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 . Pre-miRNAs, exported into the cytoplasm by Exportin-5 and Ran-GTP , 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  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 functional guide strand by AGO2 depends on the thermodynamic stabilities of the base pairs at the 5' ends of the two strands [12, 14, 15]. Duplexes of siRNA or miRNA produced by DICER can be loaded in either direction to Argonaute [16–18]. Indeed, the mature miRNA either in the 5' or 3' strands can be harboured from pre-miRNA [19–21]. On the other hand, endogenous human AGO2 can bind directly to pre-miRNAs in DICER-knockout cells . Recently, it was reported that human DICER is not essential for loading dsRNAs to AGO2 but functions in pre-selection of effective siRNAs for handoff to AGO2 .
Human DICER is a ~220 kDa protein consisting of several domains; an N-terminal DExH-box RNA helicase-like domain, a DUF283 domain, a PAZ domain, two RNase III domains (RIIIa and RIIIb), and a dsRNA binding motif domain (DARM) . The two RNase III domains of DICER form a single dsRNA processing center via intramolecular dimerization which together cleave the opposite strands of the dsRNA, generating dinucleotide-long 3' overhangs on both ends . The crystal structure of Dicer from Giardia intestinalis showed that the hydrophobic pocket of the PAZ domain was responsible for the binding of the 3' dinucleotide overhangs of the substrate and the connector helix between the PAZ domain and RNase III domain functioned as a molecular ruler measuring the distance from the 3' end of pre-miRNA to the cleavage site [26, 27]. However, 3'-dinucleotide dsRNA overhangs are not essential for binding with DICER . When the 3' overhang is removed, DICER can still cleave dsRNA through interaction with the remaining 5' overhang . This is consistent with MacRae et al. who found that the recombinant Dicer protein of Giardia intestinalis could cleave the dsRNA with 5' overhangs . However, they used perfectly matched dsRNAs with no gap, which might resemble an endogenous siRNA precursor. An additional study by Flores-Jasso et al. showed that human recombinant DICER protein could nick either strand of a mononucleotide-5' overhanged pre-miRNA with some strand preferences . Despite this, the detailed step mechanism for pre-miRNA cleavage, especially for the pre-miRNA with 5' overhangs, is not yet elucidated.
An alternative nuclear pathway of pre-miRNA biogenesis was described where a short intron with a hairpin can be spliced and debranched into pre-miRNA hairpin mimics (mirtrons) [30–32]. This processing pathway uses intron splicing machinery instead of the Drosha endonuclease; miRNA precursors generated from intronic sequences (debranched mirtrons) are believed to be incorporated into the canonical miRNA pathway as a substrate of DICER. Interestingly, mouse pre-mir-1982 is a mirtron with an 11 nt tail at the 5' end , although most mammalian mirtron are hairpin structures with single nucleotide overhangs at both ends [32–34]. Mature mouse miR-1982* miRNA emerges without 11 nt-5' overhangs from deep sequencing data of murine cells [33, 35] while the elimination mechanism of this 11 nt-5' tail is still unknown.
In this paper, we investigated the detailed processing pattern of hairpin RNAs containing 5' overhangs by human recombinant DICER. We show here that human recombinant DICER is able to process hairpin RNA with 5' overhangs and two-step cleavage by DICER forms the mature miRNA duplex from the hairpin RNAs. Additionally, pre-mmu-mir-1982 RNA, which is a natural hairpin RNA with 5' overhangs, is also processed by a two-step cleavage mediated by human recombinant DICER protein in vitro.