Exploring Gene Function in C. elegans:Mutations and RNA Interference

Carolina Biological Supply Company

Bruce NashDolan DNA Learning CenterCold Spring Harbor Laboratory

Craig Mello

• He was awarded the 2006 Nobel Prize for Physiology or Medicine, along with Andrew Z. Fire, for the discovery of RNA interference.

• This research was conducted at the University of Massachusetts Medical School and published in 1998

A Grand Challenge

To understand how life works

How do we go from DNA to function?

Function?

Find a tractable systems:

Model organisms

What makes a good model organism?

• Ease of cultivation

• Simplicity

• Relevant biology

• Amenability(W)hen I embarked on this problem, I decided that what was needed was an experimental organism which was suitable for genetical study and in which one could determine the complete structure of the nervous system.S. Brenner Genetics 77: 71-94 May 1974

The model organism: Caenorhabditis elegans

Electron micrograph of a C. elegans hermaphrodite

Why Worms?

ProfileSoil nematodeGenome size: 100 MbNumber of chromosomes: 6Generation time: about 2 daysFemale reproductive capacity: 250 to 1000 progeny

Special characteristicsStrains Can Be FrozenHermaphroditeKnown cell lineage pattern for all 959 somatic cellsOnly 302 neuronsTransparent bodyCan be characterized geneticallyAbout 70% of Human Genes have related genes in C. elegans

=~

Worms have gut, muscle, skin and a nervous system like us

About 40% of worm genes are very related

to human genes

Identify mutants

Deduce the function of mutated genes

How do geneticists study gene function?

How do geneticists study gene function?

Dumpy mutant

The wild type gene must maintain normal body shape.

Wild type

• Students examine worm behavior and morphology

• Students identify stages of worm development

• Students examine mutant strains and compare to wild type

• Students culture C. elegans

Examining, growing and caring for worms

Anatomy of a worm

(from www.wormatlas.org)

Anatomy of a worm

Wild-type

Clear patch on L4

Adult with embryos

L4 with clear patch

Identifying hermaphrodites

Notice the large clear area on the side of the worm (a blister in the cuticle)

bli-1

Dumpy worms are shorter.

dpy-11

Lifecycle of a worm

(from www.wormatlas.org)

Identifying hermaphrodites

Clear patch on L4

A problem:

Creating mutations in specific genes is very hard

Can we go directly from sequence to function?

Function?

This can give the same phenotype as a mutant

One approach: Antisense RNA turns down specific genes

An experiment showed that the antisense model didn’t make “sense”

First noticed that sense RNA was as effective as antisense RNA for suppressing gene expression in worm

Guo S, and Kemphues KJ.1995

Antisense RNA

Turns off gene Turns off gene????

Sense RNA

Double-stranded RNA causes silencing:RNA Interference!

First described RNAi phenomenon in C. elegans by injecting dsRNA into C. elegans, which led to an efficient sequence-specific silencing and coined the term "RNA Interference".

Fire et al.1998

Negative control uninjected

mex-3B antisense RNA mex-3B dsRNA

Antisense RNA

Weak effect

dsRNA

Strong effect

Double-stranded RNA was a contaminant in antisense experiments

How can dsRNA turn of genes?

Gene OFF

dsRNA

Cell free extract

RNAi?

Biochemistry to the rescueRNAi in vitro...

Hannon Lab, Zamore Lab, Tuschl Lab, Sharp Lab

GFP – germline transgene

Dicer is required for RNAi

GFPdsRNA

wild-type

dcr-1-/-

Dicer is an evolutionarily conserved nuclease

dsRNA

RNAi functions in many different organisms

Dicer

2001

Bernstein et al.

Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as two dsRNA-binding domains.

Dicer cuts dsRNA into short RNAs

2001

Bernstein et al.

Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as two dsRNA-binding domains.

Dicer cuts dsRNA into short RNAs

Dicer

siRNA

2001

Bernstein et al.

Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as two dsRNA-binding domains.

Dicer cuts dsRNA into short RNAs

Dicer

How can an siRNA turn of genes?

