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

Abstract

In most eukaryotes, small RNA-mediated gene silencing pathways form complex interacting networks. In the ciliate Paramecium tetraurelia, at least two RNA interference (RNAi) mechanisms coexist, involving distinct but overlapping sets of protein factors 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 mutations by whole-genome sequencing. We present a set of 47 mutant alleles for five genes, revealing two previously 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 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.

Figure 1.

Mutagenesis screen and identification of unknown RNAi factors. (A) UV-irradiated cell cultures were starved to induce autogamy, a self-fertilization process that leads to complete homozygocity of the MIC genome. Re-feeding cells with K. pneumoniae medium allowed development of the new MACs from the resulting homozygous zygote within two cell divisions, leading to expression of the mutated genes. Transfer of cells into NSF dsRNA feeding medium allowed isolation of potential rnai mutants, as silencing of the essential NSF gene leads to lethality only in wild type cells (28,42,43). (B) To identify mutations in unknown RNAi factors, silencing deficient mutants were crossed with the wild type, resulting in heterozygous F1 progeny. After autogamy, F2 clones become homozygous for the rnai mutant (gray) or wild type (black) allele. Most other (UV-induced) mutations (m) are not linked with the target mutation (rnai) and segregate independently. F2 clones carrying the rnai allele were pooled for MAC DNA extraction and whole genome-sequencing. In this data set only the rnai mutation is covered by 100% of SNP-containing reads, whereas all other mutations are ideally covered by 50% SNP-containing and 50% wild type reads.

Figure 2.

Alleles identified from a screen for mutants deficient in dsRNA-inducible RNAi. Unambiguous null alleles (purple, rnai- phenotype as defined in the text) were obtained only for RDR1 and PDS1. The location of premature stop codons resulting from frameshifting mutations is shown by gray dots. The 5′ TA boundary of the only IES (76 bp) in the RDR2 gene is mutated to AA in the rdr2–1.24 allele, leading to complete retention of the IES in the MAC (Supplementary Figure S8B). Hypomorphic alleles are shown in blue (rnai+/- phenotype). Gene accession numbers are available in supplementary data (Supplementary Table S1). Co-segregation of the phenotype with the mutation in F2 was verified for alleles labelled with an asterisk (*). Alleles complemented by injection of linear DNA encoding the wild-type gene with its natural flanking regions are labeled with two asterisks (**).

Figure 3.

Two newly identified proteins involved in dsRNA-induced RNAi. Protein alignments were performed using the MUSCLE v4 software (78). (A) Cid1 is a nucleotidyl transferase. Aspartic-acid residues of the predicted catalytic triad (*) required for nucleotidyl transferase activity of Arabidopsis thaliana Heso-1 (52) and Tetrahymena thermophila Rdn1 and Rdn2 (47) are mutated in cid1–1.6 and cid1–3.4. Pt, Paramecium tetraurelia; Tt, T. thermophila; Sp, Schizosaccharomyces pombe; Ce, Caenorhabditis elegans; At, A. thaliana; Hs, Homo sapiens. (B) Pds1 is conserved in Paramecium species. Examples of missense alleles obtained for Pt-Pds1 in the RNAi mutant screen are indicated with black arrowheads (for substituting amino acids, see Supplementary Table S6). Pt, P. tetraurelia; Ppr, P. primaurelia; Pb, P. biaurelia; Ppe, P. pentaurelia; Ps, P. sexaurelia; Pm, P. multimicronucleatum, Pc, P. caudatum.

Figure 4.

Abolished siRNA accumulation in RNAi mutants. (A) Northern blot analysis of ND169 siRNAs revealed failure or strong reduction of accumulation of exogenously induced siRNAs in all mutants, consistent with previous findings from depletion of Dcr1, Rdr1 and Rdr2 by RNAi (15,22,23). This suggests a role of these RNAi factors upstream or within siRNA biosynthesis or stabilization. It is of note that silencing efficiencies can vary from one dsRNA feeding experiment to another, possibly due to contamination by traces of the standard food bacterium Klebsiella (showing partial Ampicillin resistance) which overgrows dsRNA-producing E. coli. This may explain differences in the total level of dsRNA-induced siRNAs in different wild-type cultures. The ND169 probe corresponds to a 100 bp region of the dsRNA and is sense oriented, as antisense siRNAs represent the predominant fraction of siRNAs in the dsRNA region (22). (B) Detection of endogenous siRNAs from an intergenic region of scaffold 22 (cluster22) revealed that rdr2 mutants are unable to accumulate these siRNAs. The probe is complementary to the predominant fraction of siRNAs (top strand) of this region. The lower panel shows hybridization to glutamine tRNA as a loading control.

