Alternative splicing factor hnRNPA1 induces U2AF2 association with Alu-derived RNA. Homo sapiens

Alternative splicing drives transcriptome and proteome diversification. While splicing regulatory proteins govern this process, their global mechanisms remain enigmatic. We generated high resolution transcriptome-wide protein-RNA interaction maps to determine how the splicing repressor hnRNPA1 influences the global association of spliceosome assembly factor U2AF2 and SRSF1 with pre-mRNA. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3’ splice sites of cassette exons but not constitutive exons upon hnRNPA1 overexpression. By contrast, SRSF1 crosslinking patterns relative to splice sites are independent of hnRNPA1 expression levels. We also observed an hnRNPA1-dependent increase in U2AF2 but not SRSF1 crosslinking to exon proximal antisense Alu elements. Thus Alu elements can serve as splicing factor-responsive sinks for U2AF2. These results not only demonstrate a novel mechanism for alternative splicing regulation but also implicate retrotransposon-derived sequences in the evolution of species-specific alternative splicing. Overall design: Perform iCLIP and RNA-seq from cells expressing endogenous or overexpressed levels of hnRNP A1. iCLIP was performed on hnRNP A1 and two splicing factors seen to be influenced by modulation of hnRNP A1 levels in vitro, SRSF1 and U2AF2. RNA-seq was performed to determine splicing changes between two conditions for comparison to changes seen between iCLIP datasets under same conditions. These changes are summarized globally, as well as at observed splicing changes, and on representative examples.

可变剪接 (Alternative splicing) 可驱动转录组与蛋白质组的多样化。尽管剪接调控蛋白掌控这一过程，但其全局作用机制仍不甚明晰。本研究构建了转录组全域的高分辨率蛋白-RNA互作图谱，以解析剪接阻遏蛋白异质性核核糖核蛋白A1 (hnRNPA1) 如何影响剪接体组装因子U2AF2与SRSF1与前体mRNA (pre-mRNA) 的全局结合情况。在异质性核核糖核蛋白A1过表达时，我们观察到U2AF2的交联位点在盒式外显子 (cassette exons) 的3’剪接位点 (3’ splice sites) 附近的分布发生改变，但组成型外显子 (constitutive exons) 并无此变化。与之相反，SRSF1相对于剪接位点的交联模式与异质性核核糖核蛋白A1的表达水平无关。我们还观察到，U2AF2与外显子近端反义Alu元件 (Alu elements) 的交联水平出现了异质性核核糖核蛋白A1依赖的升高，而SRSF1则无此现象。据此可知，Alu元件可作为U2AF2的剪接因子响应性结合储库。本研究结果不仅揭示了一种全新的可变剪接调控机制，还表明逆转录转座子 (retrotransposon) 衍生序列参与了物种特异性可变剪接的演化过程。
整体实验设计：从表达内源性或过表达水平异质性核核糖核蛋白A1的细胞中开展iCLIP (ultraviolet cross-linking immunoprecipitation) 与RNA测序 (RNA-seq)。针对异质性核核糖核蛋白A1，以及经体外实验证实受其水平调控的两种剪接因子SRSF1与U2AF2开展iCLIP实验。通过RNA测序测定两组实验条件下的剪接变化，以与相同条件下iCLIP数据集的变化进行对比分析。上述变化将从全局分布、已观测到的剪接改变位点以及典型示例三个层面进行总结。