Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Exportin 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells, whereas we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3′ end, yielding miRNAs from the 5′ strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research.icroRNA (miRNA) biogenesis begins with the synthesis of primary miRNA (pri-miRNA) by RNA polymerase II (1). The stem-loop structure embedded in pri-miRNA is cleaved by the Microprocessor complex composed of DROSHA and DGCR8 (2-6). The released hairpin, called precursor miRNA (pre-miRNA), is exported to the cytoplasm by Exportin 5 (XPO5) in a Ran-GTPdependent manner (7-9). In the cytoplasm, the pre-miRNA is further processed by DICER, producing a duplex RNA of ∼22 nt with its 3′ ends having a two nucleotide overhang (10-13). The duplex is loaded onto the ARGONAUTE (AGO) proteins, and one strand of the duplex remains as mature miRNA, whereas the other strand is discarded from AGO (11,14). The strand selection is dictated mainly by the relative thermodynamic stability of the two ends of the duplex: the strand whose 5′ terminal nucleotides are less stable is selected as mature miRNA (15,16). miRNAs originating from the 5′ and 3′ strands of pre-miRNA are referred to as the 5p and 3p miRNAs, respectively. Mammals have four closely related AGO proteins (AGO1-4) that interact with deadenylation factors and translational machinery to induce mRNA degradation and translational repression.Although the aforementioned canonical pathway accounts for the production of most miRNAs (1), it has also been shown that there exist alternative (noncanonical) pathways for miRNA biogenesis, which bypass a part of the biogenesis steps mentioned above. Mirtrons are one of the first miRNA groups described as noncanonical miRNAs, which do not require DROSHA for their production (17-19). Because mirtrons are located inside short ...

Although more than 100 types of RNA modification have been described thus far, most of them were thought to be rare in mRNAs and in regulatory noncoding RNAs. Recent developments have unveiled that at least some of the modifications are considerably abundant and widely conserved. This Minireview summarizes the molecular machineries and biological functions of methylation (N6-methyladenosine, m(6)A) and uridylation (U-tail).

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