A forward genetic screen reveals essential and non-essential RNAi factors in Paramecium tetraurelia

In most eukaryotes, small RNA-mediated gene si-lencing pathways form complex interacting net-works. In the ciliate Paramecium tetraurelia, at least two RNA interference (RNAi) mechanisms coexist, involving distinct but overlapping sets of protein fac-tors and producing different types of short interfering RNAs (siRNAs). One is specifically triggered by high-copy transgenes, and the other by feeding cells with double-stranded RNA (dsRNA)-producing bacteria. In this study, we designed a forward genetic screen for mutants deficient in dsRNA-induced silencing, and a powerful method to identify the relevant muta-tions by whole-genome sequencing. We present a set of 47 mutant alleles for five genes, revealing two pre-viously unknown RNAi factors: a novel Paramecium-specific protein (Pds1) and a Cid1-like nucleotidyl transferase. Analyses of allelic diversity distinguish non-essential and essential genes and suggest that the screen is saturated for non-essential, single-copy genes. We show that non-essential genes are specif-ically involved in dsRNA-induced RNAi while es-sential ones are also involved in transgene-induced RNAi. One of the latter, the RNA-dependent RNA poly-merase RDR2, is further shown to be required for all known types of siRNAs, as well as for sexual re-production. These results open the way for the dis-section of the genetic complexity, interconnection, mechanisms and natural functions of RNAi pathways in P. tetraurelia