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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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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).
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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).

Mentions: To isolate mutants of the dsRNA-induced silencing pathway, we first treated wild-type cells with UV light to induce random mutations in the MIC genome. Cell populations were then allowed to undergo autogamy, a self-fertilization sexual process in which only one of the parental MIC alleles is retained, and made homozygous in the MICs and MACs of progeny (Figure 1A). Cultures were then screened by feeding them an E. coli strain producing dsRNA homologous to NSF, an essential gene involved in exocytosis and membrane traffic (42,43). NSF dsRNA feeding rapidly kills the wild type (28), so that only mutant cells deficient in dsRNA-induced RNAi would be able to survive. One hundred and fifty surviving cells were isolated, and the RNAi-deficient phenotypes were confirmed by dsRNA feeding targeting different genes (NSF; ND7 and ND169, two single-copy, unrelated genes involved in the exocytosis of secretory granules called trichocysts (34,37); for details, see below, Figure 7B). After sorting out non-viable, false positive or strongly hypomorphic clones, a set of 79 RNAi-deficient cell lines was established (detailed outcome of the screen in Supplementary Table 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)

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).
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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).
Mentions: To isolate mutants of the dsRNA-induced silencing pathway, we first treated wild-type cells with UV light to induce random mutations in the MIC genome. Cell populations were then allowed to undergo autogamy, a self-fertilization sexual process in which only one of the parental MIC alleles is retained, and made homozygous in the MICs and MACs of progeny (Figure 1A). Cultures were then screened by feeding them an E. coli strain producing dsRNA homologous to NSF, an essential gene involved in exocytosis and membrane traffic (42,43). NSF dsRNA feeding rapidly kills the wild type (28), so that only mutant cells deficient in dsRNA-induced RNAi would be able to survive. One hundred and fifty surviving cells were isolated, and the RNAi-deficient phenotypes were confirmed by dsRNA feeding targeting different genes (NSF; ND7 and ND169, two single-copy, unrelated genes involved in the exocytosis of secretory granules called trichocysts (34,37); for details, see below, Figure 7B). After sorting out non-viable, false positive or strongly hypomorphic clones, a set of 79 RNAi-deficient cell lines was established (detailed outcome of the screen in Supplementary Table 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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