Carradec Quentin, Götz Ulrike, Arnaiz Olivier, Pouch Juliette, Simon Martin, Meyer Eric, Marker Simone
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.
Zentrum für Human- und Molekularbiologie, Molekulare Zelldynamik, Universität des Saarlandes, Campus A2 4, 66123 Saarbrücken, Germany.
Nucleic Acids Res. 2015 Feb 18;43(3):1818-33. doi: 10.1093/nar/gku1331. Epub 2015 Jan 15.
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.
在各种生物体中,通过用产生针对内源基因的双链RNA(dsRNA)的细菌喂养细胞,可以触发有效的RNA干扰反应。然而,在大多数情况下,该途径的详细机制和自然功能尚不清楚。在这里,我们通过高通量测序研究了纤毛虫四膜虫中外源RNA的小干扰RNA(siRNA)生物合成及其与内源RNA干扰的遗传重叠。使用野生型和缺乏dsRNA喂养的突变菌株,我们发现双链的高水平初级siRNA是由Dicer Dcr1、RNA依赖性RNA聚合酶Rdr1和Rdr2以及其他因子从摄入的dsRNA触发物中加工而来的。我们进一步表明,这会诱导沿着整个内源mRNA扩散的次级siRNA的合成,首次在真核生物的SAR进化枝(不等鞭毛类、囊泡虫类、有孔虫类)中证明了3'到5'和5'到3'的传递性。次级siRNA依赖于Rdr2并表现出强烈的反义偏好;它们的产生水平比初级siRNA低得多,并且对RNA干扰效率几乎没有贡献。我们进一步提供证据表明,四膜虫的RNA干扰机制也能处理来自其细菌食物的单链RNA,拓宽了外源诱导的RNA干扰在该生物体中可能的自然功能。