MicroRNAs (miRNAs) are small noncoding RNAs that emerge as key regulators for gene expression. The expression level and sequence identity of miRNA, which determine the functionality, are mainly regulated in the Microprocessor-mediated primary miRNA (pri-miRNA) processing step. Multiple RNA elements that interact with Microprocessor or its co-factor and regulate the pri-miRNA processing were found in the basal junction, basal segment, lower stem, and apical loop of pri-miRNA. However, the contribution of pri-miRNA upper stem in regulating pri-miRNA processing remains unknown. In this study, high-throughput sequencing-integrated in vitro processing assays on artificial or natural human pri-miRNAs that contain randomized sequence in the upper stem were conducted using purified Mic...[ Read more ]

MicroRNAs (miRNAs) are small noncoding RNAs that emerge as key regulators for gene expression. The expression level and sequence identity of miRNA, which determine the functionality, are mainly regulated in the Microprocessor-mediated primary miRNA (pri-miRNA) processing step. Multiple RNA elements that interact with Microprocessor or its co-factor and regulate the pri-miRNA processing were found in the basal junction, basal segment, lower stem, and apical loop of pri-miRNA. However, the contribution of pri-miRNA upper stem in regulating pri-miRNA processing remains unknown. In this study, high-throughput sequencing-integrated in vitro processing assays on artificial or natural human pri-miRNAs that contain randomized sequence in the upper stem were conducted using purified Microprocessor proteins. Combining the analysis of the high-throughput assays and a series of systematic validation experiments, multiple mismatched and wobble base pairs in the upper stem of pri-miRNAs were found to be critical for the efficient and accurate pri-miRNA processing. The alternative cleavages of Microprocessor on some human pri-miRNAs, the existence of which cannot be understood by current knowledge, turn out to be relied on mismatched or wobble base pairs in the upper stem. These findings add one more layer to the pri-miRNA processing mechanisms and improve our interpretation of differential miRNA expression by several mechanisms, such as single nucleotide polymorphisms (SNPs).