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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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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.
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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.

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.


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)

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.
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Related In: Results  -  Collection

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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.
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.

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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