio of Ipg/Lpl of buffer containing Endoproteinase Lys-C (Boehringer

Mannheim)(enzyme:substrate=1:50) for 5 h). The reaction was stopped by freezing the

samples at -70"~. Different enzyme concentrations and incubation times were tested and the

optimal condition was determined by assessing the amount and the integrity of peptide

produced with no degradation as shown by SDS-PAGE followed by silver staining.

However, in al1 cases, complete digestion of LC3 cannot be achieved because longer

incubation time leads to the degradation of the peptides.

II. Cyunogen Bromide (CNBr) Digestion

CNBr was dissolved in 70% formic acid to a concentration of 50 m g M . Lyophilized

recombinant LC3 was suspended in CNBr/70% formic acid at a ratio of 1pg:lpl. The

reaction mixture was incubated at room temperature for 30 min. 10 fold volume of water

was added to stop the reaction. Complete cleavage cannot be achieved even if incubation

leaves ovemight. Therefore, 30 minute-time point was chosen to preserve the intergrity of

the generated peptides h m further non-specific degradation.

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5' end-radiolabelhg of RNA Probe

18-mer RNA oligonucleotides containing either the wild type consensus sequence

(UUAUüUAU) of the AU-rich region element (ARE) of the FN mRNA, or the mutant

consensus sequence (GGAGGGAG)(synthesized by Biotechnology Centre, University of

Calgary, Calgary, Alberta) were radiolabeled with ~y-3~P] ATP using T4 pol ynucleotide

kinase (PNK) (Pharmacia Biotech.). 50 ng of RNA oligonucleotide was incubated with

150 K i of [y-32~1 ATP(3000 Ci/mmol), 9.5 U of T4 PNK and 10x PNK B a e r to a total

volume of 20 pl- The reaction mixture was incubated at 37°C for 1 h. The enzyme was

then inactivated by heating the sample at 95°C for 2 min or by adding 1 pl of 0.M EDTA

pH8.0, followed by incubation on ice. The probe was purified using a NucTrap Probe

Purification Column (Strategene). The radioactivity of purified probe was determined by

liquid scintillation spectrometry.

In vitro Transcription of RNA Probe

The full-length 3WïR of the rat FN mRNA containing wild type or mutated ARE (al1 U's

mutated to G's) were subcloned into pBSKS4 vector (performed by Zhou). Plasmids were

linearized with XbaI. RNA was transcribed with T3 RNA Polymerase in the presence of

[ 3 2 ~ ] - U T P for 1 h at 37°C. DNA templates were removed with DNase 1 digestion for 15

min at 3 7 " ~ , and the full-length probes were obtained by 6% acrylamide/8M urea gel

purification.

North western (NW) Blot Analysis

Northwestem blot analysis was carried out as previously described (Chen, 1993) with

modifications. Enzyme-digested and control undigested wild type or mutant recombinant

LC3 samples, 2.5 pgllane, were resolved under reducing conditions on a 10-20s tricine

gel (Novex) and electroblotted ont0 a 0.2 Pm nitroceiiulose membrane. The LC3 peptides

were allowed to renatwe in RNA-binding buffer (lOmM Tris-Cl, pH7.5, 50mM NaCl,

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ImM EDTA, and l x Denhardî's solution) overnight at 4 ' ~ . Rior to RNA binding, proteins

were blocked with RNA-binding buffer containing 100 pg/d tRNA for 1 h at room

temperature. The radiolabeled ARE of the rat FN mRNA at a concentration of 107 c p d d

was also incubated with RNA-binding b m e r containing 100 pghl tRNA for 1 h at room

temperature. The blot was theu incubated with the radiolabeled RNA mixture at room

temperature for 1 h, and finally washed with RNA-binding buffer twice for 5 min. The

dried nitrocellulose membrane was exposexi to X-ray film for autoradiography at -70°C.

Site-directed Mutagenesis

Three mutant pGEX-LC3 vectors were generated by PCR to produce mutant recombinant

LC3 for NW immunoblotting analysis : i, pGEX-LC3/R68-70Q, ii, pGEX-LC3/R68-70K,

and iii, pGEX-LC3lF79-80A. Three pairs of oligonucleotides carrying mismatched bp

corresponding to the speciiic mutations were used The upper and lower primers were: i,

5'-ATTCAACAGCAACTGCAGCTCAAT-3' and S-CAGTTGCTGTTGAA'ITATC-

TTGAT-3'; ii, 5'-ATTAAAAAGAAACTGCAGTCAAT-3' and 5'-CAGTTTCTTTTT-

AATTATCTTGAT-3'; iii, GCCGCGGCCCTCCTGGTGAATGGG-3' and 5'-

GAGGGCCGCGGCITGGTTAGCATT-3' (nts corresponding to the mutated amino

acids shown in bold). A S'-end primer, 5'-CGGGATCCCATATGCCGTCCGAG-

AAGACC-3', located upstream of the mutated site and a downstrearn 3knd primer, 5'-

CTGGATCCGAATTCAAGCATGGCTCTCITCC-3', were also used. Three 450-bp

products with specific mutations were generated by PCR and subjected to restriction

enzyme digestions. Three 435-bp Ba--EcoRI fragments containing the mutated sites

were then used to replace the corresponding fragment within @EX-LC3. These mutant

pGEX-LC3 vectors were transformed into E-coli and expressed as mutant recombinant

LC3 as mentioned above.

Another three mutant pCR3-LC3 vectors containing the full length LC3 sequence were

generated by the same method for stable transfection into HTlO8O human fibrosarcoma

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cells: i, pCR3-LC3/R68-70Q. ii, pCR3-LC3/R68-7OK, and iii, pCRÎLC3/F79-8OA. The

same sets of p h e r s carrying the specific mutations were used. The S'-end primer and 3'-

end primer flankuig the mutated site w m SIGAGCKGGATCCACTAGTCCAGTGTG-

GTGG-3' and 5'-GTCACCGCCGGCGAGCTCAGATCTCCCGGG-3'. Three 976-bp

products with specific mutations were generated by PCR and subjected to restriction

enzyme digestions. Three 963-bp BamHl-XbaI fragments containing the mutated sites

were then used to replace the corresponding fragment within wild type pCR3-LC3. Al1

constnicts were confirmed by restriction enzyme mapping, and the mutations were verined

by DNA sequencing.

Gel Mobility Sirift Assays

10 pg of cytoplasmic extracts from WT and mutant LC3-transfected ElTl080 cells was

incubated with 1x1@ cpm of the 3WïR of the rat FN mKNA in RNA-binding buffer

(1 5rnM Hepes, pH 7.9, lOOmM KCl, 5mM MgCL2, 10% giycerol, 0.2mM DTT) in a total

volume of 20 pl containing 2 pg of tRNA for 30 min at 3 0 " ~ . 0 . N of RNase A and 400U

of Rnase Tl were added to each sample and incubated for 15 min at 3 7 " ~ . Samples were

then run on a 6% native polyacrylamide gel in 0 . 2 5 ~ TBE (Tris-borate-EDTA) buffer

(90rnM Tris, 90mM br ic acid, 2mM EDTA) at 250V on ice, dried and exposed to X-ray

film for autoradiography at -70°C. For cornpetition and specificity studies, 500x of

unlabeled RNA probes containing either WT or mutant ARE were incubated with

cytoplasmic extracts for 10 min before adding the labeled RNA transaïpts. (Note: 0.2mM

DTT were added to the RNA-binding buffer to reduce the disulphide bonds between

individual LC3 proteins to generate monomeric LC3 with higher ARE-binding affinity, as

shown in UV cross-linking analysis in a previous published paper (Zhou er al., 1997).

However, the potentiai inactivation of RNase A by DTï was not observed, probably

because the disulphide bonds in RNase A do not contribute to its RNA digestion activity.)

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Stable Transf ection

24 h prior to transfection, HTlO8O ceUs were plated at a density of 10s cells 1100-mm dish.

HT1080 cells were stably transfected using SuperFect Reagent (QIAGEN Inc. Valencia,

CA). 10 pg of empty vector (pCR3), wild type (pCR3-LC3/WT) or mutant (pCR3-

LC3/R68-70Q, pCR3-LC3/R68-70K, and pCR3-LC3IF79-80A) LC3 plasmids were used

to transfect each dish for 3 h. The cells were then fed with fresh complete medium

containing the aminoglycoside G418 (200 pg/ml, GIBCO). The media were changed

every two days with gradually increasing concentrations of G418 up to 800 pglml. 8

clones transfected with empty vector and 24 clones each transfected with pCR-LCJ/WT,

pCR3-LC3/R68-70Q, pCR3-LC3R68-70K and pCR3-LC3#9-80A were selected on the

basis of resistance to G418 (800 pglml) by selective trypsinization and screened for LC3

expression using westem immunoblot analysis. 4 clones each were venfied to express

WT-LC3, R/K-LC3 and F/A-LC3, while 3 clones were verified to express R/Q-LC3.

These clones, together with 3 vector-transfected clones (which were shown to express no

LC3 by westem inimunoblot), were expanded individually and passaged at least 3 times

before use.

