PubMed · 15242584
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Leemor Joshua-Tor. 2004. siRNAs at RISC.. https://doi.org/10.1016/j.str.2004.06.008
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Dicer is an RNase III which processes two classes of cellular small RNAs: the microRNAs (miRNA) and short interfering RNAs (siRNA). Previously, we observed that over-expressed HIV-1 Tat protein can suppress the processing of small RNAs inside cells. Here, we have investigated the requirements for Tat interaction with Dicer. We report that Tat-Dicer interaction depends on RNA, requires the helicase domain of Dicer, and is independent of Tat's transactivation domain.
Argonaute proteins associate with microRNAs (miRNAs) that bind mRNAs through partial base-pairings to primarily repress translation in animals. A fraction of Argonaute proteins and miRNAs biochemically cosediment with polyribosomes, yet another fraction paradoxically accumulates in ribosome-free processing bodies (PBs) in the cytoplasm. In this report, we give a quantitative account of the Argonaute protein localization and dynamics in living cells in different cellular states. We find that the majority of Argonaute is distributed diffusely in the cytoplasm, and, when cells are subjected to stress, Argonaute proteins accumulate to newly assembled structures known as stress granules (SGs) in addition to PBs. Argonaute proteins displayed distinct kinetics at different structures: exchange faster at SGs and much slower at PBs. Further, miRNAs are required for the Argonaute protein localization to SGs but not PBs. These quantitative kinetic data provide insights into miRNA-mediated repression.
Recent studies have shown that small noncoding RNAs, such as microRNAs and siRNAs, regulate gene expression at multiple levels including chromatin architecture, transcription, RNA editing, RNA stability, and translation. Each form of RNA-dependent regulation has been generally found to silence homologous sequences and collectively called RNAi. To further study the regulatory role of small RNAs at the transcriptional level, we designed and synthesized 21-nt dsRNAs targeting selected promoter regions of human genes E-cadherin, p21(WAF1/CIP1) (p21), and VEGF. Surprisingly, transfection of these dsRNAs into human cell lines caused long-lasting and sequence-specific induction of targeted genes. dsRNA mutation studies reveal that the 5' end of the antisense strand, or "seed" sequence, is critical for activity. Mechanistically, the dsRNA-induced gene activation requires the Argonaute 2 (Ago2) protein and is associated with a loss of lysine-9 methylation on histone 3 at dsRNA-target sites. In conclusion, we have identified several dsRNAs that activate gene expression by targeting noncoding regulatory regions in gene promoters. These findings reveal a more diverse role for small RNA molecules in the regulation of gene expression than previously recognized and identify a potential therapeutic use for dsRNA in targeted gene activation.