Structure of pre-miR-31 reveals an active role in Dicer processing

Abstract

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.

Use of steric blocking antisense oligonucleotides for the targeted inhibition of junction containing precursor microRNAs

“…The DNA template for preparation of the HH-pre-miR-31-HDV was generated as previously described (30). The DNA template for preparation of the pre-miR-144, pre-miR-21, pre-let-7c, GG-pre-miR-19a and pre-miR-143 were generated by overlap-extension (OE) polymerase chain reaction (PCR) using EconoTaq PLUS 2x Master Mix (Lucigen) with primers listed in Table S1.…”

“…Human Dicer/TRBP protein processing assays were performed as previously described with minimal modifications (30). Concentrated recombinant human Dicer/TRBP protein was diluted in 1× Dicing buffer (24 mM HEPES pH = 7.5, 100 mM NaCl, 5 mM MgCl 2 , 4 μM EDTA).…”

“…1a) (30). The open structure of pre-miR-31, which does not contain a pre-formed junction region, is a poor substrate for Dicer/TRBP cleavage (30).…”

“…Previously, we found that the junction region of pre-miR-31 is essential for efficient Dicer/TRBP processing and that disrupting junction base pairing inhibits Dicer processing (30). Here, we examined the effect of ASOs that can disrupt the pre-miR-31 junction, promoting an open structure.…”

Monitoring Anthracycline Cancer Drug‐Nucleosome Interaction by NMR Using a Specific Isotope Labeling Approach for Nucleosomal DNA

“…NMR studies can provide such detailed insight for nucleic acids, so far most clearly demonstrated on RNA. [17][18][19][20][21] Application to the nucleosomal DNA has so far been limited as the size (~200 kDa with histone proteins included) and the length (at least 145 bp [22] ) poses significant challenges.…”