Western Irnmunoblot Analysis

Confluent cells were harvested by scraping into phosphate-buffered saline (FBS) and spun

at 3,000 rpm for 10 min at 4°C. Pellets were resuspended in twice the volume of

h ypo tonic bu ffer (30 mM Tris-Cl, pH7.9) with proteinase inhibi tors aprotinin, pepstatin

and leupeptin (1 pg/d each) and lysed by 3 cycles of freeze-thaw, followed by a 1-h

centrifugation at 16,000g at 4°C Cytosolic extracts (S) were isolated and pellets were fmt

digested with RNase-free DNase 1 (Phannacia Biotech) for 30 min at 37°C. Supernatant

(PI) was extracted after a 10-min spin at 16,000g at m m temperature. Pellets were M e r

extracted by resuspending in 2 peliet volumes of 1% SDS, and supernatant (Pz) was

isolated &ter a 10-min spin at 16,000g at RT. Protein concentrations in supernatant (S)

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and pellets (Pl and Pd were measured by the BCA protein assay kit (Bio-Rad Laboratories,

Hercules, CA) followed by spectrophotometry at 562 nm. Rotein samples were separated

by reduced sodium dodecyl sulfate (SDS)-PAGE (10-20% tricine gel, NOVEX) and

electrotransferred onto a PVDF membrane. The membranes were bloçked for 1 h at m m

temperature in TBS-T (Tris-buffered saline with 0.5% Tween-20) containing 5% non-fat

milk and then probed with rabbit-anti-LC3 antismim (1:2Oûû dilution in TBS-T) (kindy

supplied by Dr. J. Hammarback, Department of Neurobiology and Anatomy, Bowman

Gray School of Medicine, Winston-Salem, NC) or mouse monoclonal anti-tubulin IgG

(12000 dilution in TBS-T, Sigma) overnight at 4°C. The membranes were washed 4 x 5

min with TBS-T followed by incubation with HRPconjugated goat-anti-rabbit or donkey-

anti-mouse IgG (1:3000) (Amersham. Buckinghamshire, England) for 1 h at room

temperature, and then washed 4 x 5 min with TBS-T and developed using an enhanced

chemiluminescence western blotting detection reagents (Arneaham). For the quantitative

analysis of LC3, the 16kD immunoreactive band for each sample was assessed by

densitometric analysis.

Cell Growth Cumes

HT1080 cells stably expressing blank vector, wild-type (WT) or mutant (WQ, R/K and

F/A) LC3 were plated on 6-well dishes at a density of 1 x 105 cells/well. The media

containing G418 were changed every day. Cells were trypsinized every 24 h and the ce11

number was determined using an improved Neubauber hemacytometer (Amencan Optical

Scientific Instrument Division, Buffalo, NY) by taking the average of two separate counts

for each well.

Indirect Immunofluorescence

HTlO80 cells were plated on 2.2 cm* coverslips at a density of 105 cells/well and cultured

for 3 days. Cells were fixed in 100% methanol at -20°C for 3 min and allowed to air dry.

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Following rehydration in phosphate-buffercd saiine (PBS) for 30 min, celi were blocked

with PBS containing 1 % normal goat s e m and O. 1% bovine senmi albumin @SA) for 1

h at room temperature. For immunofluorescence staining of fibronectin 0, tubulin and

LC3, cells were probed with a monoclonal rabbit anti-FN IgG (1:100 dilution;

Neomarker), a monoclonal mouse anti-tubulin IgG (1: 1000 dilution; Sigma Chemicai Co.)

or a rabbit anti-LC3 antisenun (1:100 diiution) in PBS containing 0.1% BSA oveniight at

4°C. After 3 x 5 min washes with 0.1% BSA/PBS, cells were then incubated with

secondary antibodies: fluorescein-conjugated goat-anti-mouse IgG for FN and tubulin

staining and fluorescein-conjugated goat-anti-rabbit IgG for staining of LC3 (aii dilutions at

150) for 30 min at room temperature, foiiowed by 3 x 5 min washes and mounted with

antifade reagent (Molecular Robes Inc., Eugene, OR). Cell nuclei were stained with 1: 150

diluted DAPI (Sigma) in distilled water for 30 min a€ter the incubation with secondary

antibody. For negative controls, normal mouse IgG or 1% normal goat semm were used

instead of the primary antibodies.

FN Biosynthesis

HT1080 cells individually expanded from vector-transfected, wild-type (WT) or mutant

(WQ, R/K and F/A) LC3-transfected clones were plated on 6-well dishes at a density of

5x 1 d cells/well. After 24 h, cells were labeled with OsSI-methionine (10 pCi/ml) for 5 or

20 h (as indicated) in 2 ml media rnixed with 3 vol of methionine-free and 1 vol of complete

EMEM containing 20% FBS. The conditioned media were collected for analysis of FN

protein. Triplicate assessments of total protein synthesis were obtained from 50p1 aliquots

of culture medium precipitated in 1% BSA/15% aichloroacetic Md (TCA) and analyzed by

liquid scintillation spectmmetry. Measurement of FN protein production was perfonned by

incubating the conditioned media containing equal counts of total TCA precipitated protein

with 50 pl of Gelatin 4B-Sepharose (Pharmacia Biotech Inc., Pixataway, NJ) overnight at

4 " ~ . The FN retained on the beads after 3 washes with 1 ml of TBS containing 0.5%

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Tween-20 was eluted by boilïng for 5 min in 60 pl of nducing SDS-sample bmer (5%

mercaptoethanol, 2% SDS, 10% glycerol, 62.5mM Tris-AC1 pH6.8) and resolved by 6%

SDS-PAGE. Gels were fmed in 5% acetic acid1096 mthanol for 30 min and prepared for

fluorography by treatment with ~ n f ~ a n c e (DuPont-NEN, Boston, MA), dried, and

exposed to the film. Using the autoradiograph as a template, the corresponding bands were

cut h m the gel and the radioactivity determineci by a liquid scintillation counter.

RNA Isolation and Northern Blot Anabsis

Total RNA was extracted from the cells using QIAGEN RNA extractkg kit (QLAGEN)

following manufacturer's instructions. lOpg of total RNA from each sample was resolved

on a 1% agarose gel, transferred ont0 a Hybond-N membrane by capillary elution

overnight, and fixed by W-irradiation. After blocking, the membranes were probed with

a [ 3 2 ~ ] - d ~ ~ ~ random labeled human FN cDNA (106cpm/ml) overnight at 5 0 " ~ . followed

by 2 washes with 2x SSC/O.l% SDS at 55°C for 30 min and 2 washes at 6 5 " ~ for 1 h.

Autoradiographs of northem blots were analyzed by relative densitometry. Ethidium

bromide staining of 28s and 18s ribosome RNAs served to control for loading conditions.

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RESULTS

LC3 Binds the ARE of FN mRNA at the lOkD N-terminai Region

When LC3 binds to the ARE at the 3'UTR of the FN mRNA, the efficiency with which

this mRNA is translated to protein is increased. To identiQ the ARE-binding site(s) on

LC3, LC3 peptides were generated by enzymatic or non-erizymatic cleavage of recombinant

LC3. Their differential ARE-binding activity was then assessed by northwestem (NW)

blot analysis. The endoproteinases used incluàe Cathepsin D, which cleaves spcificall y

between Phe-Phe Linkage; Endoproteinase Asp-N, which cleaves specifically at the N-

terminal of Aspartate acid residues; and Endoproteinase Lys-C, which cleaves specificdy

at the C-terminal of the Lysine residues. Cynogen Bromide cleaves by reacting specifically

with methionine residues to produce peptides with C-terminai homoserine lactone residues

and new N-terminal residues. The peptides generated were separated by SDS-PAGE (Fig

4A), transferred ont0 nitroceilulose membranes, and probed with a [32P]-radiolabeled RNA

probe containing the FN ARE. ARE-binding activity of these peptides was detected by

autoradiography (Fig 4B).

Cathepsin D (CD) cleaves specifically between Phe-Phe residues at amino acid 79 and 80

of LC3, generating 2 peptides: the 8.9 kD N-terminal peptide and the 7.1 kD C-terminal

peptide (Fig 4Aa). Two Iower bands around 7 kD were observed. They might represent 2

different C-terminal peptides due to a post-translational modification near the C-terminal

end of the protein, or one of these peptides might represent the degradation product of the

N-terminal peptide or the whole LC3 proteins. The [ 3 2 ~ ] - ~ ~ ~ was able to bind to the

undigested 16kD recombinant LC3, but none of the CD-generated peptides (Fig 4Ba).

Complete CNBr cleavage should generate 5 peptides, al1 smailer than 6.8 kD. However,

incomplete cleavage led to the production of 2 bands above 7 kD and a smail band about 4

kD (Fig 4Ab). The highest molecular weight 10 kD peptide showed strong binding to the

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Figure 4. Northwestern (NW) blot analyses show LC3-ARE binding

localizes to a 10 kD N-terminal region on LC3.

Recombinant LC3 was enzymaticdly or non-enzymah'cdy digested into discrete peptides,

and their binding activity to the radiolabeled ARE was compared by northwestem (NW)

analycir. A. Silver staining of the SDS-PAGE shows the LC3 peptides generated by the

cleavage of recombinant LC3 by Cathepsin D (CD)(a), Cyanogen Bromide (CNBr)(b),

Endoproteinase Asp-N (EPAN)(c) and Endoproteinase Lys-C (EPLC)(d), as well as the

control undigested LC3 protein (control). B. Representative autoradiographs demonstrate

the binding activity of the [32P]-radiolabeled FN ARE to the LC3 peptides and undigested

LC3 proteins. ARE bound to al1 undigested 16kD LC3 proteins. Note the absence of

ARE-binding in peptides generated by CD (a), EPAN @) and EPLC (c), while strong

binding is observed in the -1OkD peptide generated by CNBr indicated by arrows in both A

and B. Since this lOkD peptide was confirrned to contain the N-terminal sequence, this

suggests that the 1 OkD N-terminal region of LC3 possesses ARE-binding activity .

