Center for RNA Research, Institute for Basic Science, Seoul, 08826, Korea.
School of Biological Sciences, Seoul National University, Seoul, 08826, Korea.
Nat Commun. 2021 Oct 15;12(1):6026. doi: 10.1038/s41467-021-26317-5.
RNA-protein interaction can be captured by crosslinking and enrichment followed by tandem mass spectrometry, but it remains challenging to pinpoint RNA-binding sites (RBSs) or provide direct evidence for RNA-binding. To overcome these limitations, we here developed pRBS-ID, by incorporating the benefits of UVA-based photoactivatable ribonucleoside (PAR; 4-thiouridine and 6-thioguanosine) crosslinking and chemical RNA cleavage. pRBS-ID robustly detects peptides crosslinked to PAR adducts, offering direct RNA-binding evidence and identifying RBSs at single amino acid-resolution with base-specificity (U or G). Using pRBS-ID, we could profile uridine-contacting RBSs globally and discover guanosine-contacting RBSs, which allowed us to characterize the base-specific interactions. We also applied the search pipeline to analyze the datasets from UVC-based RBS-ID experiments, altogether offering a comprehensive list of human RBSs with high coverage (3,077 RBSs in 532 proteins in total). pRBS-ID is a widely applicable platform to investigate the molecular basis of posttranscriptional regulation.
RNA-蛋白质相互作用可以通过交联和富集,然后进行串联质谱分析来捕获,但确定 RNA 结合位点(RBS)或提供 RNA 结合的直接证据仍然具有挑战性。为了克服这些限制,我们在这里开发了 pRBS-ID,它结合了基于 UVA 的光活化核苷酸(PAR;4-硫代尿嘧啶和 6-硫代鸟嘌呤)交联和化学 RNA 切割的优点。pRBS-ID 可以可靠地检测与 PAR 加合物交联的肽,提供直接的 RNA 结合证据,并以单个氨基酸分辨率识别具有碱基特异性(U 或 G)的 RBS。使用 pRBS-ID,我们可以全局分析尿嘧啶接触的 RBS,并发现与鸟嘌呤接触的 RBS,这使我们能够描述碱基特异性相互作用。我们还应用搜索管道分析基于 UVC 的 RBS-ID 实验的数据集,总共提供了具有高覆盖率的全面人类 RBS 列表(总共 532 个蛋白质中有 3077 个 RBS)。pRBS-ID 是一个广泛适用的平台,可以研究转录后调控的分子基础。