Electrostatic Interactions between CSTF2 and pre-mRNA Drive Cleavage and Polyadenylation.
Electrostatic Interactions between CSTF2 and pre-mRNA Drive Cleavage and Polyadenylation.
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DOI：
10.1016/j.bpj.2022.01.005
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发表时间：
2022-02-15
影响因子：
3.4
通讯作者：
Latham MP
中科院分区：
文献类型：
--
作者：
Masoumzadeh E;Grozdanov PN;Jetly A;MacDonald CC;Latham MP
新生前mRNA 3′-末端切割和多聚腺苷酸化（C/P）涉及许多识别多个RNA元件的蛋白质。人CSTF 2通过其RNA识别基序（RRM）调节C/P与下游富含U或G/U的序列结合。我们以前报道了唯一已知的疾病相关的CSTF 2 RRM突变体（CSTF 2D 50 A），并表明它改变了RNA结合的速率，导致携带相同突变的小鼠脑中的交替聚腺苷酸化。在这项研究中，我们进一步研究了静电相互作用的作用，在热力学和动力学的RNA结合的CSTF 2 RRM和下游的C/P调节的后果。通过结合诱变与NMR光谱和生物物理测定，我们证实，静电吸引力是RRM结合到一个天然存在的U-丰富的RNA序列的主导因素。此外，我们证明，RNA结合伴随着一个pico-nanosecond时间尺度RRM蛋白质动力学的变化所支持的熵补偿机制。我们认为，RRM的动态绑定到富含U的RNA支持它在细胞核中遇到的序列的多样性。最后，在体内C/P测定表明之间的竞争快速，高亲和力的RNA结合和有效的，正确的C/P。这些结果突出了RRM的表面电荷在RNA结合和新生的mRNA结合和C/P之间的平衡在体内的重要性。
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.
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