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.

 Fluorescent microscopy of a seam cell expressing green fluorescent protein (GFP)–NRDE-3. Arrows indicate nuclei. NRDE-3 binds siRNAs and, in response, localizes to the nucleus similarly in both wild-type (left) and nrde-2 (gg091) mutant cells (right). eri-1 (mg366) mutant animals (middle) fail to express endo-siRNAs and, consequently, NRDE-3 is mislocalized to the cytoplasm. Image is reproduced, with permission, from Guang, S. et al. © (2010) Macmillan Publishers Ltd. All rights reserved.

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.