Structure and dynamics of CstF-64 RNA recognition motif drive cleavage and polyadenylation



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RNA-binding proteins are one of the most abundant types of protein family in the cell, and they are crucial for cell survival. Protein-RNA interactions have important roles in the regulation of gene expression and post-transcriptional processes in all eukaryotes, such as splicing regulation, mRNA transport, and modulation of mRNA translation. Cleavage and polyadenylation (C/P) of mRNA is an important cellular processes which occurs to the nascent mRNA promoting increased diversity of mRNA isoforms. The cleavage stimulation factor (CstF) complex is part of the C/P machinery and includes three protein subunits: CstF-64, CstF-77, and CstF-50. CstF binds to U- and G/U-rich sequences located downstream from the cleavage site through its RNA-binding subunit, CstF-64. Less is known about the function of the other two subunits of CstF; however, the CstF complex has a hexameric architecture, consisting of a dimer of two trimeric CstF oligomers, which seems to play an important role in the recognition of the downstream elements. CSTF2 encodes an RNA-binding protein (CstF-64 in human) that is essential for mRNA cleavage and polyadenylation (C/P). No disease-related mutations had been described for this gene. Here, we reported a mutation in the RNA recognition motif (RRM) of CstF-64 that changes an aspartic acid at position 50 to alanine, resulting in intellectual disability in male patients. This mutation was sufficient to alter polyadenylation sites in over 1300 genes critical for brain development in mice. To account for this, we determined that the D50A mutation changed the position of amino acid side chains altering RNA binding sites in the RRM. Side chain repositioning upon the D50A mutation modified the electrostatic potential of the RRM leading to a greater affinity for RNA. With the use of fast (psec – nsec) timescale Nuclear Magnetic Resonance (NMR) relaxation experiments, we observed motions for the RNA-free and -bound states of the RRM, which underlines the necessity of studying the RNA binding mechanism for both wildtype and D50A mutated RRM. Next, we investigated the role of electrostatic interactions in the thermodynamics and kinetics of RNA-binding by the RRM domain of CstF-64. By combining mutagenesis with biophysical assays and NMR spectroscopy, we confirmed that electrostatic attraction is the dominant factor in the RRM binding to a G/U-rich RNA sequence. Moreover, we demonstrated that RNA-binding is accompanied by an enthalpy-entropy compensation mechanism supported by changes in fast timescale RRM protein dynamics. We hypothesized that the global surface charge has an essential role in RNA binding during the C/P in vivo. In addition to the canonical RNA-binding motifs, it appears that conformational changes to the C-terminal -helix of the RRM domain, which has been shown to play an important role in RNA binding in other RRM contain proteins, also functions in RNA binding within CstF-64. Although the CstF-64 RRM is a well-studied protein, the mechanism for how the RRM achieves preference for G/U- or U-rich sequences and the role of conformational changes within the domain lacks because of the absence of RRM-RNA complex structural models. To address the ambiguity of RNA binding in CstF-64 RRM, we employed NMR spectroscopy and docking techniques to model the CstF-64 RRM structure bound to a G/U rich 16-mer RNA. Instead of generating a single traditional structure, we proposed an ensemble of structures for the complex selected based on their correlation to the experimental restraints. Having an ensemble of conformers represents a more natural RNA-bound state and allows us to look at the binding mechanisms in a more dynamic way. Our data proposed a new RNA binding function for RRM loops. In addition, the ensemble model confirmed that the conformational changes within C-terminal -helix upon RNA binding are related to RNA binding. The mechanisms by which CstF-64 and CstF-77 cooperate to regulate cleavage and polyadenylation were the subject of our next study. One role of CstF-77 in the complex is to act as a scaffold linking CstF-64 and CstF-50 to the cleavage and polyadenylation specificity factor CPSF. CstF-77 consist of twelve half-a-tetratricopeptide repeats (HAT) domains and a proline-rich protein-protein interaction domain called the “monkeytail”. The monkeytail has been shown to interact with the Hinge domain of CstF-64. To investigate the molecular details of the CstF 64-77 domain interactions, we turned to NMR spectroscopy and Small-Angle Xray Scattering (SAXS) to propose a structural model of the complex. Although our data showed that the C-Terminal Domain (CTD) of CstF-77 does not interact with the RRM domain of CstF-64 and does not directly bind to the mRNA, it affects the RNA binding by partially blocking the RRM binding sites. Therefore, calculating the structure of the CstF complex structure provides a better picture of the RNA-binding mechanism in CstF RRM. Moreover, solving the structure of the CstF complex by NMR offers a great opportunity to study the dynamics of RNA binding and interaction within the CstF domains. Overall, this study provides deeper insights into the structure and function of the CstF protein complex and how these structures and functions regulate the cleavage and polyadenylation process in the cell.

Embargo status: Restricted until 06/2022. To request the author grant access, click on the PDF link to the left.



RNA-binding, CstF-64, Structure, NMR Spectroscopy