Physical and functional coupling of RNA-dependent RNA polymerase and Dicer in the biogenesis of endogenous siRNAs

Abstract

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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Figure 1: T. thermophila RDR1 is essential.
Figure 2: Rdr1 assembles as an RDRC.
Figure 3: Dcr2 associates with RDRC.
Figure 4: Dcr2 generates 23- and 24-nt sRNA duplexes from dsRNA products of Rdr1.
Figure 5: RDRC stimulates Dcr2 activity.
Figure 6: Duplex end structure influences Dcr2 cleavage specificity.
Figure 7

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Acknowledgements

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.

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Contributions

S.R.L. performed the experiments, and K.C. and S.R.L. wrote the manuscript.

Corresponding author

Correspondence to Kathleen Collins.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

The RDRC and Dcr2 generate characteristic products on different templates. (PDF 8775 kb)

Supplementary Fig. 2

RDRC products can be diced by Dcr2 after double-stranded RNA synthesis. (PDF 1473 kb)

Supplementary Fig. 3

Intact RDRC stimulates dicing by Dcr2. (PDF 3249 kb)

Supplementary Table 1

Summary of mass spectrometry analysis. (PDF 2772 kb)

Supplementary Methods (PDF 87 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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