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C D-d iges ted

con t rol

CNBr-digested

cont r d

EPAN-digested

control

control

EPLC-digested

a control

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probe, as marked by an amow, while the middle (-7kD) and the lowest peptides (-4kD)

showed no binding at ail (Fig 4Bb). These upper 2 bands were sent for amino acid N-

tenninal sequence analysis. (The sequencing report for l O k D and 6.8kD peptides are

shown in appendix 1 and II respectively). The major peptides corresponded to the N-

terminal of LC3 although the minor peptides generated did not correspond to any part of

LC3. The higher band, about 10 kD, therefore represents the N-terminal peptide spanning

the sequence from P2 to M88, while the lower band about 6.8 kD, represents the N-

terminai peptide spanning the sequence from PZ to M60. This suggested that the RNA

binding site(s) was within the C-terminai portion of the 10 kD peptide, from residue S61 to

M88. This region (as shown in boid in Fig 5) contains 2 possible RNA-binding sites: the

positively-charged Arginine-Rich Motif (ARM) and a predicted Psheet region (p). Since

the conformation of the protein plays a significant role in RNA-protein interaction, Circular

Dichroism can be perforrned to examine the secondary structure of the peptide, and

determine whether the CNBr cleavage alters the conformation of the peptides and results in

the increased ARE-binding.

To further confirm the ARE-binding activity of the region S61-M88, endoproteinase AspN

(EPAN) digestion was performed to generate peptides spanning residues D56 to R103,

w hich contain the speculated ARE-binding region (S6 1 -M88) (Fig 4Ac). However,

without the N-teminal sequence, none of the peptides showed ARE-binding (Fig 4Bc).

Endoproteinase Lys-C (EPLC) digestion was also perfomed to generate a 7.5 k D peptide

which contained the speculated ARE-binding region plus the C-terminal of LC3 (Fig 4Ad).

Again, no ARE-binding was observed (Fig 4Bd). Therefore, these results suggested that

while the region S61-M88 contained the ARE-binding site(s), the N-terminal region of LC3

was also mandatory for RNA-binding, either by influencing the charge andfor the

secondary structure of the protein. A sumrnary of the northwestern analyses is shown in

Fig 6.

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Figure 5. Amino acid sequence of rat LC3 showhg the 2 candidate RNA

binding sites.

The bold region spanning residues 61 to 88 represents the speculated RNA binding region

circled in Fig 6. It contains 2 candidate RNA binding motifs: the arginine-rich motif

(ARM) and a predicted bsheet region @).

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. . . . , . . . . 1....,....2....,....3....,....4...., . . . . 5 . . . . , . . . . 6 MPSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPVLDKTKFLVPDHVNM

u ARM

O... , o . . . 13 . . . , . . . . 14. TALAVTYMSALKATATGREPCL

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Figure 6. Summary diagram of NW blot analysis of LC3-ARE binding.

The bars represent the whole LC3 amino acid sequence while the saipped regions represent

the regions of the peptides generated by enzymatic or CNBr cleavage using listed on the

Ieft. The numbers below the bars represent the N- and C-terminal residues of the peptides

and the arrows above represent the LC3 cleavage sites used by the various reagents. The

size and ARE-binding property of each peptide are Listeci on the right. The lOkD CNBr-

generated peptide bound ARE while the 6.8 kD peptide did not, leading to the speculation

that the C-terminal region of the lOkD peptide (circled) contains the RNA binding site(s).

This circled region represents the bold sequence as shown in Fig 5.

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REAGENTS USED DIGESTED LC3 FRAGMENTS

Cathepsin D

Endoproteinase

Endoproteinase

FRAGMENT BINDING(Y1N) SIZES

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LC3 Binds Specifically to ARE of FN mRNA

To confirm the specificity of LC3 peptide binding to the ARE in the FN 3'UTR, NW

analyses were carried out using both wild type (WT) and mutant ARE. Equal

countsfamounts of wild type and mutant probes (U's in ARE mutated to G's) were

incubated with blots containing equal amounts of transferred protein. Both blots were then

exposed on the same film. The autoradiograph is shown in Fig 7. In this way, any

difference in the intensity of bands wouId likely reflect the differential afnnity of the probes

for the proteins. By comparing the densitometric analysis of the binding intensity of the

16k.D protein and the l O k D peptide to the WT and mutant ARE, the respective affinity to

the WT ARE was about 6 times and 2 thes higher than that of the mutant ARE (Fig. 7).

Although we cannot conclude that LC3 binds exclusively to the ARE, the binding to ARE

is preferentïal compared to 'poly G' in mutant probe. The binding between the LC3

peptide and the mutant ARE might be nonspecific in nature, based on solely charge

interaction. The difference between the binding of the WT compared to the mutant ARE

may reflect the sequence specificity of the protein-RNA interaction, and this can be m e r

tested using other mutants or mutant oligonucleotides as cornpetitors. Interestingly, 16kD

whole LC3 protein showed a bwer ARE-binding affinity compared to the lOkD peptide.

There are 2 possibilites contributing to this differential binding: fmt, there might be an

ARE-binding inhibitory element located at the C-terminal half of the protein. The absence

of this element in the lOkD peptide therefore enhances its ARE-binding afftnity; second, the

conformation of the l O k D peptide rnight be different from the N-terminal region of the LC3

protein, which somehow favors the ARE-binding. We can further investigated these

possibilities by circular dichroism or mass spectroscopy.

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Figure 7. RNA binding of LC3 protein and l O k D peptide is preferential for

the ARE.

The specificity of ARE-LC3 binding was investigated by NW analyses using WT or mutant

ARE-RNA oiigonucleotide probes. Representative autoradiographs show stronger binding

of 16kD and 1OkD LC3 proteins and peptides to the WT compared to the mutant FN ARE

probes. Bottom shows the corresponding densitometric analysis comparing the binding

afinity of WT and mutant FN ARE region to the 16kD LC3 protein and 10kD peptide.

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lOkD CNBr-generated LC3 peptide

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Arginine-Rich Motif (ARM) is Critical in LC3 Binding to ARE of FN

mRNA

To M e r examine the importance of the 2 candidate RNA-binding regions within thel0kD

N-terminal LC3 peptide, we used site-directed mutagenesis to mutate these 2 regions.

Arginine nch motifs (ARM) are important in RNA-binding since they increase the non-

specific affïnity of proteins for RNA (Tan et al., 1993) and make specific hydrogen

bonding networks with the RNA sugar-phosphate backbone and bases (Iwai et al., 1992).

We therefore rnutated the 3 arginines (R) in the ARM either to 3 glutamines (Q) to examine

the significance of positive charge, or to 3 lysines (K) to examine the significance of

structure. Phenylalanines play a cntical role in RNA binding in hr&V A l by forming

stacking and hydrogen-bonding interactions with the guanine nucleotides (Merill et al.,

1988; Ghetti et al., 1990; Ishida et al., L986). We therefore also mutated the 2

phenylalanines (F) at the predicted beta sheet strtucutre to 2 alanines (A), to determine

whether the 2 benzene rings in phenylalanines are involved in ring stacking interaction with

the ribose component of the RNA.

Site-directed mutagenesis was performed using PCR, followed by subcloning the mutated

LC3 into the @EX-2T vector to generate GST-fusion proteins as described ir, detail in the

Methods. Wild type (WT) and mutant LC3 were subjected to N W analysis as above, and

results are shown in Fig 8. Comparable amounts of WT and mutant recombinant LC3

(though somewhat less in IUQ LC3) were loaded ont0 SDS-PAGE and transferred, as

illustrated by silver staining in Fig 8A. Densitornetric analyses of the autoradiograph

representing the NW analyses (Fig 8B) normalized to the amount of proteins loaded were

shown in Fig 8C. These quantitative results demonstrated that WT LC3 showed the

strongest ARE-binding compared to al1 other mutant LC3. LC3 carrying an R to K

substitution (RK) in the ARM and an F to A substitution (HA) in a possible sheet

structure showed about two-thirds of the ARE-binding affinity as compared to the WT

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Figure 8. LC3 binds ARE at the arginine-rlch motif (ARM) via a charge-

charge interaction.

A. Silver staining of the SDS-PAGE shows the amounts of WT and mutant recombinant

LC3 proteins loaded and transferred B. Representative autoradiograph shows the NW

analyses of WT and mutant recombinant LC3 binding to the [32~]-radiolabeled ARE probe.

C. Densitometric analyses normalized to the amount of proteins tested (as shown in A)

demonstrated the strongest binding of WT LC3 to ARE, while mutant LC3 carrying the R

to Q substitution in the ARM (R/Q) showed lower binding to the ARE compared to the WT

and other mutant LC3 species. LC3 with an R to K substitution in the ARM (WK) and a F

to A substitution in the potential fl sheet structure @/A) showed about two-third of binding

affinity to ARE compared to WT LC3. Therefore, the 3 arginine residues constituting the

ARM are critical for LC3-ARE binding, probably via a charge-charge interaction.

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WT R/Q Wb: FIA

WT R/Q R/K FIA

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LC3, while LC3 carrying the R to Q substieon (R./Q) in the ARM showed a significantly

lower affinity to the ARE compared to the WT and other mutant LC3. Therefore. the

positive charge of the 3 arginine residues in the ARM is aitical for ARE binding of the

LC3. This RNA-binding is more lîkely a chargecharge interaction which is not sequence

specific, because the substitution of arginines to lysines, which is another positively

charged residue, does not significantly lower ARE-binding affinity.

The Importance of A R M in LC3-ARE Binding Confirmed in Stably-

transfected HTlOSO Cells

One of the disadvantages of N W analyses is that proteins have been denatured, transferred

renatured during the process, and their ability to retain their original conformation is

therefore questionable. Since secondary and tertiary structure might play a cri tical roIe in

RNA-protein interaction, it is very important to maintain the original conformation of the

proteins when investigating their RNA-binding activity. To c o n f i our NW results, we

therefore carried out electrophoretic mobility shift assay (EMSA) using the [ 3 2 ~ ] -

radiolabeled rat FN whole 3'UTR under non-denaturing conditions and cytosolic extracts

from H T I O S O cells expressing stably-transfected WT and mutant LC3.

