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 email@example.com.Show MeSH
Mentions: Our screen for RNAi-deficient mutants was unlikely to reveal genes with functionally redundant WGD1 ohnologs, unless disruption of one copy led to a dosage effect. For single-copy genes, we could expect alleles of non-essential genes and hypomorphic alleles of essential genes. Unambiguous alleles were here defined as those containing nonsense mutations or frameshifts resulting in premature stop codons, due to indels or mutations in intron splice sites (48), except in cases where the best part of the protein is conserved and where there is experimental evidence for a partial RNAi deficiency. An allele was categorized as hypomorphic if the RNAi deficient phenotype was only partial and/or dsRNA feeding-induced siRNAs were reduced, but still detectable on northern blots. Indeed, the diversity of alleles obtained for each of the genes hit appeared to reflect their importance for cellular viability (Figure 2): 41 rdr1 mutants were found, representing 26 different alleles. Among these were eight putative alleles (e.g. rdr1–5.28 or rdr1–5.7). Missense alleles were mostly non-conservative substitutions in conserved residues, two of which in the putative catalytic core region (rdr1–1.4 D1021N and rdr1–3.16 D1021Y) (49–51) (Supplementary Table S6). Similarly, the 10 alleles obtained for PDS1 included alleles, suggesting that this gene, like RDR1, is not required for viability. Although no unambiguous allele was found among the seven cid1 alleles, some of the mutations changed highly conserved amino acids (cid1–3.4 D68N and cid1–1.6 D70N) shown to be required for uridylyl transferase activity (47,52). In all putative mutants tested, a complete loss of dsRNA-induced siRNAs was observed on northern blots (Figure 4A and Supplementary Figure S6). RNAi-deficient rdr1, pds1 and cid1 mutants are fully viable throughout the life cycle (vegetative growth and sexual events), and no other phenotypic anomaly was observed. Furthermore, the F2 (Supplementary Table S4B) and F3 generations of an rdr1–3.1/cid1–1.8 double homozygote showed normal vegetative growth in standard conditions and at high temperature (34°C). We conclude that the capacity to synthesize siRNAs from dsRNA ingested with food is not essential in laboratory conditions.
Affiliation: Ecole Normale Supérieure, Institut de Biologie de l'ENS, IBENS, Inserm, U1024, CNRS, UMR 8197, Paris F-75005, France firstname.lastname@example.org.