Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

In this study, we show that human recombinant DICER protein (rDICER) processes a hairpin RNA with 5' overhangs in vitro and generates an intermediate duplex with a 29 nt-5' strand and a 23 nt-3' strand, which was eventually cleaved into a canonical miRNA duplex via a two-step cleavage. The previously identified endogenous pre-miRNA with 5' overhangs, pre-mmu-mir-1982 RNA, is also determined to be a substrate of rDICER through the same two-step cleavage.The two-step cleavage of a hairpin RNA with 5' overhangs shows that DICER releases double-stranded RNAs after the first cleavage and binds them again in the inverse direction for a second cleavage. These findings have implications for how DICER may be able to interact with or process differing precursor structures.DICER plays a key role in RNA interference pathways through the biogenesis of microRNA (miRNA) and small interfering RNA (siRNA) [1-3]. Most miRNA genes are transcribed as long primary transcripts (pri-miRNAs) where stem-loop structures with mature miRNA sequences embedded in the arm of a stem are cleaved by the Drosha nuclear microprocessor complex releasing a precursor miRNA (pre-miRNA) hairpin [4,5]. The cleavage site is determined mainly by the distance (~11 bp) from the stem-single stranded RNA junction of pri-miRNA and most pre-miRNAs have 2 nt-3' overhangs [6]. Pre-miRNAs, exported into the cytoplasm by Exportin-5 and Ran-GTP [7], are processed by the RISC loading complex (RLC) into 20~23 nt duplexes where the RNase III enzyme DICER plays a central role together with the double stranded (ds) RNA-binding proteins TRBP and PACT and the miRNA-associated RNA-induced silencing complex (miRISC) core component Argonaute-2 (AGO2) [8-10]. miRNA duplexes processed by RLC are finally loaded to miRISC as a double stranded-structure [11] and separated into the functional guide strand, which is complementary to the target, and the passenger strand, which is subsequently degraded [12,13]. Strand selection of the fun