Human CSTF2 RNA Recognition Motif Domain Binds to a U-Rich RNA Sequence through a Multistep Binding Process

The RNA recognition motif (RRM) is a conserved and ubiquitous RNA-binding domain that plays essential roles in mRNA splicing, polyadenylation, transport, and stability. RRM domains exhibit remarkable diversity in binding partners, interacting with various sequences of single- and double-stranded RNA, despite their small size and compact fold. During pre-mRNA cleavage and polyadenylation, the RRM domain from CSTF2 recognizes U- or G/U-rich RNA sequences downstream from the cleavage and polyadenylation site to regulate the process. Given the importance of alternative cleavage and polyadenylation in increasing the diversity of mRNAs, the exact mechanism of binding of RNA to the RRM of CSTF2 remains unclear, particularly in the absence of a structure of this RRM bound to a native RNA substrate. Here, we performed a series of NMR titration and spin relaxation experiments, which were complemented by paramagnetic relaxation enhancement measurements and rigid-body docking, to characterize the interactions of the CSTF2 RRM with a U-rich ligand. Our results reveal a multistep binding process involving differences in ps-ns time scale dynamics and potential structural changes, particularly in the C-terminal alpha-helix. These results provide insights into how the CSTF2 RRM domain binds to U-rich RNA ligands and offer a greater understanding for the molecular basis of the regulation of pre-mRNA cleavage and polyadenylation.