Figure 5.

Saturation of the screen for non-essential single-copy genes involved in dsRNA-induced RNAi. Gene sizes of non-essential RNAi factors (A) plotted against the number of identified mutant alleles obtained (B). Extrapolation by linear regression determined the mutation frequency to 1 in 172 bp of coding sequence.

Figure 6.

Cid2 is also involved in dsRNA-induced silencing. (A) Phylogenetic relationship of the Cid1-like proteins clustering with Pt-Cid1 (all Cid1-like genes identified in the P. tetraurelia MAC genome and generation of phylogenetic trees, see Supplementary Figure S2 and Table S7). (B) Double knock down experiment of Pt-Cid1-like genes and ND169 by dsRNA feeding. ND169 was used as a reporter for silencing; the gene was considered as silenced when cells showed complete deficiency of trichocyst discharge (tric-) and inhibition of silencing was measured as proportion of cells in the culture showing partial (tric+/−) or complete (tric+) reversion of silencing. Silencing was significantly inhibited upon knock down of CID1 and CID2 (P-values < 0.03 (*), Mann–Whitney U test, significance level 0.05; n = 3; standard deviation is shown). Phenotypes were recorded 72 h after the first feeding. Bacteria were fed in equal amounts. Triple knock down experiments (CID3+CID4+ND169; CID3+CID5+ND169; CID4+CID5+ND169) did not show inhibition of ND169 reporter silencing (data not shown). Cross-silencing between CID1 and CID2 genes is unlikely, as the maximum length of perfect homology is 12 nt, and 18 nt with one central mismatch. (C) Northern blot analysis of associated ND169 siRNAs. The ND169 probe corresponds to a 100 bp region of the dsRNA and is sense oriented. The lower panel shows hybridization to glutamine tRNA as a loading control.

Figure 7.

dsRNA-induced RNAi factors sharing functions with the transgene-induced silencing pathway. (A)ND169 transgene construct pTI- carrying a 3′ truncated version of the ND169 coding sequence driven by a bidirectional constitutive promotor. Green fluorescent protein (GFP) was monitored as control for injection and expression (not shown). (B) Nd169 is involved in the exocytosis of secretory granules (trichocysts) docked at the plasma membrane. Wild-type cells expell trichocysts upon treatment with picric acid (tric+). By microinjection of pTI- into the MAC, strongly silenced clones showing complete trichocyst discharge defects were obtained (tric−) (not shown), as well as three clones with moderate silencing effect (tric+/−), c2, c5, and c7. Note that clone c2 showed up to 18% of cells with tric+ phenotypes in control cultures (ICL7a feeding). (C) Knock down of RNAi factor genes by dsRNA feeding in clones with moderate transgene-induced silencing phenotypes. De-repression of ND169 silencing was measured as the percentage of cells showing a complete wild type phenotype (tric+). A significant difference to the ICL7a control was detected upon silencing of CID2, RDR2, RDR3 and PTIWI13 (P-values < 10e-5 (***), one-way ANOVA, significance level 0.005). Phenotypes recorded 120 h after the first feeding are shown. Note that pTI- induced silencing efficiencies varied slightly in different cultures of the same injected clone. (D) The effect of RNAi factor depletion on dsRNA-induced silencing was verified in parallel as a positive control by double knock down, mixing RNAi factor and ND169 dsRNA-producing bacteria in equal amounts. The percentage of tric+ cells, i.e. the degree of inhibition of ND169 silencing, is shown (mean of two replicates for CID1 and PDS1). ICL7a control dsRNA feeding does neither inhibit dsRNA- nor transgene-induced silencing; PTIWI13 is involved in both pathways, whereas RDR3 is specific for transgene- and RDR1 for dsRNA-induced silencing, as previously described (22,23).

Figure 8.

Sharing of RNAi core factors by non-essential exogenously and essential endogenously triggered RNAi pathways in P. tetraurelia. In this study, mutants were obtained for genes labelled with an asterisk, and genes shared between the transgene-induced (dark gray circle) and the dsRNA-induced pathway (blue circle) were found to be essential. Brackets connecting two genes symbolize WGD1 duplicate genes. CID2 was identified by recursive RNAi in this study. Other genes were identified previously (15,22,23). The RDR2 gene is required for the accumulation of endogenous siRNAs (purple) of the cluster22 locus, as previously found for RDR3 (22). Note that production of dsRNA from this locus is speculative, and that other endogenous siRNAs may require different factors. The figure is only meant to depict genetic requirements, not the subcellular localization or mechanistic roles of the corresponding proteins.