Department of Chemistry, College of Arts and Sciences, University of Alabama at Birmingham, CH266, 901 14th Street South, Birmingham, Alabama35294-1240, United States.
Biochemistry. 2022 Dec 20;61(24):2922-2932. doi: 10.1021/acs.biochem.2c00565. Epub 2022 Dec 1.
Ser/Arg-rich splicing factor 1 (SRSF1 or ASF/SF2) is the prototypical member of SR proteins. SRSF1 binds to exonic splicing enhancers, which prompts inclusion of corresponding exons in the mature mRNA. The RNA-binding domain of SRSF1 consists of tandem RNA-recognition motifs (RRM1 and RRM2) separated by a 30 amino acid long linker. In this study, we investigate roles of RRM1, RRM2, and the linker in RNA binding. We find that although both RRMs are crucial to RNA binding, RRM2 plays the dominant role. The linker mildly contributes to RNA binding and remains flexible in the RNA-bound state. Flexibility of the linker allows the RRM1-cognate motif to be either upstream or downstream of the RRM2-cognate motif. In addition, we find that the spacer length between the bipartite motifs varies from 0 to 10 nucleotides. Our binding assays reveal that SRSF1 prefers RNA sequences with shorter spacers and the RRM1-cognate motif being placed upstream. Restrained by nuclear magnetic resonance data, we simulate RNA-bound complexes and demonstrate how tandem RRMs bind to RNA of different spacer lengths and swapped bipartite motifs. We find that when the RRM1-cognate motif is placed downstream, either the RRM1/RRM2 linker needs to be more extended or RNA needs to form a U turn, which may reduce conformational entropy. Our study suggests that the RNA-binding specificity of SRSF1 is broader than traditionally recapitulated by consensus sequences of 7 to 8 nucleotides. Instead, centered on the RRM2-cognate motif, an RNA fragment encompassing 10-nucleotide upstream and downstream should be scrutinized.
丝氨酸/精氨酸丰富剪接因子 1（SRSF1 或 ASF/SF2）是 SR 蛋白的典型成员。SRSF1 与外显子剪接增强子结合，促使相应的外显子包含在成熟的 mRNA 中。SRSF1 的 RNA 结合结构域由串联的 RNA 识别基序（RRM1 和 RRM2）组成，中间由 30 个氨基酸长的连接子隔开。在本研究中，我们研究了 RRM1、RRM2 和连接子在 RNA 结合中的作用。我们发现，虽然两个 RRMs 对 RNA 结合都很重要，但 RRM2 起主导作用。连接子对 RNA 结合的贡献较小，但在 RNA 结合状态下保持灵活。连接子的灵活性允许 RRM1 识别基序位于 RRM2 识别基序的上游或下游。此外，我们发现二聚体基序之间的间隔长度从 0 到 10 个核苷酸不等。我们的结合实验表明，SRSF1 优先结合间隔较短且 RRM1 识别基序位于上游的 RNA 序列。受核磁共振数据的限制，我们模拟了 RNA 结合复合物，并演示了串联 RRMs 如何结合具有不同间隔和交换二聚体基序的 RNA。我们发现，当 RRM1 识别基序位于下游时，要么 RRM1/RRM2 连接子需要更长，要么 RNA 需要形成 U 形转弯，这可能会降低构象熵。我们的研究表明，SRSF1 的 RNA 结合特异性比传统的 7 到 8 个核苷酸的共识序列所概括的更广泛。相反，以 RRM2 识别基序为中心，应仔细研究包含上下游 10 个核苷酸的 RNA 片段。
