A forward genetic screen reveals essential and non-essential RNAi factors in Paramecium tetraurelia.
Bottom Line: We show that non-essential genes are specifically involved in dsRNA-induced RNAi while essential ones are also involved in transgene-induced RNAi.One of the latter, the RNA-dependent RNA polymerase RDR2, is further shown to be required for all known types of siRNAs, as well as for sexual reproduction.These results open the way for the dissection of the genetic complexity, interconnection, mechanisms and natural functions of RNAi pathways in P. tetraurelia.
Affiliation: Ecole Normale Supérieure, Institut de Biologie de l'ENS, IBENS, Inserm, U1024, CNRS, UMR 8197, Paris F-75005, France firstname.lastname@example.org.Show MeSH
Mentions: To isolate mutants of the dsRNA-induced silencing pathway, we first treated wild-type cells with UV light to induce random mutations in the MIC genome. Cell populations were then allowed to undergo autogamy, a self-fertilization sexual process in which only one of the parental MIC alleles is retained, and made homozygous in the MICs and MACs of progeny (Figure 1A). Cultures were then screened by feeding them an E. coli strain producing dsRNA homologous to NSF, an essential gene involved in exocytosis and membrane traffic (42,43). NSF dsRNA feeding rapidly kills the wild type (28), so that only mutant cells deficient in dsRNA-induced RNAi would be able to survive. One hundred and fifty surviving cells were isolated, and the RNAi-deficient phenotypes were confirmed by dsRNA feeding targeting different genes (NSF; ND7 and ND169, two single-copy, unrelated genes involved in the exocytosis of secretory granules called trichocysts (34,37); for details, see below, Figure 7B). After sorting out non-viable, false positive or strongly hypomorphic clones, a set of 79 RNAi-deficient cell lines was established (detailed outcome of the screen in Supplementary Table S3).
Affiliation: Ecole Normale Supérieure, Institut de Biologie de l'ENS, IBENS, Inserm, U1024, CNRS, UMR 8197, Paris F-75005, France email@example.com.