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

Abstract

As an essential posttranscriptional regulator of gene expression, microRNA (miRNA) levels must be strictly maintained. The biogenesis of many miRNAs 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 the precursor element of miR-31 (pre-miR-31) can provide a mechanism by which its biogenesis is regulated. We determined the solution structure of pre-miR-31 to investigate the role of distinct structural elements in regulating processing by the Dicer-TRBP complex. We found that the presence or absence of mismatches within the helical stem does not strongly influence Dicer-TRBP processing of the pre-miRNAs. However, 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 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 influences processing by the Dicer-TRBP complex. Our results 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.

Keywords: NMR spectroscopy; RNA; microRNA; structural biology.

Fig. 1.

Conflicting secondary structure models for pre-miR-31 apical loop. (A) Secondary structure derived from in vitro DMS-MapSeq where coloring denotes reactivity of given bases. Red=high reactivity, orange=medium reactivity, black=low reactivity, gray=no data available. (B) Secondary structure derived from NMR characterization. Coloring is based on the identification of A-U base pairs (see panel E). (C) Portion of a 2D ¹H–¹H NOESY spectrum of an A^2rG^rU^r-labeled FL pre-miR-31. Adenosine cross-strand NOEs consistent with helical stacking in the junction region are indicated. (D) Secondary structure of the apical loop region highlighting NOEs noted in C with red arrows. (E) Best-selective long-range HNN-COSY spectrum identifying A-U base pairs within FL pre-miR-31. Black peaks are adenosine H2–N1 correlations, red peaks are adenosine H2–uracil N3 correlations. Vertical lines indicate the detection of A-U base pairs. Unpaired adenosines are denoted in green, A-U base pairs in the stem region are denoted in black, junction A-U base pairs are denoted in cyan and purple.

Fig. 2.

Tertiary structure of pre-miR-31. (A) NMR-derived secondary structure of FL-pre-miR-31. Dicer cleavage sites are indicated with scissors. Gray nucleotides were included in structural studies but are not present in a Dicing-competent WT pre-miR-31. (B) Ensemble of 10 lowest energy structures after RDC refinement superimposed over residues 1–13 and 59–71. (C) The lowest energy structure of pre-miR-31 with a transparent surface rendering. (D) Enlarged view of the dicing site, colored orange. (E) Enlarged view of the C•A mismatch, colored pink. (F) Enlarged view of the G•A mismatch, colored teal. (G) Enlarged view of the A•A mismatch, colored green.

Fig. 3.

Structure at the dicing site serves as an important feature for Dicer-TRBP processing. (A) Predicted secondary structures of constructs designed to minimize the internal loop at the dicing site. Mutations are indicated with red lettering. (B) Minimization of the internal loop at the Dicing site enhances the processing by the Dicer–TRBP complex. (C) Secondary structures of dicing site mutants with expanded internal loop structures. Mutations are indicated with red lettering. (D) Pre-miR-31 RNAs with larger internal loops at the Dicer cleavage site have reduced Dicer-TRBP processing efficiencies, relative to WT. For all processing assays, average and SD from n = 3 independent assays are presented.

Fig. 4.

Pre-miR-31 requires a greater than 4-nt apical loop for efficient processing. (A) Secondary structures of pre-miR-31 RNAs engineered to contain smaller apical loops. Sites of mutation are denoted with red lettering. (B) In vitro processing assays with the Dicer–TRBP complex reveal a significant reduction in substrate cleavage for the G32C/A33C RNA. (C) Secondary structures of mutants designed to extend the pre-miR-31 apical loop size. Nonnative nucleotide insertions are indicated with red lettering. (D) Dicer–TRBP processing of pre-miR-31 RNAs with larger apical loops was largely unchanged relative to WT. For all processing assays, average and SD from n = 3 independent assays are presented.

Fig. 5.

The junction region is a regulatory element within pre-miR-31. (A) Design of pre-miR-31 RNAs with varying junction stabilities. Mutations are indicated with red lettering. (B) Time-dependent Dicer–TRBP processing of pre-miR-31 junction RNAs reveals the importance of junction stability. (C) Correlation between Dicer–TRBP processing (% substrate cleaved and k_obs) and measured thermal stability (melting temperature, T_m) for WT and junction region mutations reveals the need for a moderately stable junction for efficient processing. For all thermal denaturation experiments and processing assays, the average and SD from n = 3 independent assays are presented.

Fig. 6.

Secondary structure elements and their contribution to the regulation of pre-miR-31 processing. The presence or absence of mismatches within the stem of pre-miR-31 had no impact on Dicer–TRBP processing. RNAs with more stabilized Dicing sites were processed more efficiently than the WT sequence, while pre-miRNAs with larger internal loops were poorly processed. Similarly, pre-miRNAs with too small of an apical loop was processed less efficiently than WT pre-miR-31. Interestingly, the WT pre-miR-31 has an inherently encoded structural switch at the junction region. Pre-miR-31 appears to sample both an open loop structure and a closed loop structure. Only RNAs with marginally stable junction regions were maximally processed by Dicer–TRBP. The ability of pre-miR-31 to sample both states promotes processing of pre-miR-31 by the Dicer–TRBP complex.