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?

Why study mutants in model organisms?

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

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 animal End point: Determine gene function

What makes a good model organism? Ease of cultivation Rapid reproduction Small size

The model organism: Caenorhabditis elegans Electron micrograph of a C. elegans hermaphrodite

Caenorhabditis elegans Profile Soil nematode Genome size: 100 Mb Number of chromosomes: 6 Generation time: about 2 days Female reproductive capacity: 250 to 1000 progeny Special characteristics Strains Can Be Frozen Hermaphrodite Known cell lineage pattern for all 959 somatic cells Only 302 neurons Transparent body Can be characterized genetically About 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: Nuclei and DNA can be visualized

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 organismHaploid genome size (Mb) Estimated # of genes S. cerevisiae136,022 C. elegans10014,000 A. thaliana120 (estimated)13,000-60,000 D. melanogaster17015,000 M. musculus3,000100,000 Homo sapien (not a model) 3,000100,000 The Genome Sequencing Project

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?

Let’s see how similar our genes are to model organisms

Species Number of Genes HomoloGene InputGroupedgroups H.sapiens23,516 * 19,33618,480 P.troglodytes21,526 13,00912,949 C.familiaris19,766 16,76116,324 M.musculus31,503 21,36419,421 R.norvegicus22,694 18,70717,307 G.gallus18,029 12,22611,400 D.melanogaster14,017 8,0937,888 A.gambiae13,909 8,4177,882 C.elegans20,063 * 5,1374,909 S.pombe5,043 3,2103,174 S.cerevisiae5,863 4,7334,583 K.lactis5,335 4,4544,422 E.gossypii4,726 3,9443,935 M.grisea11,109 6,2905,884 N.crassa10,079 5,9085,902 A.thaliana26,659 11,18010,857 O.sativa33,553 11,0229,446 P.falciparum5, Many genes are conserved in model organisms

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

Model organismHaploid genome size (Mb) Estimated # of genes S. cerevisiae136,022 C. elegans10014,000 A. thaliana120 (estimated)13,000-60,000 D. melanogaster17015,000 M. musculus3,000100,000 Homo sapien (not a model) 3,000100,000 Genome sequencing has identified many genes

Reverse Genetics Starting point: Gene sequence End 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

With the completion of the genome sequencing project, a quicker, less expensive reverse genetics method was needed. Luckily scientists discovered... RNAi

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? 1995Guo 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 controluninjected mex-3B antisense RNAmex-3B dsRNA Double-stranded RNA injection reduces the levels of mRNA Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Andrew Fire*, SiQun Xu*, Mary K. Montgomery*, Steven A. Kostas*†, Samuel E. Driver‡ & Craig C. Mello‡

dsRNA hypothesis explains the white petunias Hypothesis: addition of extra purple pigment genes should produce darker flowers. Results: Instead, the flowers became whiter. New hypothesis: the multiple transgene copies of the pigment gene made double stranded RNA.

C. elegans is amenable to many forms of RNAi treament 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)