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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-terminalα-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.

Proline isomerization is widely recognized as a kinetic bottleneck in protein folding, amplified for proteins rich in Pro residues. We introduced repeated hydrostatic pressure jumps between native and pressure-denaturing conditions inside an NMR sample cell to study proline isomerization in the pressure-sensitized L50A ubiquitin mutant. Whereas in two unfolded heptapeptides, X-Pro peptide bonds isomerized ca 1.6-fold faster at 1 bar than at 2.5 kbar, for ubiquitin ca eight-fold faster isomerization was observed for Pro-38 and ca two-fold for Pro-19 and Pro-37 relative to rates measured in the pressure-denatured state. Activation energies for isomerization in pressure-denatured ubiquitin were close to literature values of 20 kcal/mole for denatured polypeptides but showed a substantial drop to 12.7 kcal/mole for Pro-38 at atmospheric pressure. For ubiquitin isomers with a cis E18-P19 peptide bond, the 1-bar NMR spectrum showed sharp resonances with near random coil chemical shifts for the C-terminal half of the protein, characteristic of an unfolded chain, while most of the N-terminal residues were invisible due to exchange broadening, pointing to a metastable partially folded state for this previously recognized 'folding nucleus'. For cis-P37 isomers, a drop in pressure resulted in the rapid loss of nearly all unfolded-state NMR resonances, while the recovery of native state intensity revealed a slow component attributed to cis → trans isomerization of P37. This result implies that the NMR-invisible cis-P37 isomer adopts a molten globule state that encompasses the entire length of the ubiquitin chain, suggestive of a structure that mostly resembles the folded state., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Charged residues on the surface of proteins are critical for both protein stability and interactions. However, many proteins contain binding regions with a high net charge that may destabilize the protein but are useful for binding to oppositely charged targets. We hypothesized that these domains would be marginally stable, as electrostatic repulsion would compete with favorable hydrophobic collapse during folding. Furthermore, by increasing the salt concentration, we predict that these protein folds would be stabilized by mimicking some of the favorable electrostatic interactions that take place during target binding. We varied the salt and urea concentrations to probe the contributions of electrostatic and hydrophobic interactions for the folding of the yeast SH3 domain found in Abp1p. The SH3 domain was significantly stabilized with increased salt concentrations due to Debye-Huckel screening and a nonspecific territorial ion-binding effect. Molecular dynamics and NMR show that sodium ions interact with all 15 acidic residues but do little to change backbone dynamics or overall structure. Folding kinetics experiments show that the addition of urea or salt primarily affects the folding rate, indicating that almost all the hydrophobic collapse and electrostatic repulsion occur in the transition state. After the transition state formation, modest yet favorable short-range salt bridges are formed along with hydrogen bonds, as the native state fully folds. Thus, hydrophobic collapse offsets electrostatic repulsion to ensure this highly charged binding domain can still fold and be ready to bind to its charged peptide targets, a property that is likely evolutionarily conserved over 1 billion years., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Autoimmunity develops when extracellular DNA released from dying cells is not cleared from serum. While serum DNA is primarily digested by Dnase1 and Dnase1L3, Dnase1 cannot rescue autoimmunity arising from Dnase1L3 deficiencies. Dnase1L3 uniquely degrades antigenic forms of cell-free DNA, including DNA complexed with lipids and proteins. The distinct activity of Dnase1L3 relies on its unique C-terminal Domain (CTD), but the mechanism is unknown. We used multiple biophysical techniques and functional assays to study the interplay between the core catalytic domain and the CTD. While the core domain resembles Dnase1, there are key structural differences between the two enzymes. First, Dnase1L3 is not inhibited by actin due to multiple differences in the actin recognition site. Second, the CTD augments the ability of the core to bind DNA, thereby facilitating the degradation of complexed DNA. Together, these structural insights will inform the development of Dnase1L3-based therapies for autoimmunity., (© 2022. The Author(s).)

Nascent pre-mRNA 3'-end cleavage and polyadenylation (C/P) involves numerous proteins that recognize multiple RNA elements. Human CSTF2 binds to a downstream U- or G/U-rich sequence through its RNA recognition motif (RRM) regulating C/P. We previously reported the only known disease-related CSTF2 RRM mutant (CSTF2 D50A ) and showed that it changed the on-rate of RNA binding, leading to alternative polyadenylation in brains of mice carrying the same mutation. In this study, we further investigated the role of electrostatic interactions in the thermodynamics and kinetics of RNA binding for the CSTF2 RRM and the downstream consequences for regulation of C/P. By combining mutagenesis with NMR spectroscopy and biophysical assays, we confirmed that electrostatic attraction is the dominant factor in RRM binding to a naturally occurring U-rich RNA sequence. Moreover, we demonstrate that RNA binding is accompanied by an enthalpy-entropy compensation mechanism that is supported by changes in pico-to-nanosecond timescale RRM protein dynamics. We suggest that the dynamic binding of the RRM to U-rich RNA supports the diversity of sequences it encounters in the nucleus. Lastly, in vivo C/P assays demonstrate a competition between fast, high affinity RNA binding and efficient, correct C/P. These results highlight the importance of the surface charge of the RRM in RNA binding and the balance between nascent mRNA binding and C/P in vivo., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

CSTF2 encodes an RNA-binding protein that is essential for mRNA cleavage and polyadenylation (C/P). No disease-associated mutations have been described for this gene. Here, we report a mutation in the RNA recognition motif (RRM) of CSTF2 that changes an aspartic acid at position 50 to alanine (p.D50A), resulting in intellectual disability in male patients. In mice, this mutation was sufficient to alter polyadenylation sites in over 1300 genes critical for brain development. Using a reporter gene assay, we demonstrated that C/P efficiency of CSTF2D50A was lower than wild type. To account for this, we determined that p.D50A changed locations of amino acid side chains altering RNA binding sites in the RRM. The changes modified the electrostatic potential of the RRM leading to a greater affinity for RNA. These results highlight the significance of 3' end mRNA processing in expression of genes important for brain plasticity and neuronal development., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cleavage and polyadenylation (C/P) of mRNA is an important cellular process that promotes increased diversity of mRNA isoforms and could change their stability in different cell types. The cleavage stimulation factor (CstF) complex, part of the C/P machinery, binds to U- and GU-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, CstF-77 and CstF-50. Here, we show that the carboxy-terminus of CstF-77 plays a previously unrecognized role in enhancing C/P by altering how the RNA recognition motif (RRM) of CstF-64 binds RNA. In support of this finding, we also show that CstF-64 relies on CstF-77 to be transported to the nucleus; excess CstF-64 localizes to the cytoplasm, possibly via interaction with cytoplasmic RNAs. Reverse genetics and nuclear magnetic resonance studies of recombinant CstF-64 (RRM-Hinge) and CstF-77 (monkeytail-carboxy-terminal domain) indicate that the last 30 amino acids of CstF-77 increases the stability of the RRM, thus altering the affinity of the complex for RNA. These results provide new insights into the mechanism by which CstF regulates the location of the RNA cleavage site during C/P.