Ma Sicong, Kotar Anita, Grote Scott, Rouskin Silvi, Keane Sarah C
Biophysics Program, University of Michigan, 930 N. University Avenue, Ann Arbor, MI 48109, USA.
Department of Microbiology, Harvard Medical School, Boston, MA 02115, USA.
bioRxiv. 2023 Jan 3:2023.01.03.519659. doi: 10.1101/2023.01.03.519659.
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.
作为基因表达至关重要的转录后调节因子，微小RNA（miR）的水平必须严格维持。许多（但并非全部）miR的生物合成由反式作用蛋白伴侣通过多种机制介导，包括RNA结构重塑。miR-31在多种癌症中发挥癌基因作用，有趣的是，其生物合成并不受蛋白结合伴侣的调控。因此，前体miR-31的内在结构特性可能为其生物合成的调控提供一种机制。我们测定了miR-31前体元件（前体miR-31）的溶液结构，以研究不同结构元件在调节Dicer加工过程中的作用。我们发现螺旋茎内错配的有无对前体miR的Dicer加工影响不大。然而，切割位点处的顶端环大小和结构是Dicer识别的关键元件。有趣 的是，我们通过核磁共振得出的结构揭示了连接Dicer切割位点和顶端环 的三联碱基对的存在。该区域的突变分析表明，连接区域的稳定性对Dicer结合和加工均有强烈影响。我们的结果丰富了我们对RNA结构在调节Dicer加工过程中所起积极作用的理解，这对基因表达的控制具有直接影响。
