Identification of genes that protect the C. elegans genome against mutations by genome-wide RNAi

Abstract

An RNA interference (RNAi)-based genome-wide screen was performed to detect genes that contribute to genome stability in somatic cells of Caenorhabditis elegans. We identified 61 such genes; these also affect spontaneous mutagenesis in the germ line. Their sequence suggests a role in DNA repair and/or replication, in chromatin remodeling, or in cell cycle control; there are also many novel genes that are highly conserved from yeast to human. Because known mutator genes are causally involved in many hereditary and sporadic human cancers, it is likely that some of these new mutators are equally relevant in cancer etiology.

Figure 1

RNAi-mediated DNA instability in C. elegans. Strains that contain heat shock promoter-driven out-of-frame gfp-lacZ reporter gene fusions were assayed on dsRNA homologous to mlh-1, pms-2, msh-2, msh-6, M04F3.1, Y71F9AL.18 (pme-1), R06C7.7, and the empty vector control L4440. Two different out-of-frame constructs were tested, one containing a mononucleotide (A₁₇) repeat placed immediately downstream of the ORF's ATG (this reporter has been used to screen the RNAi library), the other identical except that it lacks the mononucleotide repeat.

Figure 2

RNAi-mediated germline mutator phenotype in C. elegans. An endogenous mutational target, unc-93 (e1500), in which mutations can be recognized based on altered movement of the animal (Greenwald and Horvitz 1980) is used to determine the level of spontaneous mutagenesis levels in the C. elegans germline. This assay was performed for 24 genes (Y-axis), several from each category listed in Table 1. As a control, the empty vector L4440 was assayed. The X-axis represents the number of independent unc-93 (e1500) cultures that showed revertant worms. *, p < 0.05; **, p < 0.001; ***, p < 0.0001; using the Chi-square test. The absolute number of cultures that contained revertants versus the total number of cultures analyzed is also indicated. The mutational loads in msh-2(RNAi), mlh-1(RNAi), and pms-2(RNAi) being 18/187, 16/183, and 16/168, respectively, were determined in a separate experiment with a reduced background level of mutations: 3/177.