Introduction to C. elegans and RNA interference

Why study model organisms?

The problem: 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

How do geneticists study gene function?

Disrupt the gene and analyze the resulting phenotype How do geneticists study gene function? Disrupt the gene and analyze the resulting phenotype Forward genetics: Classical approach A gene is identified by studying mutant phenotype and mutant alleles The gene must be cloned for further functional analysis

Why study mutants in model organisms?

How do geneticists identify genes? 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

Sort through the mutations identified Linkage mapping and complementation analysis.

Forward Genetics Starting point: A mutant animal End point: Determine gene function 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

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

Caenorhabditis elegans 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

Nuclei and DNA can be visualized Kelly, W. G. et al. Development 2002;129:479-492

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

The Genome Sequencing Project 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)

Disrupt the gene and analyze the resulting phenotype How do geneticists study gene function? Disrupt the gene and analyze the resulting phenotype Reverse genetics: Start with gene sequence information Engineer a loss of function phenotype to evaluate gene to function

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

Many genes are conserved in model organisms Species   Number of Genes   HomoloGene Input Grouped groups                                                                                                                                                                           H.sapiens 23,516* 19,336 18,480 P.troglodytes 21,526  13,009 12,949 C.familiaris 19,766  16,761 16,324 M.musculus 31,503  21,364 19,421 R.norvegicus 22,694  18,707 17,307 G.gallus 18,029  12,226 11,400 D.melanogaster 14,017  8,093 7,888 A.gambiae 13,909  8,417 7,882 C.elegans 20,063* 5,137 4,909 S.pombe 5,043  3,210 3,174 S.cerevisiae 5,863  4,733 4,583 K.lactis 5,335  4,454 4,422 E.gossypii 4,726  3,944 3,935 M.grisea 11,109  6,290 5,884 N.crassa 10,079  5,908 5,902 A.thaliana 26,659  11,180 10,857 O.sativa 33,553  11,022 9,446 P.falciparum 5,222  971 950 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=homologene

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

Genome sequencing has identified many genes 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)

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

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

The old model: Antisense RNA leads to translational inhibition This can give the same phenotype as a mutant

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! Negative control uninjected 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‡ mex-3B antisense RNA mex-3B dsRNA Double-stranded RNA injection reduces the levels of mRNA 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".

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)