Poster abstracts
Poster number 135 submitted by Sydney Steele
Probing functional differences in pri-miR-20a binding by the 2 RRMs of hnRNPA2B1
Sydney M. Steele (Department of Chemistry, University of Michigan), Cade T. Harkner (Department of Chemistry, University of Michigan), Yaping Liu (Biophysics Program, University of Michigan), Sarah C. Keane (Department of Chemistry, University of Michigan and Biophysics Program, University of Michigan)
Abstract:
MicroRNAs are short (~22 nt) RNAs which function to regulate gene expression. Primary microRNA-20a (pri-miR-20a) is an oncogenic miRNA whose enzymatic processing is post-transcriptionally regulated by RNA-binding proteins (RBPs). The apical loop of pri-miR-20a is known to be a critical binding site for RBPs, including heterogeneous nuclear ribonucleoproteins (hnRNPs). Our laboratory has shown that hnRNPA2/B1 binds pri-miR-20a and forms a 1:1 complex. However, the molecular details of the intermolecular interaction remain unknown. For example, it is not clear whether one or both of the tandem RNA recognition motifs (RRMs) preferentially binds pri-miR-20a or if the RRMs have functionally distinct or redundant roles. Isothermal titration calorimetry (ITC) data revealed that each isolated RRM bound pri-miR-20a, with similar affinity. Interestingly, the binding of each isolated RRM was similar to that measured for the intact RNA-binding domain (RBD), which contains both RRMs. To elucidate potential functional differences between the RRMs, we used site-directed mutagenesis to mutate residues in each RRM predicted to be important for RNA binding. We are currently investigating how these mutations, in the isolated RRMs and in the full-length RBD, impact the binding of pri-miR-20a using ITC. Our studies will uncover important functional differences between the RRMs and reveal the mechanism by which hnRNPA2/B1 binds pri-miR-20a.
Keywords: microRNA, Protein-RNA interaction, hnRNP