Summary RNAi is widely appreciated as a powerful regulator of mRNA translation in the cytoplasm of mammalian cells. However, the presence and activity of RNAi factors in the mammalian nucleus has been the subject of considerable debate. Here we show that Argonaute-2 (Ago2) and RNAi factors Dicer, TRBP and TRNC6A/GW182 are in the human nucleus and associate together in multi-protein complexes. Small RNAs can silence nuclear RNA and guide site-specific cleavage of the targeted RNA, demonstrating that RNAi can function in the human nucleus. Nuclear Dicer is active and miRNAs are bound to nuclear Ago2, consistent with the existence of nuclear miRNA pathways. Notably, we do not detect loading of duplex small RNAs in nuclear extracts and known loading factors are absent. These results extend RNAi into the mammalian nucleus and suggest that regulation of RNAi via small RNA loading of Ago2 differs between the cytoplasm and the nucleus.

Many neurological disorders are caused by expanded trinucleotide repeats1, including Machado-Joseph Disease (MJD)2 and Huntington Disease (HD)3. MJD and HD are caused by expanded CAG repeats within the ataxin-3 (ATXN3) and huntingtin (HTT) genes. Inhibiting expression of ATXN3 or HTT are promising therapeutic strategies, but indiscriminant inhibition of wild-type and mutant alleles may lead to toxicity. We hypothesized that expanded triplet repeat mRNA might be preferentially recognized by complementary oligomers. We observe selective inhibition of mutant ataxin-3 and HTT protein expression by peptide nucleic acid (PNA) and locked nucleic acid (LNA) oligomers targeting CAG repeats. Duplex RNAs were less selective, suggesting an advantage for single-stranded oligomers. Inhibiting mutant HTT expression protected cultured striatal neurons from an HD mouse model against glutamate-induced toxicity. Antisense oligomers that discriminate between wild-type and mutant genes on the basis of repeat length offer new options for treating MJD, HD, and other hereditary diseases.

Although many long non-coding RNAs (lncRNAs) have been discovered, their function and their association with RNAi factors in the nucleus have remained obscure. Here, we identify RNA transcripts that overlap the cyclooxygenase-2 (COX-2) promoter and contain two adjacent binding sites for an endogenous miRNA, miR-589. We find that miR-589 binds the promoter RNA and activates COX-2 transcription. In addition to miR-589, fully complementary duplex RNAs that target the COX-2 promoter transcript activate COX-2 transcription. Activation by small RNA requires RNAi factors argonaute-2 (AGO2) and GW182, but does not require AGO2-mediated cleavage of the promoter RNA. Instead, the promoter RNA functions as a scaffold. Binding of AGO2 protein/small RNA complexes to the promoter RNA triggers gene activation. Gene looping allows interactions between the promoters of COX-2 and phospholipase A2 (PLA2G4A), an adjacent pro-inflammatory pathway gene that produces arachidonic acid, the substrate for COX-2 protein. miR-589 and fully complementary small RNAs regulate both COX-2 and PLA2G4A gene expression, revealing an unexpected connection between key steps of the eicosanoid signaling pathway. The work demonstrates the potential for RNA to coordinate locus-dependent assembly of related genes to form functional operons through cis-looping.