MicroRNAs (miRNAs, miRs) are short, ∼20 nts long RNAs which function in conjunction with Argonaut proteins (Agos) to regulate gene expression through translational repression of messenger-RNA (mRNA) targets. However, functional microRNAs are the products of a multi-step enzymatic processing pathway involving cleavage of primary microRNA transcripts (pri-miRNAs) by the microprocessor (MP, comprised of the RNase III enzyme Drosha and partner protein DGCR8) into a precursor microRNA (pre-miRNA), which is further processed RNase II enzyme Dicer. Importantly, these enzymatic processing steps must remain tightly regulated to prevent disease states and are commonly regulated by protein binding partners. Here, we investigate the interaction between pri-miR-20a—a component of the oncomiR-1 cluster whose dysregulation is associated with numerous cancers—and heterogeneous nuclear ribonuclear protein A2B1 (hnRNPA2B1, A2B1). We find that A2B1 binds to a UAG motif within the apical loop of pri-miR-20a with moderate (∼6 μM) affinity. Interestingly, despite having two RNA recognition motifs (RRMs) capable of binding the UAG motif in isolation, A2B1 binds pri-miR-20a in a 1:1 stoichiometry. Additionally, small-angle X-ray scattering (SAXS) data best fits with a A2B1 engaged through a single RRM. Probing further, we show that mutations on A2B1 within RRM2 are deleterious to binding whereas mutation of RRM1 shows little effect. We additionally observe NMR chemical shift perturbations throughout both RRMs of A2B1 upon binding pri-miR-20a. Mutating the salt bridge interaction between RRMs, shown to be important for allostery in other hnRNPs, such as hnRNPA1, confers a loss of binding activity. Collectively, these results allude to potential structural/allosteric regulation across A2B1 RRMs which alter or regulate their RNA-binding capabilities.
Harkner et al. (Sun,) studied this question.