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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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Alleles identified from a screen for mutants deficient in dsRNA-inducible RNAi. Unambiguous  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 (**).
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Figure 2: Alleles identified from a screen for mutants deficient in dsRNA-inducible RNAi. Unambiguous 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 (**).

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)

Alleles identified from a screen for mutants deficient in dsRNA-inducible RNAi. Unambiguous  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 (**).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4066745&req=5

Figure 2: Alleles identified from a screen for mutants deficient in dsRNA-inducible RNAi. Unambiguous 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 (**).
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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