The H T l O S O human fibrosarcoma cells, with undetectable LC3 expression and very low

levels of FN synthesiç, were stably transfected with a PCR mammalian expression vector

carrying WT or mutant LC3 sequences. Positive clones were selected by increasing

concentration of neomycin G418 up to 800 pg/ml and selected clones were confirmed by

western immunoblot for the expression of transfected LC3, while vector-transfected clones

were confirmed for the absence of LC3 expression. Binding reactions were canied out

with 10 ~g of HT1080 cytosolic extract and 105 cpm radiolabeled FN 3'UTR with intact

ARE. The results are shown in Fig 9. When extracts obtained from HT1080 cells with

stably-transfected WT, R/K and F/A LC3 were added to the radiolabeled FN 3WïR, three

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Figure 9. Gel mobility shift assay on HTlOSO cytosolic extracts with

stably-transfected WT and mutant LC3 confiims the significance of the

ARM in ARE-binding

A. Representative autoradiograph of gel mobility shift assay shows the binding between

the [32~]-radiolabeled FN 3'UTR and the cytosolic extracts from HT1080 cells stably

transfected with wild type and mutant LC3-encodîng plasmids. No complex formation was

observed with only FN 3'UTR (FP). Three binding complexes were formed in ceil

extracts with stably-transfected WT, R/K and F/A LC3 (indicated by arrows), while the

lowest complex was absent in ceil extracts with empty vector and WQ LC3 (indicated by

*). These top 2 complexes were specific for ARE, which could be partially competed by

500x cold FN 3WïR with intact ARE (cold FN) but not mutated ARE (cold FNA). B.

Densitometric analysis of the lowest complex illustrates complex formation between FN

3'UTR and WT, R/K and F/A LC3-containing ce11 extracts, but not R/Q LC3 or empty

vector transfec ted ce11 extrac ts. Compiex formation in WT and R/K LC3 can be competed

by cold FN with intact ARE (cold FN) but not mutant ARE (cold FNA), while FIA was

only slightiy competed by both.

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FP Vect FIA

d -3 z 7 Z Z k iL Cr, CL

FP Vect WT WQ R/K FIA

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binding complexes were formed (indicated by amows). The Iowest complex was absent in

cell extracts with empty vector and WQ LC3 (indicated by *). Since the upper two bands

were present in d l WT and mutant LC3 transfected HTlOSO cells as well as empty vector

transfected cells, they might represent complex formation between FN 3'UTR and other

proteins endogenously expressed in HT1080 celis. These 2 complexes seemed to be

specific for the ARE since they could be partially competed by 500x cold FN 3'UTR with

intact ARE (cold FN) but not with mutated ARE (cold FNA). On the other hand, the

lowest band appears to represent the complex fomied between LC3 and FN 3'UTR, since

it is absent in vector transfected controls. Its absence in R/Q LC3 transfected celis was

consistent with the NW results mentioned above, and m e r confirmed the significance of

ARM in LC3-ARE binding via chargecharge interaction. Demsitometnc analysis was then

performed on the lowest complex formed between FN 3'UTR and WT, R/K and FIA LC3

(Fig 9B). Interestingly, when we compared the specificity of ARE-binding between WT,

R K and F/A mutant LC3 by cornpetition studies, the lowest complexes in both WT and

R/K LC3 could be partïaily competed by 500x cold FN 3- with intact ARE (cold FN)

but not with mutated ARE (cold FNA), while the complex fonned between F/A LC3 and

FN 3'UTR could only be slightly competed by both cold WT and mutant FN 3'UTR,

leading to the speculation that F to A substitution downstream of the ARE-binding site

results in the loss of ARE binding specificity. The RNA binding specificity imposed by

sequences flanking the RNA binding site has also been shown in hnRNP A l (Burd and

Dreyfus, 1995) and nucleolin (Senin et al., 1997). These flanking sequences might

contribute to RNA binding specificity by increasing the structural stability of the RNA-

protein complex or by increasing the contact surface between the protein and the RNA.

Though the ARE-binding specificity of LC3 is removed by the F/A mutation, the ARE-

affmity of LC3 is probably contributeci by the ARM since the inmase in affinity of LC3 to

the WT FN 3'UTR Vs mutant was sirnilar when comparing the lowest complex fomed

with W T and R K mutant,

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Effect of WT and Mutant LC3 on FN Synthesis

Previous studies show that IlTl080 cells synthesize low level of FW and do not exhibit ceil

surface FN matrix deposits, a feature associated with the tumorigenicity of the cells.

However, the transformeci phenotype of the cells can be reverted by the upregulation of FN

synthesis induced by different mechanisms of FN gene regulation. For example,

dexarnathasome upregulates FN expression by increasing FN mRNA stability while TGF-

$ increases FN gene transcription (Dean el al., 1988). Previous work in our laboratory

shows that FN can be upregulated in HT1080 cells by stable transfection with LC3 which

enhances translational efficiency of FN mRNA (Zhou et al., 1999, manuscript submirted).

To elucidate if LC3-ARE binding plays the key role in the LC3-rnediated upregulation of

FN synthesis, WT and mutant LC3 constructs were stably transfected into HT1080 cells

and their FN biosynthesis was compared.

Indirect immunofluorescent staining of FN showed a filamentous pattern on the ceil surface

of WT, R/K and F/A LC3 transfectants (Fig lob, d and e), and FIA LC3 showed the most

intense staining. R/Q LC3 transfectants showed diffuse cytoplasmic staining instead of

punctate surface staining (Fig lOc), indicating that less FN was deposited on the surface of

R/Q transfectants compared to WT and other mutant transfectants. Vector transfectants

showed no punctate staining at al1 (Fig lOa), which was consistent with the previous

finding that HT1080 cells show no surface FN matrix deposits.

To confirm that increased FN surface deposition correlates with increased FN biosynthesis

in LC3 transfectants, cells were metabolically labeled with [3SS]-methionine for 5 h and

newly-synthesized FN secreted into the cultured media were measured. Determined by

TCA precipi tation, equal amounts of proteins in cuitured media were incubated with gelatin

4B sepharose beads to purify FN, which was then eluted and resolved by SDS-PAGE. As

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Figure 10. Immunofîuorescence Iabeling of ceIl surface FN deposit in WT

and mutant LC3 transfected HTlOSO cells.

Vector transfectants (a) showed weak FN sîaining while WT (b). R/K (d) and F/A (e) LC3

transfectants showed a punctate and filamentous pattern of FN staining on the c d surface.

F/A LC3 (e) showed the most intense staining among the three. In contrast, WQ LC3

transfectants (e) showed a diffuse cytoplasmic staining of FN instead of a punctate surface

staining. ( f ) corresponds to negative control with secondary antibody alone.

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shown in Fig 1 1, FN synthesis in LC3 WT transfectant clones was increased by about 3

fold when compared with vector transfectants. WQ transfectants did not show an increase

in FN synthesis when compared to vector transfèctants, but with the R/K mutant clone,

synthesis was increased about 4 fold (not significantly different h m wild type LC3). In

the FIA transfectant, FN synthesis was increased by about 5 fold compared to the vector

transfectant and was significantiy higher than in WT LC3 transfectants. This demonstrates

that the F to A substitution in the f3 sheet region enhances the LC3-mediated FN

upregulation. The clones used in assessing FN synthesis were expanded from those used

in the gel shift assays.

WT LC3 upregulates FN synthesis by enhancing FN mRNA transiation, since no

difference was observed in the steady state level of FN mRNA in LC3 compared to vector-

transfected HTlOSO cells and LC3 shifted FN mRNA ont0 heavy polysornes (Zhou et al.,

1999, manuscript submifted). To exclude the possibility that the mutant clones were acting

at different levels of regulation (e.g., mRNA transcription or stabili ty), northem blot

analyses were performed to measut the steady-state level of FN mRNA as shown in Fig

12. No significant difference was observed when comparing FN mRNA leveIs in the

different transfectants, supporting the proposal that RIQ and F/A LC3 regulates FN

synthesis by acting on the translation of FN -A. Translational efficiency, as defined

by the ratio of FN synthesis to FN mRNA Ievel, was compared among different

transfectants in Fig 13. The F/A transfectant was about 4 times more efficient than vector

transfectant in FN mRNA transIation, while the WT and WK transfectant were about 3 and

3.5 times more efficient than vector transfectant. The R/Q transfectant showed a àecrease

in mRNA translation compared to the WT LC3 transfectant, to a level similar to vector

transfectant. When the ARM, the component critical to LC3-ARE binding, is mutated as in

R/Q LC3, FN synthesis decreases probably by failing to help dock FN mRNA ont0

ribosomes for transIation. In contrast, FIA LC3 enhances FN synthesis probably by

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Figure 11. FN expression in specific clones of WT and mutant LC3 stabiy-

transfected HTlO8O cells.

FN synthesis by specifc clones, whose ARE-binding affinity has been c o n f i e d by gel

mobility shift assays, is shown. A. An autoradiograph shows 5-h [35S]-methionine

labeled newl y synthesized FN from culture medium of vector-transfectants, WT LC3

transfec tan ts, R/Q LC3 trans fectants, R/K LC3 transfectants and F/A LC3 transfectants

containing equal total TCA-precipitated proteins counts. B. A graph shows a significant

increase of FN synthesis in WT, R/K and F/A LC3 transfectants cornpared to vector and

R/Q LC3 transfectants (*P<0.05 compared to vector). F/A LC3 transfectant also shows a

significant increase in FN synthesis compared to WT LC3 transfectant (+P<O.OS compared

to WT LC3). n= 4 for each transfectant. Bars reflect SEM.

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Vect WT R/Q R/K FIA

Vect WT R/Q R/K FIA

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Figure 12. Steady-state levels of FN mRNA in WT and mutant LC3 stably-

transfected HTlOSO cells.

