siRNA in human cells selectively localizes to target RNA sites

Abstract

Recent observations of RNA interference (RNAi) in the nuclei of human cells raise key questions about the extent to which nuclear and cytoplasmic RNAi pathways are shared. By directly visualizing the localization of small interfering RNA (siRNA) in live human cells, we show here that siRNA either selectively localizes in the cytoplasm or translocates into the nucleus, depending on where the silencing target RNA resides. Two siRNAs that target the small nuclear 7SK and U6 RNAs localize into the nucleus as duplexes. In contrast, an siRNA targeting the cytoplasmic hepatitis C virus replicon RNA dissociates, and only antisense strand distributes in the cytoplasm of the cells harboring the target RNA, whereas sense strand gets degraded. At the same time, both strands of the latter siRNA are distributed throughout the cytoplasm and nucleus in cells lacking the silencing target RNA. These results suggest the existence of a mechanism by which the RNAi machinery orchestrates a target-determined localization of the siRNA and the corresponding RNAi activity, and also provide evidence for formation of nuclear-programmed active RNA induced silencing complexes directly in the nucleus.confocal imaging ͉ nuclear͞cytoplasmic localization ͉ RNA-induced silencing complex ͉ RNA interference mechanism ͉ small interfering RNA N early a decade after the discovery that double-stranded RNA can trigger an RNAi response that inhibits gene expression in a sequence-specific manner (1), the complexity of the mechanisms by which small RNAs regulate gene expression continues to unfold (2-11). RNA interference (RNAi) has generally been defined as a cellular pathway that mediates posttranscriptional gene silencing either by sequence-specific degradation of targeted RNAs or via sequence-specific inhibition of translation. Thus, RNAi studies in mammalian cells have mainly focused on the cytoplasm, where mature mRNA is translated and key proteins of RNA-induced silencing complexes (RISCs) were thought to localize and function. These RISCs, by which the RNAi machinery implements silencing of gene expression, are composed of several proteins (including Ago1 and Ago2) and one strand of small interfering RNA (siRNA) (12, 13). During the course of RISC assembly, the siRNA͞ microRNA duplex dissociates, and the guide strand enters active RISCs, allowing binding and degradation of the complementary target mRNA.Target specificity in RNAi is achieved through RNA-RNA sequence recognition and base pairing. Because RNA can also recognize and form duplexes with DNA, RNAi should be capable of affecting gene function at the level of genomic DNA, extending the realm of RNAi function into the nucleus. Indeed, recent demonstrations of siRNA-induced transcriptional gene silencing through involvement of DNA methylation (2, 3) in various human cell types, siRNA-dependent knock-down of nucleus-restricted transcripts (4, 5), and a direct documentation of potent and specific down-regulation of 7SK and U6 small nuclear RNAs (6) have uncovered such nuclear RNAi pathways in ...

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