Many classes of small RNA (sRNA) involved in RNA silencing are generated by double-stranded RNA (dsRNA) processing. Although principles of sRNA biogenesis have emerged, newly identified classes of sRNAs have features that suggest additional biogenesis mechanisms. Tetrahymena thermophila expresses one such class, comprising sRNAs of 23 and 24 nucleotides (nt) with an absolute strand bias in accumulation. Here we demonstrate sRNA production by the T. thermophila Dicer Dcr2 and the RNA-dependent RNA polymerase Rdr1, which purifies as a multisubunit RNA-dependent RNA polymerase complex (RDRC). Dcr2 and RDRC interact, stimulating Dcr2 activity. Moreover, Dcr2 specificity is influenced by RDRC beyond this physical interaction, as Dcr2 generates discrete 23- and 24-nt sRNAs only from dsRNA with a 5′-triphosphate. These findings suggest that sRNA strand bias arises from Dcr2 processing polarity, conferred by physical and functional coupling of RDRC and Dicer enzymes.
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We thank the Collins laboratory for discussions and comments on this manuscript, and I. Macrae and J. Doudna for technical suggestions. This research was supported by a Howard Hughes Medical Institute Predoctoral Fellowship to S.R.L.
The authors declare no competing financial interests.
The RDRC and Dcr2 generate characteristic products on different templates. (PDF 8775 kb)
RDRC products can be diced by Dcr2 after double-stranded RNA synthesis. (PDF 1473 kb)
Intact RDRC stimulates dicing by Dcr2. (PDF 3249 kb)
Summary of mass spectrometry analysis. (PDF 2772 kb)
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Lee, S., Collins, K. Physical and functional coupling of RNA-dependent RNA polymerase and Dicer in the biogenesis of endogenous siRNAs. Nat Struct Mol Biol 14, 604–610 (2007). https://doi.org/10.1038/nsmb1262
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