1. RNA processing and regulation

2. Network interactions in gene expression

3. RASL-DASL the genome: approaches for systems biology

Topics

rRNA Processing Pathway: Involvement of A Series of Endo- and Exonucleases

45S18S 5S 28S

41S

20S 32S

28S

5S

18S

Maturation of tRNAs

• tRNA is transcribed by RNA Pol III.

• The 5’ end is generated by RNase P, an RNA enzyme.

• The 3’ end is generated by RNase D followed by post-transcriptional addition of CCA.

• Many nucleotides are modified, which are critical for matured tRNAs to function in translation.

• Some tRNA genes contain a single intron, which is removed byendonuclease and RNA ligase.

Discovery of Introns

Transcription Unit in Eukaryotes

5’ 5’ 3’3’

Exon (100 - 300 nts)

Intron (103 - 104 nts)

Transcription Polyadenylation

Promoter

Addition of a Cap to the 5’ End of Transcript

pppNpNp

ppNpNp

ppNpNp

ppNpNp

Gp

CH3-Gp

Pi

GTP

PPi

Transfer of methyl group to the cap

CBP80/20

Phosphatase

Guanyl transferase

Methylase

Polyadenylation signals

Cap AAUAAA GU-rich

160K30K

100K73K

Cleavage & polyadenylation specificity factor(CPSF)

64K

77K

50K

Cleavage stimulation factor(CstF)

PAP

Cleavage site

Keller and Minvielle-Sebastia, Curr. Opion, Cell Biol. 9:329-336, 1997

The Polyadenylation Pathway

•PAP stimulates cleavage by CPSF•Bound PAP adds A residues at a slow rate to the 3'OH group•Binding of poly(A) binding protein II accelerates A addition•PBBII plays a role in signaling poly(A) of about 200-250 A residues

From Mole. Cell Biol., Lodish et al., 2000

Consensus Splicing Signals

A/CAG GURAGU YNYURAY--Y10-20--YAG

exon exonintron

5’ splice site 3’ splice sitebranchpoint

polypyrimidine tract exonic enhancer

R: PurineY: PyrimidineN: Any base

exonic silencer

The Nuclear pre-mRNA Splicing Pathway

P P

A 2’HO

3’OH

P

pA

P pA 3’HO

Pre-mRNA

Lariat intermediate

Ligated exons Released lariat intron

Step I: 5’ splice site cleavage and branch formation

Step II: 3’ splice site cleavage and exon ligation

+

The Spliceosome Assembly Pathway

U1

U1 U2A

U6

U5U4

U2

U1

U4U6

U5

U2

ATP

E(Commitment Complex)

A(Pre-spliceosome)

B(spliceosome)

C(Activated Spliceosome)

U6

U5

U2mRNA

Nuclear ExportExon 1

Exon 1

Exon 2

Exon 2

Mechanistic Similarity between Splicing of Nuclear pre-mRNAs and Self-splicing of Group II Introns

Establishment of the Catalytic Core in the Spliceosome and Similarity to the Catalytic Core in Group II Introns

Nilsen, T.W. In RNA Structure and function, 1998

Human:

% of alternatively spliced genes

# of isoforms per gene

Worm:

59%

22%

3.17

1.34

Source: Nature 409:898, 2001

Alternative Splicing: A Genomic Issue

Different Types of Alternative Splicing

1. Balanced Splicing:

2. Alt. 5' Splicing:

3. Alt. 3' Splicing:

4. Exon Skipping:

5. Mutual Exclusion:

5'ss 3'ss

5'ss 3'ss5'ss

5'ss 3'ss3'ss

5'ss 3'ss3'ss 5'ss

5'ss 3'ss

21

VASE7 8

Embryonic brain

Adult brain

NCAM

AUGF

AUGM

2 3

(a) (b)

(c)

(e)

(d)

fruitless

FGFR-2

Binds KGF

Binds FGF

7(IIIa) 10IIIb IIIc

Thyroid

Neurons

34 5 6

A

A

msl-2

Calcitonin/CGRP

Examples of Biologically Important Alternative Splicing Events

Smith and Valcarcel, TIBS 25:381-388, 2000

Genomic DNA

1 12 1 48 1 33 1 2

Exon 4 Exon 6 Exon 9 Exon 17

TM

Protein

Dscam: An Exon Guidance Receptor with 38,016 Isoforms Generated by Alternative Splicing

