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

Marker S, Carradec Q, Tanty V, Arnaiz O, Meyer E - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Ecole Normale Supérieure, Institut de Biologie de l'ENS, IBENS, Inserm, U1024, CNRS, UMR 8197, Paris F-75005, France simone.marker@uni-saarland.de.

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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.
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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.

Mentions: The remaining 26 lines were found to carry mutations in either of two new genes, for which one allele was initially identified by the whole-genome sequencing procedure. The missense mutation 1.8 (Figure 2; Supplementary Table S6) is located in the predicted gene PTETG9100013001 (ParameciumDB), encoding a protein of the nucleotidyl transferase family (also referred to as non-canonical polyA/U RNA polymerases (44)) (Figure 3A). It is related to the first described non-canonical polyA polymerase, Cid1 of S. pombe, as well as to S. pombe's Cid12 (Supplementary Figure S2), the first protein among others of that family shown to be involved in small RNA mediated silencing (45). The gene was therefore named CID1. Cid1 is closely related to Rdn2 of T. thermophila (Supplementary Figure S2; Figure 6A), which physically interacts with the RdRP Rdr1 in an ‘RdRC’ complex (46,47). CID1 is expressed at constant levels throughout the life cycle (Supplementary Figure S3).


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

Marker S, Carradec Q, Tanty V, Arnaiz O, Meyer E - Nucleic Acids Res. (2014)

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.
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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.
Mentions: The remaining 26 lines were found to carry mutations in either of two new genes, for which one allele was initially identified by the whole-genome sequencing procedure. The missense mutation 1.8 (Figure 2; Supplementary Table S6) is located in the predicted gene PTETG9100013001 (ParameciumDB), encoding a protein of the nucleotidyl transferase family (also referred to as non-canonical polyA/U RNA polymerases (44)) (Figure 3A). It is related to the first described non-canonical polyA polymerase, Cid1 of S. pombe, as well as to S. pombe's Cid12 (Supplementary Figure S2), the first protein among others of that family shown to be involved in small RNA mediated silencing (45). The gene was therefore named CID1. Cid1 is closely related to Rdn2 of T. thermophila (Supplementary Figure S2; Figure 6A), which physically interacts with the RdRP Rdr1 in an ‘RdRC’ complex (46,47). CID1 is expressed at constant levels throughout the life cycle (Supplementary Figure S3).

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.

View Article: PubMed Central - PubMed

Affiliation: Ecole Normale Supérieure, Institut de Biologie de l'ENS, IBENS, Inserm, U1024, CNRS, UMR 8197, Paris F-75005, France simone.marker@uni-saarland.de.

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