Human CSTF2\nRNA Recognition Motif Domain Binds to\na U‑Rich RNA Sequence through a Multistep Binding Process

The RNA recognition motif (RRM) is a conserved and ubiquitous\nRNA-binding\ndomain that plays essential roles in mRNA splicing, polyadenylation,\ntransport, and stability. RRM domains exhibit remarkable diversity\nin binding partners, interacting with various sequences of single-\nand double-stranded RNA, despite their small size and compact fold.\nDuring pre-mRNA cleavage and polyadenylation, the RRM domain from\nCSTF2 recognizes U- or G/U-rich RNA sequences downstream from the\ncleavage and polyadenylation site to regulate the process. Given the\nimportance of alternative cleavage and polyadenylation in increasing\nthe diversity of mRNAs, the exact mechanism of binding of RNA to the\nRRM of CSTF2 remains unclear, particularly in the absence of a structure\nof this RRM bound to a native RNA substrate. Here, we performed a\nseries of NMR titration and spin relaxation experiments, which were\ncomplemented by paramagnetic relaxation enhancement measurements and\nrigid-body docking, to characterize the interactions of the CSTF2\nRRM with a U-rich ligand. Our results reveal a multistep binding process\ninvolving differences in ps-ns time scale dynamics and potential structural\nchanges, particularly in the C-terminalα-helix. These results\nprovide insights into how the CSTF2 RRM domain binds to U-rich RNA\nligands and offer a greater understanding for the molecular basis\nof the regulation of pre-mRNA cleavage and polyadenylation.