Mechanisms of biogenesis and function
- MicroRNA: Biogenetic and Functional Mechanisms and Involvements in Cell Differentiation and Cancer
Most miRNA genes are located in the introns of host genes or outside genes. Unlike Drosophila, most of the human miRNA genes individually exist, although some human miRNAs are found in polycistronic clusters (5, 8, 9).
The miRNAs are synthesized through multiple steps (Fig. 1). Initially, the miRNAs are transcribed as long RNA precursors (pri-miRNAs) (11). As pri-miRNAs usually contain the cap structure and the poly(A) tail, it is suggested that the transcription of miRNAs is carried out by RNA polymerase II (12). The primiRNAs are processed into the precursors of approximately 70 nucleotides (pre-miRNAs) with a stem-loop structure and a two nucleotide 3' overhang by the RNase III enzyme Drosha and the double-stranded- RNA-binding protein DGCR8/Pasha (13, 14), and premiRNAs are exported from the nucleus to the cytoplasm by Exportin-5 in a Ran guanosine triphosphatedependent manner (15). Pre-miRNAs exported in the cytoplasm are processed by another RNase III enzyme, Dicer, and only one strand (guide strand) as a mature miRNA is incorporated into a RNA-induced silencing complex (RISC) that mediates either target RNA cleavage or translational inhibition, while the another strand (passenger strand) is excluded. Which strand is incorporated in RISC is determined by the stability of the base pairs at the 5' end of the duplex (16, 17). The incorporated guide strand guides the RISC to the complementary sequence in the 3'UTR of target mRNA. When the guide strand shares perfect or near perfect base pairing with the 3'UTR of target mRNA, the target mRNA is degraded by Argonaute2 (Ago2), a component of RISC (18). On the contrary, when the guide strand shares partial base pairing, translation is target-specifically repressed without the target mRNA degradation (19). Recent studies have revealed that RISC is at least composed of Dicer, Ago2, and the double-strand RNA binding protein TRBP, and RISC efficiently processes pre-miRNAs to mature miRNAs (20). Furthermore, RISC more efficiently cleaves target mRNAs by using the pre-miRNAs than the duplex miRNAs that do not have the stem-loop. These results suggest that miRNA processing by Dicer, assembly of the mature miRNA into RISC, and target RNA cleavage by Ago2 are coupled. Compared to the RNA cleavage mechanism by Ago2, the translational repression mechanism by miRNAs had been poorly understood. Recently, it was revealed that the target mRNAs binding to RISC through partial base pairing are accumulated in the cytoplasmic foci referred to as processing bodies (P-bodies) (21, 22). P-bodies, in which the mRNAs are stored or degraded by the decapping enzymes and exonucleases, do not contain the translational machinery (23). Furthermore, the disruption of P-bodies by the silencing of GW182, a key protein in P-body, inhibits translational silencing in not only partial base pairing but also perfect base pairing (24), although the localization of target mRNA with perfect base pairing is not detected in P-bodies (21). These results suggest that, at least in part, translational repression appears to be caused by the recruitment of target mRNAs to P-bodies. However, whether localization of the RISC-target mRNA complex in P-bodies is a cause or a result of the translational repression and whether the target mRNA cleavage by RISC occurs in the cytoplasm or P-bodies remain controversial issues.
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