tRIP-seq reveals repression of premature polyadenylation by co-transcriptional FUS-U1 snRNP assembly
tRIP-seq reveals repression of premature polyadenylation by co-transcriptional FUS-U1 snRNP assembly
复制标题
DOI：
10.15252/embr.201949890
复制
发表时间：
2020-03-18
期刊：
影响因子：
7.7
通讯作者：
Ohno, Kinji
中科院分区：
文献类型：
--
作者：
Masuda, Akio;Kawachi, Toshihiko;Ohno, Kinji
RNA 加工是通过将 RNA 加工因子动态招募到 RNA 聚合酶 II (RNAPII) 来进行共转录的。然而，在转录组范围内鉴定在转录 RNAPII 上专门组装的蛋白质-RNA 相互作用具有挑战性。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 结合与 RNAPII 结合的新生 RNA 的替代多腺苷酸化 (APA) 位点的上游，从而延迟 RNAPII 并抑制 CPSF 对多腺苷酸化信号的识别。进一步的 tRIP-seq 分析表明，APA 的抑制是通过 RNAPII 上的 FUS 和 U1 snRNP 组成的复合物实现的，而不是单独通过其中任何一个来实现。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。
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.