A. A representative autoradiograph of northem blot shows a comparable amount of

steady-state FN mRNA in vector (vect), WT, RIQ, R E and FIA LC3 transfected cells.

GAPDH serves as a positive control and ethidium bromide staining of 28s and 18s

ribosome RNAs serve as controls for loading conditions. B. A graph of relative

densitometric units of FN mRNA normalized for 18s ribosomal RNA confîrms similar

levels in al1 transfectants. n=3 for each transfectant. Bars represent SEM and significance

was tested by Student t-test.

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Vect WT R/E F/A

7

Vect

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Figure 13. Translational efficiency of FN mRNA in WT and mutant LC3

stably-transfected HTlOSO cells.

Using the ratio of FN protein synthesis / FN mRNA, the increase in LC3 WT Vs vector is

not apparent with the R/Q mutants but is observeci with R/K and F/A mutants.

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Vect WT R/Q R/K FIA

LC3

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façilitating the dockïng of FN mRNA onto translational machinery, via inmasecl affinity :O

ribosomes or other ribosornal proteins. However, increased affinity to FN mRNA was not

observed in NW or gel shift analyses.

LC3 Regulates CeU Growth and Morphology via ARM-ARE Binding

Upregulation of FN in tumor cells has been reported to revert the transforrned phenotype

by enhancing adhesion of cells to the substrate, changing the morphology of the celis from

rounded to a more spreading shape as well as decreasing growth rate (Akamatsu et al.,

1996). Previous studies have show that WT LC3 transfected HT1080 ceUs adopted a

more flattened cell shape and slower growth rate compared to vector transfectants (Zhou et

al., 1999, manuscript submiîîed). To elucidate whether these phenotypic changes are

related to LC3-ARE interaction and upregulation of FN synthesis, ce11 morphology and

growth rate of vector, WT and mutant LC3 transfectants were examined Each transfectant

was plated at the same density in 6-weil dishes and the number of cells per well was

counted every 24 hours. Ce11 counts were shown in Table 1 and growth curves were

plotted in Fig 14. After 4 days, WT and F/A LC3 transfectants showed significantly

slower growth compared to vector, RIQ and R/K LC3 transfectants, while R/Q showed the

highest growth rate even compared to the vector transfectant Therefore, LC3 inhibits

HTlOSO ce11 growth probably via its interaction with the ARE of FN mRNA, resulting in

the upregulation of FN synthesis which in turn reverts the transformed phenotype of these

tumor cells. It is also possible that LC3 suppresses cell growth via binding to the ARE of

other mRNAs w hic h encode growth-regulatory elements.

The morphology of these different transfectants was compared 24 h after plating at the

sarne density. Representative phase contrast photornicrographs are shown in Fig 15. WT

and FIA LC3 transfected celIs (Fig 1% and f) were more e l o n g d , spread better and were

less phase-dense compared to the non-transfected and vector-transfected cells (Fig 15a and

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Table 1. Ce11 count of WT and mutant LC3 transfected HTlOSO ceiis.

Values are mean f SEM. n= number of clones. *Pd .05 compared with the vector.

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Vector (n=3)

WT LC3 (n=4)

iUQ LC3 (n=3)

R/K LC3 (n=4)

N A LC3 (n=4)

Day 1 Day 2 Day 3 Day 4

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Figure 14. Growth curves of WT and mutant LC3 transfmted ET1080

cells.

Growth curves of empty vector, WT and mutant LC3 transfected HTIOSO cells were

plotted with the average number of cells per weii in 4 consecutive days (as s h o w in Table

1) after plating at the same density on &y O. Significantly reduced growth of WT and F/A

LC3 transfectants compared to vector and other mutant LC3 transfectants is observed 4

days after plawig. (*Pc0.05 compared to the vector-transfected cells.)

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Figure 15. Effect of WT and mutant LC3 expression on HTlOSO eell

morphology.

A representative phase-contrast photomicrograph showing the WT and mutant LC3

transfected HTf 080 cells 24 hours after plating. Non-tïansfected (a) and vector-transfected

(b) cells were more phase-dense and showed a rounded ce11 shape compared to the

elongated and flattened ce11 shape of the W T (c) and F/A (f) LC3 transfected cells. R/Q (d)

and R/K (e) msfected cells showed an intermediate phenotype between the rounded and

the fi attened celi shape.

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b), while WQ and WK transfectants (Fig 1Sd and e) showed an intermediate phenotype

between the vector and WT LC3 transfectants. These morphological differences were

further examined by indirect immunofluorescence of mimtubules with an anti-tubulin

antibody (Fig 16). Consistent with the previous photomicmgraph (Fig 15), WT, F/A as

weii as WK LC3 transfected cells (Fig 16b, e and d respectively) showed an eiongated cell

shape with densely organized microtubule arrays. WQ LC3 transfectants were similar to

vector transfectants (Fig 16c and a), with less well organized microtubules. Therefore,

besides decreasing celi growth, L a - A R E interaction also induces a flattened ceU shape in

HT 1080 cells, probably by influencing microtubule organization.

SubcelIular Localization of WT and Mutant LC3 in Stable-transfectants

Since LC3 bas a dual role as both a microtubule-binding protein and an RNA-binding

protein, its subcellular distribution might affect its different functions in regulating

microtubule dynamics and FN mRNA translation, or the coordination of both. From the

RNA binding studies above, WQ LC3 shows lower RNA-binding activity compared to WT

or other mutant LC3. It would be interesting to see if the decrease in RNA-binding of WQ

LC3 would shift the distribution of the protein away from the FN mRNA and translational

rnachinery, resulting in increased LC3 CO-localization with mimtubules.

Subcellular distribution of LC3 was first examined by indirect immunofiuorescence of LC3

with an anti-LC3 antiserurn as s h o w in Fig 17. Al1 WT and mutant LC3 transfectants

were expressed along the microtubules as granules as well as in perinuclear region

associated with ribosomes. WT LC3 transfectants showed very intense granular staining in

the perinuclear region (Fig 17a) evident as bright yellow dots while al1 mutant transfectants

showed less intense perinuclear staining (Fig 17c. e and g). Interestingly, at a different

focal plane, al1 W T and mutant aansfectants showed a network pattern of intranuclear

staining (Fig 17b, d, f and h), most prominent in F/A and R K transfectants (Fig 17h and f

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Figure 16. Immunofluorescence labelhg of tubuiin in WT and mutant LC3

transfected HTlOSO cells.

Consistent with the celi morphology shown in Fig 15, vector-msfected (a) and WQ LC3

@) transfected cells showed a rounded cell shape and less organized tubulin staining, while

the WT (c), R K (d) and FIA (e) LC3 transfected cells showed a more elongated shape with

densely organized fdamentous microtubule staining. (f) corresponds to negative conml

with secondary antibody alone.

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Figure 17. Immunofluorescence Iabeihg of WT and mutant LC3 in distinct

subcellular locations in stably-transfected HTlOSO cells.

Al1 WT (a) and mutant LC3 (R/Q(c), R/K(e) and F/A(g)) transfectants showed dual

staining in the cytoplasm dong the microtubules as granules as well as in the pennuclear

region associated with ribosomes. WT LC3 aansfectants showed a very strong granular

staining in the perinuclear region as bright yellow dots while al1 mutant transfectants

showed a less intense perinuclear staining. Interestingly, at a different focal plane on the

nght column (b, ci, f and h respectively), al1 mutant transfectants showed a network pattern

of intranuclear staining, most prominent in F/A (h) and WK ( f ) transfectants. Thus

intranuclear LC3 might be associated with some unknown nuclear cytoskeletal components

or with the nuclear membrane dong with pretranslational machinery. (i) and (j) correspond

to vector-transfectants and negative control with secondary antibody alone.

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respectively). This intranuclear LC3 might be associated with some unknown nuclear

cytoskeletal component or with the nuclear membrane dong with pretranslational

machinery. This should be further investigated by comparing LC3 concentration in

purified nuclear extracts €rom these cells. However, there is no notable

immunohistochemical difference in LC3 distribution when comparing WT and mutant LC3-

transfected cells.

To quantify the differential distribution of LC3 associated with the microtubules or the

ribosomes respectively, the supernatant and peilet fractions of the ceil lysate fiom the empty

vector, WT and mutant LC3-transfected HT1080 cells were assessed for LC3 by western

immunoblot. Since microtubules are mostly concentrated in the supematant fraçtions, as

confirmed by western imrnunoblot using anti-tubulin antibody as shown in Fig 18A, LC3

present in the supematant represents the fraction both free in the cytoplasm and bound to

the microtubules, while that present in the pellet represents the fraction in the nucleus or

bound to membranes including the translational machinery, ribosomes and B A .

Twenty pg of protein from the supernatant and each of the 2 pellet fractions were loaded

ont0 the gels and transferred ont0 membranes. For the cell lysate from empty vector

transfected HT LOS0 cells, no LC3 was detected (data not shown); whereas for the WT and

mutant LC3 transfectants, more LC3 was found in the 2 pellet fractions compared to the

supernatant (Fig 18A). Densitometry was performed on the LC3 immunoreactive bands to

quanti fy the difference between the supernatant and the pellet. The pellet/supematant (PIS)

ratio was obtained by dividing the sum of the densitometric units from the 2 pellet bands by

the densitornetric unit of the supernatant band. As shown in Fig 18B, there is no

significant difference in the P/S ratio amring the WT and the mutant LC3, even the R/Q

tC3, illustrating that the lack of ARE-binding does not unload RIQ LC3 from the

translational machinery or the ribosomes, probably kcause of the interactions between

LC3 and other membrane-bound components besides FN mRNA. From our previous

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Figure 18. Quantitative analyses comparing the subcellular distribution of

WT and mutant LC3 in stably-transfected HT1080 cells by western

imrnunoblot.

