Structure and Targeting of Precursor MicroRNA-31: From Mechanism to Application

Abstract

As an essential post-transcriptional regulator of gene expression, microRNA (miRNA) levels must be strictly maintained. The biogenesis of many miRNAs is regulated by trans-acting protein partners, which exert control through a variety of mechanisms, including remodeling of the RNA structure and recruitment of RNA processing or modifying enzymes. MicroRNA-31 (miR-31) functions as an oncogene in numerous cancers, and interestingly, its biogenesis is not known to be regulated by protein factors. Therefore, I investigated if the intrinsic structural and dynamical properties of the miR-31 precursor element, pre-miR-31, can provide a mechanism by which its biogenesis is regulated. Base pair mismatches are a common feature of primary and precursor miRNAs. In this thesis, I characterized the base pair mismatches within pre-miR-31 and found the C•A mismatch within the stem of pre-miR-31 to be strongly pH sensitive and, stabilizing the RNA structure at near physiological pH. Next, I investigated the role of distinct structural elements within pre-miR-31 in regulating processing by the Dicer/TRBP complex. I found that both the apical loop size and structure at the Dicing site are key elements for discrimination by the Dicer/TRBP complex. Interestingly, our NMR-derived structure revealed the presence of a triplet of base pairs, or junction region, that link the Dicer cleavage site and the apical loop. My mutational analysis in this region revealed that the stability of the junction region strongly influenced processing by the Dicer/TRBP complex. Based on these findings, I developed a new type of antisense oligonucleotide (ASO) that specifically targets the junction region of pre-miR-31 to inhibit Dicer/TRBP processing. Furthermore, I demonstrated that this new type of ASO design is broadly applicable to other junction containing pre-miRNAs, which account for ~20 % of human pre-miRNAs, and function to reduce the Dicer/TRBP cleavage of this family of pre-miRNAs. These studies enhance our understanding of RNA structure based ASO design and development. The results in this thesis enrich our understanding of the active role that RNA structure plays in regulating miRNA biogenesis, which has direct implications for the control of gene expression. This study further points out that RNA structure is not a passive element in the protein enzymatic steps. Rather, the RNA structural elements play important roles in regulating processing by Dicer/TRBP. Furthermore, my thesis work provides a new approach for antisense oligonucleotide design by targeting microRNA biogenesis at a step upstream of the traditional anti-microRNA antisense oligonucleotide design strategy. This new type of antisense oligonucleotide allows intervention at an early stage of miRNA biogenesis and may lead to a novel treatment by selectively inhibiting disease-related pre-miRNAs.