As an essential post-transcriptional regulator of gene expression, microRNA (miR) levels must be strictly maintained. The biogenesis of many, but not all, miRs is mediated by trans-acting protein partners through a variety of mechanisms, including remodeling of the RNA structure. miR-31 functions as an oncogene in numerous cancers and interestingly, its biogenesis is not known to be regulated by protein binding partners. Therefore, the intrinsic structural properties of pre-miR-31 can provide a mechanism by which its biogenesis is regulated. We determined the solution structure of the precursor element of miR-31 (pre-miR-31) to investigate the role of distinct structural elements in regulating Dicer processing. We found that the presence or absence of mismatches within the helical stem do not strongly influence Dicer processing of the pre-miR. However, both the apical loop size and structure at the Dicing site are key elements for discrimination by Dicer. Interestingly, our NMR-derived structure reveals the presence of a triplet of base pairs that link the Dicer cleavage site and the apical loop. Mutational analysis in this region suggests that the stability of the junction region strongly influence both Dicer binding and processing. Our results enrich our understanding of the active role that RNA structure plays in regulating Dicer processing which has direct implications for control of gene expression.
MicroRNAs (miRNAs) are important regulators of post-transcriptional gene expression. Mature miRNAs are generated from longer transcripts (primary, pri- and precursor, pre-miRNAs) through a series of highly coordinated enzymatic processing steps. The sequence and structure of these pri- and pre-miRNAs play important roles in controlling their processing. Both pri- and pre-miRNAs adopt hairpin structures with imperfect base pairing in the helical stem. Here, we investigated the role of three base pair mismatches (A·A, G·A, and C·A) present in pre-miRNA-31. Using a combination of NMR spectroscopy and thermal denaturation, we found that the three base pair mismatches displayed unique structural properties, including varying dynamics and sensitivity to solution pH. These studies deepen our understanding of how the physical and chemical properties of base pair mismatches influence RNA structural stability.
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