A. Representative western immunoblots showing LC3 and tubulin expression in the

supernatant (S) and the 2 pellet (Pl and Pd fractions of the cytoplasmic extract from

HT1080 cells stably transfected with WT or mutant (R/Q, R/K and F/A) LC3 constructs.

Densi tome try was perforrned on the LC3 immunoreac tive bands to calculate the

pellet/supernatant ratio (P1+P2/S) as shown in B. No significant difference is observed in

the pellet~supernatant ratio of cell extracts fiom WT and different mutant LC3 transfectants.

Bars refiect standard deviations fiom n=4 WT LC3 transfectants, n=3 R/Q LC3

transfectants, n=4 R/K LC3 transfectants and n d F/A LC3 transfectants.

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WT RIQ R/K FIA

FIA

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studies, LC3 was found to bind 40s and 60s ribosomal subunits, suggesting that LC3

facilitates the docking of FN mRNA onto the translational machinery by acting as a iinkage

between these 2 elements (Zhou et al., 1999, manusmpt subrnitted).

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DISCUSSION

In this thesis we investigated the site on LC3 which binds the AU-rich element (ARE) on

the 3' UTR of the FN mRNA, and how this specific RNA-protein interaction contributes to

the regulation of M synthesis, as well as the tumorigenicity and the morphology of LC3

stably-transfected HT1080 cells. To narrow down the potential ARE binding site to a

smaller region within the molecule, we used proteases and CNBr to generate discrete LC3

peptides from the 16 kD tecombinant LC3 protein, foliowed by NW blot analysis to assess

the ARE-binding activity of these peptides using a [32P]-radiolabeled ARE oligonucleotide

probe. A 10 kD N-terminal peptide generated by CNBr cleavage showed strong ARE-

binding activity while the 6.8 kD N-terminal peptide showed no binding. This narrowed

the ARE binding site down to residues S61 to M88, which was confirmed by proteolytic

digests using other enzymes. Since peptides containing only the S61-MS8 region show no

binding to the ARE, the upstream Pl-M60 sequence was also judged to be important for

ARE-binding, probably by contributing to the charge andor the secondary structure of the

protein. Based upon these results, we tried to generate a tmncated recombinant 10 kD N

terminal peptide, but could not show that it possessed ARE binding ability, perhaps

because it was not folded properly or not glycosylated in E. coli comptent ceils. We

therefore perfonned site-dîrected mutagenesis to determine the specific residues within

S61-M88 which bind the ARE. There are 2 possible RNA binding sites: the arginine-rich

motif (ARM) containing 3 consecutive arginine residues and a predicted sheet region

containing 2 consecutive phenylalanine residues. We mutated the 3 consecutive arginines

or the 2 consecutive phenylalanines: R to Q substitution in the ARM to alter charge

abolished the ARE binding activity upon W anaiysis, while R to K substitution which did

not alter charge, did not appear to decrease ARE binding. F to A substitution in the

predicted f3 sheet region also did not affect ARE binding. This led to our conclusion that

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the positive charge of the 3 R's in ARM are critical for ARE binding of LC3, probably by

contributing to the charge-charge interaction.

Since N W analysis rnight distort the original conformation of the protein, we set out to

confirm our N W resulu by gel shift analysis in which the protein and the RNA are in intact

state. However, we were unable to produce convincing gel shift results using recombinant

intact wild type or mutant LC3 proteins with either the ARE oligonucleotide or the whole

FN 3'UTR probe. This is likely because pst-transiational modification of the recombinant

proteins might be different in E-coli compared to mammalian cells. Considering that

distinct complex formation is easily shown using the ARE oligonucleotides or the FN

3'UTR and endogenously expressed LC3 present in cultured smooth muscle cells from

sheep ductus arteriosus and aorta and piglet coronary artery (Zhou et al., 1998; Mason et

al., 1999, manuscript in preparation), we stably transfected Hi' 1080 cells with plasmids

encoding the WT and the mutant LC3, and used the cell extracts to perform gel shift

analyses. Zhou has shown by NW analysis that the 16kD recombinant LC3 expressed in

HT1080 transfected cells maintains its RNA-binding capacity with preference for ARE

(Zhou et al., 1999, manuscri' submined). From the gel shift anaiysis shown in Fig 9,

WT, RIK and FIA LC3-containing HT1080 ce11 extracts formed 3 ARE-dependent

complexes witb the [32~]-radiolabeled FN 3'UTR, whereas in cell extracts containing WQ

LC3 and empty vector, only the top 2 complexes were formed. Therefore, the lowest

complex represents the recombinant LC3 and the ARE- FN 3'UTR, while the top 2

complexes might represent cornplex formation between FN 3'UTR and other endogenously

expressed proteins in ml080 cells. Previous NW analysis using [32~]-radiolabeled FN

3'UTR and HT108O ce11 extracts containing WT LC3 or empty vector demonstrated that

beside the l6kD recombinant LC3, a 200kD and a 551d) proteins also showed ARE-

binding in both LC3 and vector transfectants (Zhou et al., 1999, munuscript submined).

These 2 proteins which in previous studies were also immunoreactive with the LC3

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antibody might represent the top 2 complexes observed in our gel shift analysis. The

absence of lower complex formation when extracts h m R/Q-LC3 transfected cells were

used, further cofirmed the significance of the 3 R's in the ARM of intact LC3 in ARE-

binding.

When investigating the ARE-binding specificity of the WT and mutant recombinant LC3

expressed in HTlOSO celis by cornpetition studies, binding between FN 321TR probe and

WT or R/K LC3 could be partially competed by cold EN 3WTR with intact ARE but not

mutated ARE; whereas binding between FN 3'UTR and F/A LC3 could only be slightly

competed if at al1 by cold FN 3WïR with either intact or mutated ARE. This showed that

binding of WT and R/K LC3 to FN mRNA is ARE-specific, consistent with the NW

results in Fig 7 which showed higher affinity binding of WT LC3 to WT ARE

oligonucleotides compared to mutant ARE oligonucleotides. However, F to A substitution

in the predicted B sheet stnicture resulted in the loss of ARE-specificity in mRNA binding,

dernonstrating that the 2 phenyldanines downstrearn of the ARE-binding sites are important

in detemiinkg the ARE-specificity of LC3 in RNA binding.

The importance of sequences flanking the RNA binding domain in confaring binding-

specificity has been shown with other RNA-binding proteins. The required flanking

sequences v q h m 5 residues in Ul-A and U2-B" to 1 11 residues in La proteins (Kenan,

199 1). This irnplies that motif alone rnay not contain sufficient information to function as a

sequence-specific RNA binding domain. The requirement for flanking sequence suggests o

function important in maintaining the secondary structure of the protein which permits the

motif to form a direct contact with the RNA. In cases where flanking sequences do not

contribute to secondary structure, they may instead provide additional RNA contacts.

hnRNP A i is similar to LC3, in that it also binds to the ARE of the 3WTR of c-fos and

GM-CSF mRNA, and this property inhibits the ARE-dependent mRNA turnover of c-fos

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mRNA (Hamilton et al., 1993). hnRNP A l acts as both a pre-mRNA binding protein

involved in nuclear RNA processing as well as a trans-acting factor involved in modulatuig

cytoplasrnic mRNA turnover and translation (Hamüton et al., 1993). It contains 2 RNA

recognition motifs (RRMs) tandemly arranged at the N-terminus, followed by a C-terminal

glycine-rich region (Ghetti et al., 1990). It was proposed that the first RRM binds to the

RNA target, and the interaction is stabilized by the second RRM (Burd and Dreyfuss,

1995). To better understand how LC3 interacts with the ARE of FN mRNA, it would be

very helpful to have the crystallized structure of LC3 or the FN mRNA-LC3 complex.

To study the association between ARM-ARE interaction and FN synthesis, WT and mutant

LC3encoding plasmids were stably-transfected into HT1080 cells, which are LC3-nul1 and

express low levels of FN. Previously, Zhou stably transfected HT1080 cells with

plasmids containing empty vector or WT LC3, and showed upregulation of FN mRNA

translation in WT LC3 transfectants by enhanceci ribosome recruitrnent (Zhou et al., 1999,

manuscript submined). Consistent with Zhou's nsults, we showed a 2.5 times increase in

FN synthesis and secretion into condition medium in WT LC3 compared to vector

transfectants. Consistent with the inability of R/Q LC3 to f o m a complex with the FN

3'UTR was its inability to upregulate FN synthesis compared to WT LC3. This was

evident both by immunofluorescent staining of FN deposition on the surfaces of cultured

cells and by [%]-metabolic labeling to detect newly synthesized and secreted FN. W e

confimed that the inability of R/Q LC3 to upregulate FN was not due to a decrease in FN

mRNA level or stability, leading to the conclusion that WQ LC3 is inefficient in docking

F'N mRNA onto polyxibosomes for translation. This can be further investigated by

polysorne profile andysis.

In contrast, F/A LC3 showed a significant increase in FN synthesis compared to the WT

LC3. Since the FN mRNA level is unchanged in FIA transfectants, this upregulation in FN

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synthesis, manifest both as enhanced FN deposition in the ECM and secretion in

conditioned medium might be due to the ability of FIA LC3 to facilitate the docking of FN

mRNA ont0 heavy poiysomes. F/A LC3 might have a stronger affinity for ribosomes or

other ribosomal proteins, such as 40s and 60s, compared to the WT LC3. F/A LC3 might

also enhance FN synthesis via increased FN mRNA binding. Aithough binding between

F/A LC3 and ARE oligonucleotide or intact FN 3VTR is unchanged compared to W T

LC3, as illustrated in both NW and gel shift analyses, we cannot exclude the possibility

that FIA LC3 has a stronger affinity for other regions of the FN mRNA, such as the S'UTR

or the coding region. The Ioss of ARE-specificity of FIA LC3 as shown in cornpetition

studies might lead to the enhanced affinity for other regions of the FN M A , or even to

other mRNAs which might indirectly contribute to the upregulation of FN synthesis, as

well as the other phenotypic changes observed To examine the binding affinity of FIA

LC3 to ribosomal proteins, total ce11 lysates from HT1080 cells can be incubated with

agarose beads conjugated with GST-FIA LC3 fusion proteins and bound extracts can be

tested for the presence of 6ûs and 40s ribosomal subunits using 28s and 18s RNA probes

in northern blot analysis, or tested for the presence of other ribosomal proteins using

western immunoblots.

