Introduction to C. elegans and

RNA interference

Why study model organisms?

• In order to understand biology, we need to learn about the function of the underlying genes

• How can we find out what genes do?

• We need a way to uncover these functions

The problem:

How do geneticists study gene function?

Forward genetics:• Classical approach• A gene is identified by studying mutant phenotype and mutant alleles• The gene must be cloned for further functional analysis

Disrupt the gene and analyze the resulting phenotype

How do geneticists study gene function?

Reverse genetics:• Start with gene sequence information• Engineer a loss of function phenotype to evaluate gene to function

Disrupt the gene and analyze the resulting phenotype

How do geneticists study gene function?

Forward Genetics

• Have a mutant phenotype and wish to determine what gene sequence is associated with it

• Allows identification of many genes involved in a given biological process

• Mutations in essential genes are difficult to find

• Works great in model organisms

Starting point: A mutant animalEnd point: Determine gene function

What makes a good model organism?

Ease of cultivation

Rapid reproduction

Small size

Why are mutants in model organisms useful?

Let’s see how similar our genes areto model organisms

Model organism Haploid genome size (Mb)

Estimated # of genes

S. cerevisiae 13 6,022

C. elegans 100 14,000

A. thaliana 120 (estimated) 13,000-60,000

D. melanogaster 170 15,000

M. musculus 3,000 100,000

Homo sapien (not a model)

3,000 100,000

A comparison of genomes

Species   Number of Genes   HomoloGene

Input Grouped groups

                                                                                                                                                                        

H.sapiens 23,516* 19,336 18,480P.troglodytes 21,526  13,009 12,949C.familiaris 19,766  16,761 16,324M.musculus 31,503  21,364 19,421R.norvegicus 22,694  18,707 17,307G.gallus 18,029  12,226 11,400D.melanogaster 14,017  8,093 7,888A.gambiae 13,909  8,417 7,882C.elegans 20,063* 5,137 4,909S.pombe 5,043  3,210 3,174S.cerevisiae 5,863  4,733 4,583K.lactis 5,335  4,454 4,422E.gossypii 4,726  3,944 3,935M.grisea 11,109  6,290 5,884N.crassa 10,079  5,908 5,902A.thaliana 26,659  11,180 10,857O.sativa 33,553  11,022 9,446P.falciparum 5,222  971 950

Many genes are conserved in modelorganisms

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=homologene

The model organism: Caenorhabditis elegans

Electron micrograph of a C. elegans hermaphrodite

Caenorhabditis elegans

Profile

Soil 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

C. elegans cell division can be studied in the transparent egg

C. elegans cell lineage is known

Kelly, W. G. et al. Development 2002;129:479-492

Nuclei and DNA can be visualized

Answer: They perform a mutagenesis screen.

1. Mutagenize the organism to increase the likelihood of finding mutants

2. Identify mutants

3. Map the mutation

4. Determine the molecular function of the gene product

5. Figure out how the gene product interacts with other gene products in a pathway

How do geneticists identify genes?

Linkage mapping and complementation analysis.

Sort through the mutations identified

What are the limitations of Forward Genetics?

1. Some genes cannot be studied by finding mutations

• Genes performing an essential function• Genes with redundant functions

2. Finding mutants and mapping is time-consuming

3. Mutagenesis is random• Cannot start with a known gene and make a

mutant

Model organism Haploid genome size (Mb)

Estimated # of genes

S. cerevisiae 13 6,022

C. elegans 100 14,000

A. thaliana 120 (estimated) 13,000-60,000

D. melanogaster 170 15,000

M. musculus 3,000 100,000

Homo sapien (not a model)

3,000 100,000

Genome sequencing has identified many genes

Can the function of a gene be studied when all we have is the DNA sequence?

Reverse Genetics

Starting point: Gene sequenceEnd point: Determine gene function

• Have a gene in hand (genome sequence, for example), and want to know what it does.

• Can be used to correlate a predicted gene sequence to a biological function

• Goal is to use the sequence information to disrupt the function of the gene

Some approaches to Reverse Genetics

• Targeted deletion by homologous recombination

– Specific mutational changes can be made

– Time consuming and limited to certain organisms

• Mutagenesis and screening for deletions by PCR

– Likely to completely abolish gene function

– Time consuming and potentially expensive

• Antisense RNA

– Variable effects and mechanism not understood

A new, fast, generally applicable technique was needed

And the winner is…..

RNAi

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

How did we come to understand how RNAi works?

Examining the antisense RNA technique revealed that the model for how it

worked was wrong.

The old model: Antisense RNA leads to translational inhibition

mRNA is considered the sense strand

antisense RNA is complementary to the sense strand

This can give the same phenotype as a mutant

The old model: Antisense RNA leads to translational inhibition

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

• The antisense technology was used in worms...

• Puzzling results were produced: both sense and antisense RNA preparations were sufficient to cause interference.

• What could be going on?

1995 Guo S, and Kemphues KJ.First noticed that sense RNA was as effective as antisense RNA for suppressing gene expression in worm

When researchers looked closely, they found that double-stranded RNA caused the

silencing!

1998 Fire et al.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".

Negative control uninjected

mex-3B antisense RNA mex-3B dsRNA

Double-stranded RNA injection reduces the levels of mRNA

Potent and specificgenetic interference bydouble-strandedRNA inCaenorhabditis elegansAndrew Fire*, SiQun Xu*, Mary K. Montgomery*,Steven A. Kostas*†, Samuel E. Driver‡ & Craig C. Mello‡

dsRNA Hypothesis explains the white petunias

• Purple plants should become purpler...

• Instead, they became whiter.

• How could this be happening?

• The multiple inserted copies of chalcone synthase were producing double stranded RNA

RNAi in worms: easier than baking pie!

We are going to inactivate genes by 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).