![]() ![]() Processing and Ago2 loading of shRNA and AgoshRNA molecules. Finally, the miRNA or siRNA duplex is incorporated into the RNA-induced silencing complex (RISC) by association with the Argonaute (Ago) protein. Such short hairpin RNA (shRNA) molecules ( Fig 1, top) skip Microprocessor processing and enter the RNAi pathway at the Dicer processing step, resulting in the generation of a small interfering RNA (siRNA) duplex. RNAi can also be induced by man-made constructs containing RNA polymerase III gene cassettes that express a short transcript with the characteristics of a pre-miRNA. This pre-miRNA is subsequently exported from the nucleus to the cytoplasm, where the RNAse III-like Dicer endonuclease will remove the terminal loop to generate a duplex of ~20–24 base pairs (bp). The nuclear Microprocessor complex, composed of the RNAse III-like endonuclease Drosha and its double stranded RNA (dsRNA)-binding partner DGCR8, cleaves the stem-loop structure embedded in the pri-miRNA, and releases a smaller ~70 nt precursor miRNA (pre-miRNA) hairpin with a 2-nt 3’ overhang. miRNAs are expressed as primary miRNA (pri-miRNA) transcripts and subsequently processed. MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are small, non-coding RNAs (∼21 nucleotides (nt) in size) that have important post-transcriptional regulatory roles by targeting messenger RNAs (mRNAs) for cleavage or translational repression. We also demonstrate specific AgoshRNA loading in Ago2, but not Ago1/3/4, thus further reducing unwanted side effects. These results suggest the presence of a cellular co-factor involved in AgoshRNA loading into Ago2 in vivo. In contrast, only the double-stranded AgoshRNA precursor associated with Ago2 in cells, correlating with efficient intracellular processing and reporter knockdown activity. Ago2 was found to bind preferentially to partially single-stranded AgoshRNA in vitro. Here we set out to analyze the activity of different synthetic AgoshRNA processing intermediates. As a result, the mature single-stranded AgoshRNA may dock more stably into Ago2. Previously, we characterized AgoshRNA processing by deep sequencing and demonstrated that-after Ago2 cleavage-AgoshRNAs acquire a short 3’ tail of 1–3 A-nucleotides and are subsequently trimmed, likely by the poly(A)-specific ribonuclease (PARN). We named these molecules AgoshRNA, which generate only a single active RNA strand and thus avoid off-target effects that can be induced by the passenger strand of a regular shRNA. One non-canonical pathway bypasses Dicer cleavage and requires instead processing by Argonaute2 (Ago2), which also executes the subsequent silencing step. ![]() ![]() The RNA interference (RNAi) pathway was recently expanded by the discovery of multiple alternative pathways for processing of natural microRNA (miRNA) and man-made short hairpin RNA (shRNA) molecules. ![]()
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