Contributions of Folded and Disordered Domains to RNA Binding by HNRNPR.

RNA binding proteins (RBPs) interact with and tightly regulate the fate of messenger RNAs, but how RNA targets are recognized remains a challenging question. RBPs often contain multiple domains known to directly bind RNA, such as RNA recognition motifs (RRMs), as well as domains whose RNA binding capacity remains incompletely understood, e.g., low complexity domains (LCDs). Here, we dissect HNRNPR, an RBP with three RRMs and an arginine-glycine rich (RG-rich) LCD. We apply unbiased high-throughput biochemical approaches and identify critical RNA binding domains that confer specificity towards AU-rich RNA sequences. We show that not all RRMs contribute equally to binding and find that RRM3, along with a downstream C-terminal charged region, is required for RNA binding. HNRNPR also binds RNA G-quadruplexes (rG4s) and contains multiple rG4 binding sites including the C-terminal charged region within RRM3 and RG-rich regions within the LCD. We dissect rG4 specificity for the full length HNRNPR and LCD using a newly created RNA pool focused on rG4s, reveal that binding is dependent on RNA folding, and find specific rG4 features that enhance HNRNPR-rG4 interactions. Our work highlights the complex interplay of folded and disordered regions within RBPs as mediators of RNA binding.