Schmucker. D. et al., Cell 101:671-684, 2000

Drosophila Sex Determination Pathway

X:A ratio: 2:2 1:2

Sxl

+

Tra

Sxl off

tra (truncated) Tra-2

Dsx + Dsx

Negative regulator of male differentiation genes

Negative regulator of female differentiation genes

Tra/Tra-2SR

stop

stop

- Sxl

+ Sxl

Four Classes of RNA Binding Proteins implicated in Splicing Regulation

Family Name Examples Key Domain Required for Splicing

SR Proteins

SC35, ASF/SF2, 9G8,hTra2-, hTra2-, SRp20, 30c, 40, 46, 54, SRp55, 75, 86

RRM and RS

Yes

HnRNPs

hnRNP A/B, hnRNP F, H hnRNP I/PTB, nPTB

RRM, some with RGG boxes

No

KH-type

KSRP, Nova-1, PS1 KH

No

CELF Factors

CUG-BP1, 2, ETR-3,NAPOR

RRM

No

U2AF

SF2/ASFor

SC35

hnRNPA/B

3' splice site ESE ESS

Positive and Negative Influences of Splice Site Selection by Exonic Enhancers and Silencers

Dis. 5'ss Prox. 5'ss 3'ss

_ _

SC35

SC35

NE

+S100 +

Pre-mRNA

Prox.

Dist.

mRNAs

M

S100 hnRNP A1

hnRNP A1

1 2 3 4 5 6 7 8 9 10 11 12

SR and hnRNP proteins affect alternative splicing in opposite ways

Fu, X-D., et al., PNAS 89:11224-11228, 1992

Tissue-specific Alternative Splicing of the src pre-mRNA:Blockage of Splice Sites by PTB

Chou, M-Y., et al., Mole. Cell 5:949-957, 2000

Cell-specific Alternative Splicing of the FGFR2 Gene Establish an Autocrine Loop Critical for Development

7 8 9 10

KGFR (or FGFR7)

FGFR2

Fibroblastsother cells

Epithelial Cells

KGFRKGF

FGFR2 FGF

Myotonic Dystrophy: A Splicing Disease

Phenotype: Skeletal muscle hyperexcitability and progressive muscle wastingCause: CUG or CCUG expansion in 3' untranslated regions in DMPK or ZNF9 genesMechanisms: Defects in splicing of the muscle-specific chloride channel CIC-1

How?AAAAA

CUG(n)

CUG-BP1 (increased stability and nuclear localization)

DMPK

2 3 6 6b 7a 7 8

Stop

U/G(n) CIC-1

Nonsense-mediated mRNA decay & protein truncation

Reduced Cl conductance

Membrane hyperexcitability

Mankodi, A., et al., Mole. Cell 10:35-44, 2002Charlet-B, N., et al., Mole. Cell 10:45-53, 2002

Life, Sex, and WT1 Isoforms: Three Amino Acids Can Make All the Difference

(Hastie, Cell 106, 391, 2001)

Exon 9 Exon 10

CATACAG GTAAAACAA gtgcgtaaactt

K T S

c

- KTS

GC

+ KTS

+/- mice develop Frasier syndrome-/- mice die after birth with kidney defectsFunction as a transcriptional factorComplete male-to-female reversal Reduced Y-specific Sry expression

+/- mice are normal-/- mice die after birth with kidney defectsFunction in pre-mRNA processingUndifferentiated gonad

{

{

All cells express +KTS and -KTS isoforms;Double heterozygous mice are normal!

Hammes, A., et al., Cell 106:319-329, 2001

1. RNA processing and regulation

2. Network interactions in gene expression

3. RASL-DASL the genome: approaches for systems biology

Topics

Complex Network of Coupled Interactions in Gene Expression

T. Maniatis & R. Reed, Nature 416:499-506, 2002

Electron Microscopic Analysis of Chromosomal Spreads: Evidence for Co-transcriptional Splicing

Beyer and Osheim, Genes Dev. 2:754-765, 1988

Targeting of Splicing Factorsto Nascent Transcripts Depends

on Pol II CTD

Cell lines were constructed to express wt and CTD-truncated -amanitin-resistant Pol II

Endogenous Pol II was inhibited by -amanitin

Nascent transcripts (pem) was detected by in situ hybridization

Splicing factors were localized by using specific antibodies

Results show that CTD is required for the recruitment of splicing factors to the site of transcription

Misteli and Spector, Mole. Cell 3:679-705, 1999

Hyperhosphorylated Pol II (IIo) Stimulates

Pre-mRNA Splicing in vitro

Hirose et al., Genes Dev. 13:1234-1239, 1999

Pol II by itself has no splicing activity

Hyperphosphorylated pol II (Iio), but not IIA, stimulates splicing in vitro

Pol II by itself has no splicing activity (b, lanes 1, 5).

