Re evaluation of the roles of DROSHA, Exportin 5, and DICER in microRNA biogenesis
Significance MicroRNAs (miRNAs) are noncoding RNAs with diverse roles in development and pathogenesis. Biogenesis of canonical miRNA requires nuclear processing by DROSHA, nuclear export by Exportin 5, and cytoplasmic processing by DICER. To gain a deeper understanding of the maturation processes, we here ablated the DROSHA, Exportin 5, and DICER genes using the same human cell line. Canonical miRNA production was abolished in DROSHA deleted cells, revealing an irreplaceable role of DROSHA. Interestingly, however, some canonical miRNAs were still produced without DICER albeit at markedly reduced levels, and many were detected in Exportin 5 deleted cells at only modestly decreased levels. Our study allows us to understand differential contributions of key biogenesis factors, and provides valuable resources for miRNA research. 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.
Proceedings of the National Academy of Sciences