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Nucleic Acids Res. 2015 Feb 18;43(3):1818-33. doi: 10.1093/nar/gku1331. Epub 2015 Jan 15.

Primary and secondary siRNA synthesis triggered by RNAs from food bacteria in the ciliate Paramecium tetraurelia.

Author information

1
Institut de Biologie de l'ENS, IBENS, Ecole Normale Supérieure, Inserm, U1024, CNRS, UMR 8197, 75005 Paris, France UPMC, IFD, Sorbonne Universités, 4 place Jussieu, 75252 Paris cedex 05, France.
2
Zentrum für Human- und Molekularbiologie, Molekulare Zelldynamik, Universität des Saarlandes, Campus A2 4, 66123 Saarbrücken, Germany.
3
Centre de Génétique Moléculaire, CNRS UPR3404, 91198 Gif-sur-Yvette cedex, France.
4
Institut de Biologie de l'ENS, IBENS, Ecole Normale Supérieure, Inserm, U1024, CNRS, UMR 8197, 75005 Paris, France.
5
Institut de Biologie de l'ENS, IBENS, Ecole Normale Supérieure, Inserm, U1024, CNRS, UMR 8197, 75005 Paris, France emeyer@biologie.ens.fr.
6
Institut de Biologie de l'ENS, IBENS, Ecole Normale Supérieure, Inserm, U1024, CNRS, UMR 8197, 75005 Paris, France Zentrum für Human- und Molekularbiologie, Molekulare Zelldynamik, Universität des Saarlandes, Campus A2 4, 66123 Saarbrücken, Germany simone.marker@uni-saarland.de.

Abstract

In various organisms, an efficient RNAi response can be triggered by feeding cells with bacteria producing double-stranded RNA (dsRNA) against an endogenous gene. However, the detailed mechanisms and natural functions of this pathway are not well understood in most cases. Here, we studied siRNA biogenesis from exogenous RNA and its genetic overlap with endogenous RNAi in the ciliate Paramecium tetraurelia by high-throughput sequencing. Using wild-type and mutant strains deficient for dsRNA feeding we found that high levels of primary siRNAs of both strands are processed from the ingested dsRNA trigger by the Dicer Dcr1, the RNA-dependent RNA polymerases Rdr1 and Rdr2 and other factors. We further show that this induces the synthesis of secondary siRNAs spreading along the entire endogenous mRNA, demonstrating the occurrence of both 3'-to-5' and 5'-to-3' transitivity for the first time in the SAR clade of eukaryotes (Stramenopiles, Alveolates, Rhizaria). Secondary siRNAs depend on Rdr2 and show a strong antisense bias; they are produced at much lower levels than primary siRNAs and hardly contribute to RNAi efficiency. We further provide evidence that the Paramecium RNAi machinery also processes single-stranded RNAs from its bacterial food, broadening the possible natural functions of exogenously induced RNAi in this organism.

PMID:
25593325
PMCID:
PMC4330347
DOI:
10.1093/nar/gku1331
[Indexed for MEDLINE]
Free PMC Article

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