CTD is necessary, but not sufficient for the effect (c, lanes 5-8, 12-25).

Promoter-dependent Alternative Splicing

Cramer et al., Mole. Cell 4: 251-258, 1999

Splicing-derived mRNAs are More Efficiently Exported

Luo and Reed, PNAS 96:14937-14942, 1999

A. Spliced mRNA is exported more efficiently in injected Xenopus oocytes.

B. Isolation of RNP complex containing spliced mRNA or a mRNA assembled in extracts.

C. Purified mRNP is more efficiently exported than assembled mRNA-protein complexes.

RNA Metabolism in the Nucleus: Coupling RNA Splicing to Nuclear Export

Cytoplasm

Nucleus

SR SR SR

SR SR SR

Aly AlyTAP

hGle2

hGle

p15

hDbp5

Partial orMutant pre-mRNA

Normal pre-mRNA

hnRNPs

Retainedand degraded

Retainedand degraded

Exported

introns Spliced mRNP

Exportcomplex

Reed and Magni, Nature Cell Biol 3:E201-204, 2001

Circular mRNA in vivo

m7GpppAAAAAA

eIF4F (= eIF4E, eIF4A, and eIF4G)PABP

5’UTR AUG

UAA3’UTR

Implications: PolyA binding protein is required for efficient translationRNA decay in many cases are translation-dependentDeadenylation will result in decapping

Tharun and Parker, 1997

Elongation

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

PABP PABP PABP

CAP

4E

4G

eRF3

3' UTR

5' UTRATG

STOP

eRF1

Initiation

Recycling

Model for translation stimulation by 5'-3' interactions

mRNA decay pathways in eukaryotic cells

AUG UAAm7G AAAAAAAAAlong

AUG UAAm7G AAAAAAAAAlong

m7G

AUG UAAm7G

AUG UAA AAAAAAAAAlongUAA

AUG UAAm7G AAAoligo

m7G

AUG UAA AAAoligo

AUG UAA AAAoligo

Deadenylation-indep.decapping

EndonucleolyticCleavage (Rnase L ?)

PolyA shorting(PAN)

Decapping(Dcp1p)

5’ to 3’ decay(Xrn1p)

3’ to 5’ decay5’ to 3’ decay

Decapping 3’ to 5’ decay

Tharun and Parker, 1997

Decapping Mechanisms

AUG UAA AAAAAAlong

m7Gppp

Dcp1p +UAAUpf3

Upf1 Upf2

AUG UAA AAAAAAlongm7Gppp

Dcp1p

Pab1p

-

Mrt1p Mrt3p+

A. Positive and negative effect on the decapping enzyme

B. Nonsense-mediated recruitment of the decapping enzyme

Tharun and Parker, 1997

The Exosome and its Target

Rep42

Rrp43

Rrp45

Rrp44

Rrp46 Mtr3

Rrp41

Rrp40

Rrp4

Csl4

3’- 5’ exoriboucleases

ARE

KSRP TTP

m7Gppp AAAAAA

3’-5’ degradationvan Hoof and Parker, Cell 99:347-350, 1999

Chen, C-Y., et al., Cell in press 2001

Nonsense-mediated RNA Decay: Where does It Occur?

Zhang, J. et al., RNA 4:801-815, 1988

Stops at position 39, 60-61, but not at 101 and 141, render mRNAs unstable.

NMD is detectable in both the nucleus and the cytoplasm

Nonsense-mediated RNA Decay: The 50-55 nt Rule

Zhang, J. et al., RNA 4:801-815, 1988

Two Positional Rules

The 50-55 nts Rule for NMD

The 20-24 nts Rule for Post-splicing Marker

Stop Exon-exon junction

D: > 50-55 nts: NMD < 50-55 nts: no effect

Exon-exon junction

D = 20-24 nts

Exon-exon Junction Complex that Links Splicing, Export, and NMD

Kim, V.N. et al., Science293:1832-1836, 2001

Summary

1. All RNAs (rRNA, tRNA, and mRNA) are matured in a series of processing steps after transcription.

2. mRNA processing takes place in the spliceosome, a large step-wise assembled ribonucleoprotein machinery.

3. Alternative splicing is very common and plays an important role in development and disease.

4. Many reactions in the nucleus are mechanistically coupled. Some proofreading mechanisms are operating to ensure the quality of processed RNAs before they are exported out of the nucleus.

5. The EJC complex connects nuclear processing to translation and stability of mRNA in the cytoplasm.