Besides acting on the translational upreguiation of FN mRNA, F/A LC3 might also

enhance FN synthesis by increasing the cytosolic pool of FN mRNA availabie for

translation. From the immunostaining pattern of LC3 in HTlOSO cells in Fig 18, al1 WT

and mutant LC3 transfectants showed intranuclear staining of LC3, but this was most

prominent in FIA LC3 transfectants. Since the FN mRNA level is unchanged in F/A LC3

transfectants, F/A LC3 does not appear to play a role in transcriptional regulation. It might,

however, interact with other nuclear proteins to form a mRNP complex that facilitates the

export of FN rnRNA into the cytoplasm for translation. To investigate the role of LC3 in

the nucleus, nuclear extracts could be obtained and we could determine using

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immunoprecipitation with appropriate antibodies whether LC3 forms a complex with other

nuclear proteins.

FN has k e n shown to revert the transformed phenotype of tumor cells by enhancing

adhesion of these cells to the substrate, changing the morphology of the cells h m rounded

to spread and decreasing growth rate (Akamatsu et al., 19%). Zhou showed that by

stably-transfecting HT108O cells with WT LC3 which induces FN mRNA translation, he

was able to revert the transformed phenotype of the HTlOSO cells in a similar manner as

was s h o w in other studies by overexpressing FN cDNA. Here, we further showed that

LC3-mediated FN synthesis and the resulting reversion of transformed ~ 1 0 8 0 phenotype

depends on the ARM-ARE interaction. WQ LC3 transfectants, which failed to upregulate

FN synthesis due to the mutated ARE-binding site, were also unable to decrease the growth

rate of the cells or revert them to a flattened shape. They behaved Iike empty vector

transfectants. In contrast, the FIA LC3 mutant, acted like a "super" LC3 and both in

upregulating FN synthesis and in reducing growth rate and reverting cell phenotype.

While, these data directly implicate FN in reverting the transfonned phenotype of the

HT1080 cells, LC3 rnight also bind to other mRNAs which contribute to the slower growth

rate and flattened ce11 shape. To investigate whether FN alone is necessary for this

antitumor effect, we can transfect these LC3 stably-transfeçted ceils with cDNA encoding

antisense FN mRNA to see if the revenant phenotype can be overcome. It would be

interesting to fuxther investigate the significant anti-tumor effect of FIA LC3 in animals

where we could detenaine the impact of gene transfer of this mutant on fibrosarcoma

growth.

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In this thesis, we show that LC3 upregulates FN mEWA translation via the contribution of

the arginine-rich motif (ARM) to the LC3 binding to the AU-rich element (ARE) in FN

rnRNA. By site-directed mutagenesis, mutant LC3 with R to Q substitution in the ARM

was unabie to bind to the ARE of the FN mRNA and upregulate its translation. Steady

state levels of FN mRNA in HTlO8O cells transfected with RiQ LC3 are unchanged

compared to WT LC3 transfectant, confirming the significance of ARE-ARM interaction at

the level of mRNA translation, without any effect on transcription, mRNA stability or

splicing. On the other hand, the upregulation of FN mRNA translation imposed by FIA

LC3 also acts at the level of mRNA translation with no notable change in FN mRNA

compared to WT transfectants. Therefore, polysome profile analysis wiil be critical in

determining more precisely whether the EUQ mutant fails to move FN mRNA into the heavy

polysome fractions and the F/A mutant does so even more efficiently than WT LC3. By

comparing the distribution of FN mRNA with that of the WT or mutant (R/Q LC3 or FIA

LC3) we will get a better idea about whether LC3 affects the docking of FN mRNA on the

tram lational machinery .

In addition to binding FN mRNA, LC3 also appears to directly bind to ribosomd subunits

(both 40s and 60s)(Zhou et al., 1999, manuscript submitted), and may also bind other

proteins important in translation initiation. It would be interesting to Qtennine whether the

R/Q and F/A mutations influence also these protein binding properties of LC3. Previously,

Zhou used agarose beads conjugated with GST-LC3 fusion protein to extract any bound

ribosomal proteins from the ceil extracts. However, considering that there might be a

ciifference in the confoxmation and pst-translational modification of fusion LC3 compared

to the endogenously expressed protein, that might influence protein-protein interaction, it

would be more convincing to study LC3-protein interaction by immunoprecipitating

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endogenously expressed LC3 h m stably-transfected HT 1080 cells. While the affinity and

specificity of the currently available LC3 peptide antibodies does not permit

immunoprecipitation, this may be overcome by producing antibodies to the intact molecule

or by epitope-tagging LC3. HA-tagging would aüow the immunoprecipitation of LC3 by

using some commercially available antibodies towards the HA tag. Since in the HT1080

cells, additional proteins are recognized by the U33 antibody, epitope-tagging of LC3 could

facilitate the intracellular localization and compa.rtmentaüzation of LC3. This would help

address the circumstances under which LC3 might be prescrit or tramlocate to the nucleus,

and associate with other mRNAs or proteins to form a RNP cornplex- Other possibilities

include conditionally expressing LC3 tagged to green fluorescent proteins (GFP) to more

precisely follow the intraceilular dynamics of this protein.

Other mRNAs such as that for apolipoprotein D have dso been isoiated using an LC3-GST

column. The ability to imrnunoprecipitate LC3 could ailow for more direct intracellular

detection of mRNAs with which this protein associates. Yeast two hybrid systems couid

also be used to detect other proteins with which LC3 interacts. A more recent strategy

would suggest that LC3 tagged with a PKA site could be used to screen a cDNA

expression library, for protein-protein interactions.

Microtubules have k e n shown to play a critical role in FN mRNA translation. When DA

SMC are treated with colchicine which disrupts the microtubule organization, they showed

a decreasc in FN mRNA translation, associated with less FN mRNA present in the

polysome fractions compared to the untreated DA SMC. Therefore, it would be interesting

to investigate if R/Q and F/A mutant LC3 affect FN mRNA translation by altering its

association with the microtubules. This can be achieved by both in vitro and in vivo

microtubule-binding assays. Ultimately, it would be very important to identify the

microtubule binding site(s) on LC3, which also play a significant role in regulating FN

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mRNA translation. This will M e r elucidate the novel mechanism of how microtubdes

regulate niRNA translation.

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BlOTECHNOLOGY SERVICE CENTRE DEPI:. OF CUMCAL BIOCHEMISTRY

100 COLLEGE ST, ROOM 351 TORONTO. ONTARIO M5GiLS

P E m E SEOUENCE ANAI-YSIS FACILlTY Phone: (416) 978-5554

. - &K. J UY Date: ...............................~.

- / s- Hfle User: .............................~......................................................................

......................................................................................... COMMENTS: .............. ..-. . ~ c 7 z y k . . xt.. .z&ifi!. œfle~~

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BIOTECHNOLOGY SERVICE CENTRE Dqt Crinicd Biochuniztxy 100 CoiIege S t. Room 351

Toronto. Ontario MSG L I 3

/Yb-,- d ? / p DATE: -----.,-...-..-.-.--.-. --- /G' /CD @7~@30s5/" SAMPLE: ...-œ..-œ-. -.-. .-œ..-œœœ. ...œ-œ.o-CODE: .,..., ..-,.., ,,,....,,,, ,.,

Page 125: OF LC3 THE AU-RICH ELEMENT OF FIBRONECTIN · RNA-BINDING OF LC3 TO THE AU-RICH ELEMENT OF FIBRONECTIN mRNA: A STRUCTURAL AND FUNCTIONAL STUDY Master of Science, 1999 Agatha Lau Department

F i le=c:\porton\datal\G9803WP.01R froin 5-50 to 26-00 min, Low seaie = 41.5ïS1 M. nigh scale = CC-5751 av,

+***+*********++++ HSC Biotechnology Service Centte **t*+++**+tt*t**+* * * Pept ide ~equencing Facility t

* pl&R 4, 1998 18:14:37 * * * CYCLE NUMBER: 1 * * lOKD * *****************+**************************************************** Peak Ret-Time Amino Peak Amount

# ( m i n - 1 A c i d Area moles) Comments 1 6-00 3398 4-48 ' 2 6.33 181108 238 &O 3 7-12 ASP 6404 7.24 4 8 - 5 7 SER 6022 18-95 5 9.17 THR 2379 4-76 6 9 - 7 5 GLY 16481 24-07 7 12-88 ALA 57940 60-20 8 15-37 TYR 3304 3.65 9 18-10 DTT 599238 77.52 10 18.68 PRO 6646 7.67 11 21.02 DPTU 50320 2.93 12 21-90 64531 85.12 13 23-77 XLE 7458 8.70 14 2 5 - 4 3 1968 2 - 6 0

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F i Le=c:\porton\datal\C98030ZP ,02R f rom 5 -50 to 26-00 min, Lou s c a k = 41,5223 rn- Nigh scate = 44-5223 W.

