siRNAs targeting a chromatin-associated RNA induce its transcriptional silencing in human cells

Transcriptional gene silencing by small interfering RNAs (siRNAs) has been widely described in various species, such as plants or fission yeasts. In mammals, its extent remained somewhat debated. Previous studies showed that siRNAs targeting gene promoters can induce the silencing of the targeted promoter, although the involvement of off-target mechanisms was also suggested. Here, by nascent RNA capture and RNA Pol II ChIP, we show that siRNAs targeting a chromatin-associated non-coding RNA induce its transcriptional silencing. Deletion of the sequence targeted by one of these siRNAs on the two alleles by genome editing further show that this silencing is due to base pairing of the siRNA to the target. Moreover, by using cells with heterozygous deletion of the target sequence, we show that only the wild type allele, but not the deleted allele, is silenced by the siRNA, indicating that transcriptional silencing occurs only in cis. Finally, we demonstrate that both Ago1 and Ago2 are involved in this transcriptional silencing. Altogether, our data demonstrate that siRNAs targeting a chromatin-associated RNA at distance from its promoter induce its transcriptional silencing. Our results thus extend the possible repertoire of endogenous or exogenous interfering RNAs. Overall design: Strand-specific RNA-seq datasets in senescent WI38 hTERT RAF1-ER cells-treated with siRNAs against VINK vlincRNA (2 different control siRNAs, siCtle-1 and siCtle2, and 2 different VINK siRNAs, siVINK-1 and siVINK-2; 2 replicates) and in senescent WI38 hTERT RAF1-ER in which the region (210 bp) covering the siVINK1 target sequence was deleted on the two alleles using CRISPR-Cas9 (control siRNA, siCtle-1, and VINK siRNA, siVINK-1; 2 replicates).