Gene OFF

siRNA

Slicer uses siRNAs to slice transcripts

2004

Song et al. Solved the crystal structure of pyrococcus Argonaute, showing it is Slicer

Slicer

Slicer

siRNA

2004

Song et al. Solved the crystal structure of pyrococcus Argonaute, showing it is Slicer

Slicer uses siRNAs to slice transcripts

2004

Song et al. Solved the crystal structure of pyrococcus Argonaute, showing it is Slicer

Slicer uses siRNAs to slice transcripts

2004

Song et al. Solved the crystal structure of pyrococcus Argonaute, showing it is Slicer

Slicer uses siRNAs to slice transcripts

Transcripts are bound by Slicer

Gene transcripts are sliced

Gene function stopped

dsRNA silences genes via Dicer and Slicer

RNAi lets us test gene function!

Function?RNAi

C. elegans is amenable to many forms of RNAi treatment

The kit uses RNAi by feeding

Feeding worms bacteria that express dsRNAs or soaking worms in dsRNA sufficient to induce silencing (Gene 263:103, 2001; Science 282:430, 1998)

RNAi by feeding is simple

Feeding worms bacteria that express dsRNAs or soaking worms in dsRNA sufficient to induce silencing (Gene 263:103, 2001; Science 282:430, 1998)

• Simply feed C. elegans bacteria expressing double-stranded RNA complementary to the gene you want to silence!

The RNAi feeding vector has two T7 RNA polymerase promoters

• T7 RNA polymerase is a viral polymerase.• It binds to a specific T7 promoter sequence.• The L4440 vector has two T7 promoters to make RNA from both strands.

Inducing RNAi by Feeding

• Demonstrate to your students the power of silencing a single gene.

• Teach about a powerful method for determining gene function.

• Introduce your students to a model organism used for studying many aspects of biology, including development and gene function.

• Engage in bioinformatics exercises exploring protein function and C. elegans and human gene relatedness.

microRNAs regulate gene expression without cleavage

small temporal RNAs (stRNAs)

stRNA precursor

stRNA

Translational repression

RN

A o

r p

rote

inle

vel

L1 stage L2 stage L3 stage L4 stage Adult stage

LIN-14, LIN-28 proteins LIN-41 protein LIN-29 protein

let-7 RNAlin-4 RNA

Dicer

• lin-4 miRNA represses translation of lin-14 (L1 to L2 molt)

siRNA miRNA

RNAi acts to regulate gene expression by two dicer-dependent mechanisms

Model for translational repression

M7GpppG AAAAAA.........RISC

RISC recognizes a target

Model for translational repression

M7GpppG AAAAAA.........RISC

DCP GW

other

Other proteins are also recruited – either along with RISC or later

Model for translational repression

M7GpppG AAAAAA.........RISC

DCP GW

other

decapping to P-bodies

block translation?(e.g. Filipowicz)

de-adenylation?(e.g. Giraldez, Belasco, Rivas)

RISC may block through multiple mechanisms

Least well understood - Arabidopsis as a model….

DNA complexes with histones to form chromatin

Regulation of gene expression at the level of chromatin

Sequence-independentlinker histones: control DNA compaction and accessibility to trans-acting factors

post-translational modifications of histone tails: control compaction of DNA and serve as docking sites for trans-acting factors

Range: Can act at the level of a single gene, often acts over groups of genes and over larger domains (20-200kb), and can affect gene expression over an entire chromosome

Model for siRNA-dependent initiation of heterochromatic silencing by RITS

• Heterochromatin and epigenetics

Model for maintenance of heterochromatic silencing by RITS

dsRNA-mediated silencing in various organisms:Multiple mechanisms that are Dicer-dependant

Meister & Tuschl, 2004

Genome wide transcription?

• Kapranov, P. et al. Science 316, 1484–1488 (2007). 

• Cheng, J. et al. Science 308, 1149–1154 (2005). 

• Bertone, P. et al. Science 306, 2242–2246 (2004). 

• Birney, E. et al. Nature 447, 799–816 (2007). 

Numerous studies, using a variety of techniques, estimate that at minimum 63% of the genome is

transcribed into RNA, with most estimates settling at > 90%!

Also of note is that all of this detected RNA has to be stable enough in the cell to be detected and that it is

estimated that ~20% never leaves the nucleus as noted in these papers.

Moral of Story

• Lots of RNA!• Cells seem to regulate transcription, largely

in cases of differentiation, development, and response to stress

• Little else known• Conveniently, differentiation and responses

to stressors is rather key to immunology

Types of small RNA (does not encode long ncRNA’s… a whole different kind of

monster)

http://www.pbs.org/wgbh/nova/sciencenow/3210/02.html