Peptide Sequencing Facility MAR 4, 1998 19:02:07

* CYCLE NUMBER: 2 1, * lOKD * ***************+****************************************************** Peak P

1 2 3 4 5 6 7 8 9

R e t . Time ( m i n . 1

6.35 7.12 8 .55 9 - 7 3 12 87 18-10 21.02 21-88 23.73

Amino A c i d

ASP SER GLY ALA D m DPTU

ILE

Peak Area 196308

2775 4644 3737

34272 430328

38346 49450

6383

Amount moles 1 Comments O

258.95 - 3.14

1 4 - 6 1 .

5-16 35-61 55.67 2.23

65.23 7.45

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F i te=c:\portori\datal\C080U1C~.03R frairi 5.50 to 26-00 min, LOU scate = 41,4245 mv- nigh scate = 44,424s H,

Peptide Sequencing Facility MAR 4, 1998 19:49:36

* CYCLE NUHBER: 3 * * lOKD * ********************************************************************** Peak Ret.Time Amino Peak Amount # ( m i n , 1 Acid Area t~molesl Comments 1 6 - 2 7 185842 2 4 5 - 1 5 - 2 10-15 HIS 11546 14 r 0 4 3 1 2 - 8 2 ALA 29729 30.89 4 18-07 D!FT 380345 4 9 - 2 1 5 2 1 - 0 2 DPTU 32511 1 - 8 9 6 21 -87 58306 76.91 7 23 243 1506 1 - 9 9 8 25.42 2520 3.32

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F i le=c:\porton\datal \C9803(UP.OCR f rom 5-50 CO 26-00 min- L w scak =

*******+******+*++ HSC Biote&nology service C e n t r e *+***********f+t** * f

* Peptide Sequencing Facility O

* MAR 4 , 1998 20:37 :06 O

* f

r~ CYCLE NüMBER: 4 O

* lOKD f

********************************************************************** Peak Ret.Time Amino Peak Amount

# ( m i n . 1 A c i d Area ( V ~ O ~ S ) C o m m e n t s O

1 6.32 185890 245.21 - 2 8.55 SER 3588 11.29 3 10.22 HIS 1760 2.14 4 12.87 ALA 27598 28.67 5 18.08 DTT 386597 50.01 6 19.32 MET 3419 3.65 7 21-02 DPTU 26178 1.52 8 21.88 49301 65.03 9 23-73 ILE 4832 5.64 10 25.42 2891 3.81

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F i ~e=c:\porton\&tal\G9CU)30CP,05R froni 5-50 to 26-OO min. L w scale = 41,4392 nv, H i g h scate = 44.4392 mv,

***+t***********t+ HSC Biotechnology Semice Centre ************++**** * 4 * Peptide Sequencing Facility * HAR 4 , 1998 21:24:35 * * # * CYCLE NUEIBER: 5 4 * lOKD * ********************************************************************** Peak Etet-Time Amino Peak Amount

pl ( m i n - 1 A c i d Area moles) Comments 8

1 6-33 186944 246.60 - 2 9 - 1 7 THR 2557 5.12 3 12-88 ALA 32278 33 54 4 1 8 - 1 0 DTT 436769 5 6 - 5 0 5 21.03 DPTU 29173 1.70 6 2 1 - 9 0 53260 70.26 7 2 3 - 7 8 ILE 7344 8 - 5 3

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**************+*** HSC Biotechnology Semice Centre ****t*********+*t*

* a * Peptide Sequencing Pacility * * MAR 4, 1998 22:12:04 * * * * CYCLE -ER: 6 * * lOKD * **********************************************************************

Peak R e L T i m e Amino Peak Amount # (min. 1 A c i d Area moles 1 Comments . 1 6.33 190281 251 -01 - 2 10.18 H IS 1304 1.59 3 12.88 ALA 33647 34.96 4 18.10 DTT 436416 56-46 5 21.02 DPTU 27745 1 .61 6 21.90 49359 6 5 - 1 1 7 23.22 P m 3394 4 . 4 0 8 23.75 ILE 6732 7.85

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BIOTECHNOLOGY SERVICE CENTRE DEPT. OF CLiNïCAL BIOCHEMISTRY

100 COLLEGE ST., ROOM 351 TORONTO, ONT"RI0 MSG I I 5

PEPTIDE SEO1 IMCE ANALYSIS FACiLITY Phone: (416) 978-5554

. Falrnmile: (416)978-8802

Date:

-HD ........................... ......................................... S ample: .œœo.o....CODE: G%f=@~ -7- 9- Pkpe User: ....................................................................................................

COMMENTS: ....................................................................................... fl&/e. . . d y .&. +2.jpi3&&Kœ .. A%&?* 0. de~dca' . ........ ................

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BIOTECHNOLOGY SERVICE CENTRE Dept Cüniui B i o d i d w 100 Coiiegc Sr. Room 351

Toconm. Onrario M5G LL5

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41,3%7 rn, tligh scale = GC.3967 nu,

*********++*+**t+t HSC Biotechnology Service Centre ****+***+**++**t*+ * * * Peptide Sequencing Facility * * MAR 4, 1998 12:08:22 t * * * CYCLE NüMBER: 1 * * 6-8KD * ********************************************************************** Peak Ret.Time Amino Peak Amount

# (min. ) A c i d Area (~molesl Comments .. 1 5.98 9592 12-65 - 2 6.33 218576 288 -33 3 7.15 7359 9.71 _ 4 7.87 10361 13.67 5 8.55 SER 7344 23.10 6 9.73 GLY 40310 58-87 7 12.87 ALA 67366 49-99 8 15.32 TYR 1766 1.95 9 18.08 DTT 729758 94.41 10 18.67 PRO 13745 15.85 11 21.00 DPTU 48491 2.82 12 21.88 73148 96.49 13 23.77 ILE 8399 9.80

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C l ,4855 riva H i g h scale = CC.CUSS rn,

Peptide Sequencing Facility MAR 4, 1998 12:55:52

* CYCLE NUMBER: 2 * r~ 6.8KD a ********************************************************************** Peak Ret.Time Amino Peak Amount # ( m i n . I A c i d eh bmolesl Comments 1 6 - 3 3 198195 2 6 1 - 4 4 - 2 7 - 1 2 ASP 3178 3-59 3 8 . 5 5 SER 7799 2 4 - 5 3 _ 4 9 - 7 5 GLY 4125 6-02 5 12-88 ALA 30942 32-15 6 13.98 5062 6-68 7 15.67 4333 5.72 8 18-08 DTT 419459 54-27 9 21-02 DPTU 28943 1-68 10 21-88 40005 52 -77 11 2 3 - 7 5 ILE 3422 3-99

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*+***++*++**t**+** HSC Biotechnology service Centre ************+**t** * * * Peptide Sequencing Facility * * nAR 4, 1998 13:43:20 * * * * CYCLE NUMBER: 3 * 6.8KD * ********************************************************************** Peak Ret-Time Amino Peak Amount

# ( m i n . 1 A c i d Area (~molesl - Comments 1 6-33 204739 270 - 08 - 2 9 - 7 7 GLY 1637 2 - 39 3 10-18 HIS 26951 32 - 77 4 12-88 ALA 26143 27.16 5 18.10 DTT . 375674 4 8 60 6 21-02 DPTU 27785 1-62 7 21.90 38473 50.75 8 23.75 ILE 5558 6 - 4 8

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~ilc=c:~rtori\&tal\C98030C-OCR fran 5-50 to 26-00 min. LW scalt = 41,JQbl mr- H i g h scalc = CL.3961 H,

t Peptide ~equencing Facility t MAR 4, 1998 14:30:50 * * CYCLE NUMBER: 4 * * 6.8KD * ********************************************************************** Peak f

1 2 3 4 5 6 7 8 9

R e t . Time ( m i n , 1

6.35 10.15 12.87 18-08 19-28 21.02 21-90 23.70 23-98

Amino A c i d

HIS ALA DTT MET DPTU

ILE LYS

Peak Amount 2irea f ~molesl Comments . 196019 258.57,

5201 6.32 21229 22.06 -

346530 44.83 17412 18.60 27049 1.57 34407 45.39 4869 5.68 8531 14.83

Page 138: OF LC3 THE AU-RICH ELEMENT OF FIBRONECTIN · RNA-BINDING OF LC3 TO THE AU-RICH ELEMENT OF FIBRONECTIN mRNA: A STRUCTURAL AND FUNCTIONAL STUDY Master of Science, 1999 Agatha Lau Department

41,3748 RI- H igh scale = 44,3748 rv,

*****+*+*****t++*t HSC Biotechnolw service C e n t r e +**++t*+*i**+**t**

* * * Peptide Sequencing Facilfty f

* HAR 4, 1998 15:18:19 * * * * CYCLE NOMBER: 5 * * 6.8KD * **********************************************************************

Peak Ret.Time Amino Peak Amount # ( m i n . A c i d Area moles 1 Comments .. 1 6.33 194769 256-92 _ 2 9.17 THR 6676 13.37 3 12-87 AtA 21509 22.35 4 18.10 DTT 346120 4 4 - 7 8 5 18.67 PRO 7836 9.04 6 21 .02 DFTU 23149 1.35 7 21 .90 3 1965 4 2 - 17 8 23.75 ILE 4459 5.20

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File=c:\portor\\datal\t98030S,06R fran 5-50 to 26-00 min. Lou scrlc = C1,429!5 W . High scale =

Peptide ~equencing Facility MAR 4, 1998 16:05:49

* CYCLE NUMBER: 6 * * 6.8KD 4 ***************+****************************************************** Peak Ret-Time Amino Peak mount # (min ) A c i d Area (~nroles 1 Comments 1 6.32 198036 261.23 2 12.87 A U 20697 21.50 - 3 13.92 1574 2.08 4 18.10 ûTT 331341 42.87 5 19.85 1843 2 .*4 3 6 21.02 DPTU 22309 1-30 7 21.90 37841 49.92 8 23.18 PHE 6374 8.27