Electrostatic Interactions between CSTF2 and pre-mRNA Drive Cleavage and Polyadenylation

Elahe Masoumzadeh, Petar N. Grozdanov, Anushka Jetly, Clinton C. MacDonald, Michael P. Latham

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

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 (CSTF2D50A) 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.

Original languageEnglish (US)
Pages (from-to)607-619
Number of pages13
JournalBiophysical journal
Volume121
Issue number4
DOIs
StatePublished - Feb 15 2022
Externally publishedYes

Bibliographical note

Funding Information:
We would like to thank Marella Canny (TTU) and Dr. Shiva Moaven (TTU) for technical and intellectual contributions. This work was supported by NIH grant 1R35GM128906 (M.P.L.); the Presidents' Collaborative Research Initiative of Texas Tech University System (P.N.G. C.C.M, and M.P.L.); the Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center (P.N.G. and C.C.M.); South Plains Foundation (P.N.G. and C.C.M.); and the Welch Summer Scholars Program (A.J.).

Funding Information:
We would like to thank Marella Canny (TTU) and Dr. Shiva Moaven (TTU) for technical and intellectual contributions. This work was supported by NIH grant 1R35GM128906 (M.P.L.); the Presidents' Collaborative Research Initiative of Texas Tech University System (P.N.G., C.C.M, and M.P.L.); the Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center (P.N.G. and C.C.M.); South Plains Foundation (P.N.G. and C.C.M.); and the Welch Summer Scholars Program (A.J.).

Publisher Copyright:
© 2022 Biophysical Society

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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