Poster abstracts
Poster number 87 submitted by Sydney Steele
Investigating the molecular determinants of pri-miR-20a binding by the tandem RRMs of hnRNP A2/B1
Sydney M. Steele (Biophysics Program, University of Michigan), Cade T. Harkner (Department of Chemistry, University of Michigan), Yaping Liu (Biophysics Program, University of Michigan), Sarah C. Keane (Biophysics Program, University of Michigan and Department of Chemistry, University of Michigan)
Abstract:
MicroRNAs are short (~22 nt) RNAs that are crucial regulators of gene expression. Primary microRNA-20a (pri-miR-20a) is an oncogenic miRNA whose enzymatic processing is known to be post-transcriptionally regulated by RNA-binding proteins (RBPs), including heterogeneous nuclear ribonuclear proteins (hnRNPs). Our laboratory has shown that hnRNP A2/B1 binds the apical loop of pri-miR-20a to form a 1:1 complex. However, the molecular determinants of this intermolecular interaction remain unclear. For example, it is not clear whether one or both of the tandem RNA recognition motifs (RRMs) within the RNA-binding domain (RBD) preferentially binds pri-miR-20a or if the RRMs have functionally distinct or redundant roles. Interestingly, isothermal titration calorimetry (ITC) data revealed that only one of the RRMs maintains RBD-like binding affinity when isolated. To elucidate potential functional differences between the RRMs, we used site-directed mutagenesis (SDM) to mutate residues, confined to each RRM but within the context of the full-length RBD, predicted to be important for RNA binding. Prior studies have indicated that members of the hnRNP family require interdomain communication via salt bridges between tandem RRMs to allosterically regulate their function. To determine whether interdomain communication is present between the tandem RRMs of hnRNP A2/B1, or whether they are thermodynamically uncoupled, we used SDM to disrupt two salt bridges at the interface between the two RRMs. We are currently investigating how these mutations impact the binding of pri-miR-20a. Our studies will help to elucidate the mechanism by which hnRNP A2/B1 binds pri-miR-20a.
References:
Levengood, J. D.; Potoyan, D.; Penumutchu, S.; Kumar, A.; Zhou, Q.; Wang, Y.; Hansen, A. L.; Kutluay, S.; Roche, J.; Tolbert, B. S. Thermodynamic coupling of the tandem RRM domains of hnRNP A1 underlie its pleiotropic RNA binding functions. Science Advances 2024, 10 (28). https://doi.org/10.1126/sciadv.adk6580.
Keywords: microRNA, Protein-RNA interaction, hnRNP