Biology subjects

Munsayac, A.

Publications and source records attributed to Munsayac, A..

3 recordsLinked to original sources

bioRxiv2025

Structural features within precursor microRNA-20a regulate Dicer-TRBP processing

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression of target messenger (m) RNAs. To maintain proper miRNA expression levels, the enzymatic processing of primary and precursor miRNA elements must be strictly controlled. However, the molecular determinants underlying this strict regulation of miRNA biogenesis are not fully understood. Here, we determined the solution structure of pre-miR-20a, an oncogenic miRNA and component of the oncomiR-1 cluster, using nuclear magnetic spectroscopy (NMR) spectroscopy and small angle X-ray scattering (SAXS). Our structural studies informed on key secondary structure elements of pre-miR-20a which may control its enzymatic processing, namely a flexible apical loop and single-nucleotide bulge near the dicing site. We found that alternative conformations within pre-miR-20as apical loop function to self-regulate its Dicer-TRBP processing, and that a single nucleotide bulge at the -5 position from the 5'-cleavage site is critical for efficient processing. We additionally found that a disease-related single-nucleotide polymorphism in pre-miR-20a, predicted to disrupt the structure near the dicing site, resulted in reduced processing. These results further our structural understanding of the oncomiR-1 cluster and show how transient RNA conformers can function to self-regulate maturation.

biochemistry↗

bioRxiv2024

Selective deuteration of an RNA:RNA complex for structural analysis using small-angle scattering

The structures of RNA:RNA complexes regulate many biological processes. Despite their importance, protein-free RNA:RNA complexes represent a tiny fraction of experimentally-determined structures. Here, we describe a joint small-angle X-ray and neutron scattering (SAXS/SANS) approach to structurally interrogate conformational changes in a model RNA:RNA complex. Using SAXS, we measured the solution structures of the individual RNAs in their free state and of the overall RNA:RNA complex. With SANS, we demonstrate, as a proof-of-principle, that isotope labeling and contrast matching (CM) can be combined to probe the bound state structure of an RNA within a selectively deuterated RNA:RNA complex. Furthermore, we show that experimental scattering data can validate and improve predicted AlphaFold 3 RNA:RNA complex structures to reflect its solution structure. Our work demonstrates that in silico modeling, SAXS, and CM-SANS can be used in concert to directly analyze conformational changes within RNAs when in complex, enhancing our understanding of RNA structure in functional assemblies.

biophysics↗

bioRxiv2022

Solution structure of NPSL2, a regulatory element in the oncomiR-1 RNA

The miR-17~92a polycistron, also known as oncomiR-1, is commonly overexpressed in multiple cancers and has several oncogenic properties. OncomiR-1 encodes six constituent microRNAs (miRs), each enzymatically processed with different efficiencies. However, the structural mechanism that regulates this differential processing remains unclear. Chemical probing of oncomiR-1 revealed that the Drosha cleavage sites of pri-miR-92a are sequestered in a four-way junction. NPSL2, an independent stem loop element, is positioned just upstream of pri-miR-92a and sequesters a crucial part of the sequence that constitutes the basal helix of pri-miR-92a. Disruption of the NPSL2 hairpin structure could promote the formation of a pri-miR-92a structure that is primed for processing by Drosha. Thus, NPSL2 is predicted to function as a structural switch, regulating pri-miR-92a processing. Here, we determined the solution structure of NPSL2 using solution NMR spectroscopy. This is the first high-solution structure of an oncomiR-1 element. NPSL2 adopts a hairpin structure with a large, but highly structured, apical and internal loops. The 10-bp apical loop contains a pH-sensitive A+{middle dot}C mismatch. Additionally, several adenosines within the apical and internal loops have elevated pKa values. The protonation of these adenosines can stabilize the NPSL2 structure through electrostatic interactions. Our study provides fundamental insights into the secondary and tertiary structure of an important RNA hairpin proposed to regulate miR biogenesis.

biophysics↗