MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. The cleavage of primary microRNA (pri-miRNA) by Microprocessor, consisting of DROSHA and DGCR8, initiates miRNA biogenesis. Therefore, Microprocessor acts as a “gatekeeper” to control the efficiency and precision of miRNA production. Several RNA elements on pri-miRNAs have been revealed to regulate pri-miRNA processing by Microprocessor. However, the contribution of the pri-miRNA upper stem to the Microprocessor activity is still unknown. In this study, we conducted the high-throughput in vitro processing assay with purified Microprocessor proteins and ~42,300 randomized pri-miRNA variants to investigate the function of the pri-miRNA upper stem on the Microprocessor activity. We found that mismatches and wobble...[ Read more ]

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. The cleavage of primary microRNA (pri-miRNA) by Microprocessor, consisting of DROSHA and DGCR8, initiates miRNA biogenesis. Therefore, Microprocessor acts as a “gatekeeper” to control the efficiency and precision of miRNA production. Several RNA elements on pri-miRNAs have been revealed to regulate pri-miRNA processing by Microprocessor. However, the contribution of the pri-miRNA upper stem to the Microprocessor activity is still unknown. In this study, we conducted the high-throughput in vitro processing assay with purified Microprocessor proteins and ~42,300 randomized pri-miRNA variants to investigate the function of the pri-miRNA upper stem on the Microprocessor activity. We found that mismatches and wobble base pairs (seedMW) located at the seed region of miRNA sequences inhibit the cleavage of Microprocessor. In addition, we also found that seedMW are targets of single nucleotide polymorphisms (SNP) and RNA-editing for regulating miRNA biogenesis. These findings considerably improve our understanding of pri-miRNA processing mechanisms and provide a foundation for interpreting differential miRNA expression by several mechanisms, such as RNA modifications and SNPs.