Texas Tech University, Lubbock, Texas.
Texas Tech University Health Sciences Center, Lubbock, Texas.
Biophys J. 2022 Feb 15;121(4):607-619. doi: 10.1016/j.bpj.2022.01.005. Epub 2022 Jan 26.
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) 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.
初生前体 mRNA 3'端切割和多聚腺苷酸化 (C/P) 涉及许多蛋白质，这些蛋白质可以识别多种 RNA 元件。人类 CSTF2 通过其 RNA 识别基序 (RRM) 结合到下游 U 或 U/G 丰富序列，从而调节 C/P。我们之前报道了唯一已知的 CSTF2 疾病相关 RRM 突变体（CSTF2），并表明它改变了 RNA 结合的初始速率，导致携带相同突变的小鼠大脑中的选择性多聚腺苷酸化。在这项研究中，我们进一步研究了静电相互作用在 CSTF2 RRM 的 RNA 结合热力学和动力学中的作用，以及对 C/P 调节的下游影响。通过结合突变体分析与 NMR 光谱和生物物理测定，我们证实静电吸引是 RRM 与天然存在的 U 丰富 RNA 序列结合的主要因素。此外，我们证明 RNA 结合伴随着焓熵补偿机制，这得到了皮秒到纳秒时间尺度 RRM 蛋白动力学变化的支持。我们认为，RRM 与富含 U 的 RNA 的动态结合支持了它在核内遇到的序列多样性。最后，体内 C/P 测定证明了快速、高亲和力的 RNA 结合与有效、正确的 C/P 之间的竞争。这些结果突出了 RRM 的表面电荷在 RNA 结合中的重要性，以及在体内新生 mRNA 结合和 C/P 之间的平衡。
