Browsing by Author "MacDonald, Clinton C. (TTUHSC)"
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Item A missense mutation in the CSTF2 gene that impairs the function of the RNA recognition motif and causes defects in 3' end processing is associated with intellectual disability in humans(2020) Grozdanov, Petar N. (TTUHSC); Masoumzadeh, Elahe (TTU); Kalscheuer, Vera M.; Bienvenu, Thierry; Billuart, Pierre; Delrue, Marie Ange; Latham, Michael P. (TTU); MacDonald, Clinton C. (TTUHSC)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.Item Electrostatic Interactions between CSTF2 and pre-mRNA Drive Cleavage and Polyadenylation(2022) Masoumzadeh, Elahe (TTU); Grozdanov, Petar N. (TTUHSC); Jetly, Anushka (TTU); MacDonald, Clinton C. (TTUHSC); Latham, Michael P. (TTU)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.Item Specialized Ribosomes in Health and Disease(2023) Miller, Sarah C. (TTUHSC); MacDonald, Clinton C. (TTUHSC); Kellogg, Morgana K. (TTUHSC); Karamysheva, Zemfira N. (TTU); Karamyshev, Andrey L. (TTUHSC)Ribosomal heterogeneity exists within cells and between different cell types, at specific developmental stages, and occurs in response to environmental stimuli. Mounting evidence supports the existence of specialized ribosomes, or specific changes to the ribosome that regulate the translation of a specific group of transcripts. These alterations have been shown to affect the affinity of ribosomes for certain mRNAs or change the cotranslational folding of nascent polypeptides at the exit tunnel. The identification of specialized ribosomes requires evidence of the incorporation of different ribosomal proteins or of modifications to rRNA and/or protein that lead(s) to physiologically relevant changes in translation. In this review, we summarize ribosomal heterogeneity and specialization in mammals and discuss their relevance to several human diseases.Item The structural basis of CstF-77 modulation of cleavage and polyadenylation through stimulation of CstF-64 activity(2018) Grozdanov, Petar N. (TTUHSC); Masoumzadeh, Elahe (TTU); Latham, Michael P. (TTU); MacDonald, Clinton C. (TTUHSC)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 RNAbinding 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 carboxyterminus 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 (monkeytailcarboxy- 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.