Many small regulatory RNAs function in nuclei, but the mechanism of nuclear silencing in Caenorhabditis elegans has remained enigmatic. Guang et al. now identify a nuclear RNA interference (RNAi) mechanism that silences precursor mRNAs (pre-mRNAs) co-transcriptionally and inhibits RNA polymerase II (RNAPII) elongation.
A forward genetic screen for factors required for RNAi in C. elegans nuclei previously identified the Argonaute protein NRDE-3, which transports small interfering RNAs (siRNAs) from the cytoplasm to the nucleus. The same screen also identified nrde-2, which encodes a conserved and nucleus-localized protein that is required for RNAi — nrde-2 mutant animals are defective for nuclear RNAi.
a new mechanism for nuclear RNAi
Genetic analyses showed that nrde-2 and nrde-3 function in the same genetic pathway. NRDE-3-bound siRNAs localize to the nucleus in both wild-type and nrde-2 mutant animals, suggesting that NRDE-2 functions downstream of NRDE-3. A small amount of NRDE-2 associates with nuclear, but not cytoplasmic, NRDE-3. In addition, RNAi directs NRDE-2 to pre-mRNAs, suggesting that NRDE-2 might be recruited by NRDE-3–siRNA complexes to nascent transcripts that have been targeted by RNAi.
A reverse genetic screen revealed five additional putative nuclear RNAi factors, including the C. elegans orthologue of Rpb7, a subunit of RNAPII that functions in siRNA-mediated heterochromatin formation in fission yeast. Although the role of RPB-7 in C. elegans nuclear RNAi needs further characterization, it indicates the involvement of RNAPII transcription. Indeed, the association of NRDE-2 and NRDE-3 with unspliced RNAs suggests that nuclear RNAi functions during transcription. In addition, NRDE-2-dependent silencing occurs downstream to sites of RNAi, suggesting that nuclear RNAi is unlikely to occur at transcription initiation but instead during transcription elongation. Chromatin immunoprecipitation analysis revealed a decrease in AMA-1 (the C. elegans orthologue of Rpb1, the largest subunit of RNAPII) downstream of the site of RNAi, suggesting that siRNAs might inhibit RNAPII-mediated transcription. This was indeed confirmed by nuclear run-on analysis.
These findings describe a new mechanism for nuclear RNAi in C. elegans that is co-transcriptional, and show that small RNAs can regulate RNAPII during the elongation phase of transcription.
References
ORIGINAL RESEARCH PAPER
Guang, S. et al. Small regulatory RNAs inhibit RNA polymerase II during the elongation phase of transcription. Nature 13 Jun 2010 (doi:10.1038/nature09095)
FURTHER READING
Hutvagner, G. & Simard, M. J. Argonaute proteins: key players in RNA silencing. Nature Rev. Mol. Cell Biol. 9, 22–32 (2008)
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Heinrichs, A. Nuclear RNAi in worms. Nat Rev Mol Cell Biol 11, 539 (2010). https://doi.org/10.1038/nrm2939
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DOI: https://doi.org/10.1038/nrm2939