Department of Chemistry, College of Arts and Sciences, University of Alabama at Birmingham, CH266, 901 14th Street South, Birmingham, AL 35294-1240, USA.
Nucleic Acids Res. 2024 May 8;52(8):4676-4690. doi: 10.1093/nar/gkae213.
SRSF1 governs splicing of over 1500 mRNA transcripts. SRSF1 contains two RNA-recognition motifs (RRMs) and a C-terminal Arg/Ser-rich region (RS). It has been thought that SRSF1 RRMs exclusively recognize single-stranded exonic splicing enhancers, while RS lacks RNA-binding specificity. With our success in solving the insolubility problem of SRSF1, we can explore the unknown RNA-binding landscape of SRSF1. We find that SRSF1 RS prefers purine over pyrimidine. Moreover, SRSF1 binds to the G-quadruplex (GQ) from the ARPC2 mRNA, with both RRMs and RS being crucial. Our binding assays show that the traditional RNA-binding sites on the RRM tandem and the Arg in RS are responsible for GQ binding. Interestingly, our FRET and circular dichroism data reveal that SRSF1 unfolds the ARPC2 GQ, with RS leading unfolding and RRMs aiding. Our saturation transfer difference NMR results discover that Arg residues in SRSF1 RS interact with the guanine base but not other nucleobases, underscoring the uniqueness of the Arg/guanine interaction. Our luciferase assays confirm that SRSF1 can alleviate the inhibitory effect of GQ on gene expression in the cell. Given the prevalence of RNA GQ and SR proteins, our findings unveil unexplored SR protein functions with broad implications in RNA splicing and translation.
SRSF1 调控超过 1500 个 mRNA 转录本的剪接。SRSF1 含有两个 RNA 识别基序 (RRMs) 和一个 C 端富含精氨酸/丝氨酸的区域 (RS)。人们一直认为 SRSF1 的 RRMs 专门识别单链外显子剪接增强子，而 RS 缺乏 RNA 结合特异性。由于我们成功解决了 SRSF1 的不溶性问题，我们可以探索 SRSF1 未知的 RNA 结合景观。我们发现 SRSF1 RS 偏爱嘌呤而不是嘧啶。此外，SRSF1 与 ARPC2 mRNA 的 G-四链体 (GQ) 结合，RRMs 和 RS 都很重要。我们的结合实验表明，RRM 串联上的传统 RNA 结合位点和 RS 中的精氨酸负责 GQ 结合。有趣的是，我们的 FRET 和圆二色性数据显示，SRSF1 使 ARPC2 GQ 展开，RS 起主导作用，RRMs 辅助展开。我们的饱和转移差异 NMR 结果发现，SRSF1 RS 中的精氨酸残基与鸟嘌呤碱基相互作用，但不与其他核碱基相互作用，突出了精氨酸-鸟嘌呤相互作用的独特性。我们的荧光素酶实验证实，SRSF1 可以减轻 GQ 对细胞中基因表达的抑制作用。鉴于 RNA GQ 和 SR 蛋白的普遍性，我们的发现揭示了未被探索的 SR 蛋白功能，这对 RNA 剪接和翻译具有广泛的影响。
