RNA processing occurs co-transcriptionally through the dynamic recruitment of RNA processing factors to RNA polymerase II (RNAPII). However, transcriptome-wide identification of protein-RNA interactions specifically assembled on transcribing RNAPII is challenging. Here, we develop the targeted RNA immunoprecipitation sequencing (tRIP-seq) method that detects protein-RNA interaction sites in thousands of cells. The high sensitivity of tRIP-seq enables identification of protein-RNA interactions at functional subcellular levels. Application of tRIP-seq to the FUS-RNA complex in the RNAPII machinery reveals that FUS binds upstream of alternative polyadenylation (APA) sites of nascent RNA bound to RNAPII, which retards RNAPII and suppresses the recognition of the polyadenylation signal by CPSF. Further tRIP-seq analyses demonstrate that the repression of APA is achieved by a complex composed of FUS and U1 snRNP on RNAPII, but not by either one alone. Moreover, our analysis reveals that FUS mutations in familial amyotrophic lateral sclerosis (ALS) that impair the FUS-U1 snRNP interaction aberrantly activate the APA sites. tRIP-seq provides new insights into the regulatory mechanism of co-transcriptional RNA processing by RNA processing factors.

中文翻译：

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tRIP-seq 揭示了通过共转录 FUS-U1 snRNP 组装抑制过早多聚腺苷酸化。

RNA 加工通过将 RNA 加工因子动态募集到 RNA 聚合酶 II (RNAPII) 进行共转录。然而，在转录 RNAPII 上专门组装的蛋白质-RNA 相互作用的全转录组鉴定具有挑战性。在这里，我们开发了靶向 RNA 免疫沉淀测序 (tRIP-seq) 方法，可检测数千个细胞中的蛋白质-RNA 相互作用位点。tRIP-seq 的高灵敏度能够在功能性亚细胞水平上识别蛋白质-RNA 相互作用。将 tRIP-seq 应用于 RNAPII 机制中的 FUS-RNA 复合物表明，FUS 结合到与 RNAPII 结合的新生 RNA 的替代多聚腺苷酸化 (APA) 位点的上游，这会延迟 RNAPII 并抑制 CPSF 对多聚腺苷酸化信号的识别。进一步的 tRIP-seq 分析表明，APA 的抑制是由 RNAPII 上的 FUS 和 U1 snRNP 组成的复合物实现的，但不能单独通过任何一个来实现。此外，我们的分析表明，家族性肌萎缩侧索硬化症 (ALS) 中的 FUS 突变会损害 FUS-U1 snRNP 相互作用，从而异常激活 APA 位点。tRIP-seq 为 RNA 加工因子对共转录 RNA 加工的调控机制提供了新的见解。