RNA interference (RNAi) is a highly efficient regulatory process that induces post-transcriptional gene silencing in most eukaryotes. RNAi machinery factors are mostly present and active in the cytoplasm. Several studies have shown the presence of RNAi machinery factors within the cell nucleus, suggesting a nuclear role for the RNAi machinery. In order to determine whether a short hairpin RNA (shRNA)-dependent RNAi mechanism can act in the human nucleus, we tested whether shRNA specifically targeted to an inducible mRNA transcribed from a candidate gene, would affect its transcription within the mammalian cell nucleus. This cell system allows the detection of the active gene during transcription, since the transcribed mRNA is tagged using the MS2 RNA-tagging method. Thereby we are able to visually track and quantify a potential nuclear RNAi effect on the active gene using RNA FISH and live-cell imaging. The expression of the shRNA caused a significant reduction in the cellular mRNAs transcribed, as might be expected, but moreover was accompanied by a dramatic weakening of the transcriptional activity of the inducible gene. The latter demonstrated an effect of the RNAi machinery in the nucleus, and actually at the site of transcription. To examine the mechanism, we checked if the silencing activity was targeting the mRNA (classical RNAi effect), or the gene. The presence of the transcription machinery was observed at the transcription sites during shRNA expression, indicating that transcription could continue but at low levels. We did not observe a change in the levels of RNAi factors in the nucleus following shRNA treatment, nor enrichment of these factors on the active gene. Additionally, no methylation of CpGs sites was detected on the promoter and on the gene region during shRNA expression, showing that the silencing was not occurring on the DNA level. Following these results, and based on recent studies, we examined the involvement of histone methylation by histone methyltransferases (HMTs). Using specific HMT inhibitors, we could observe an increase in the transcriptional activity of the gene, including during shRNA presence. Altogether, our data demonstrate the presence of a nuclear RNAi mechanism, probably involving histone methylation of the gene region.
