High-throughput interaction proteome mapping revealed that U6 snRNA multi-dimensionally regulates mRNA processing and maturation in tumor cells

doi:10.3969/j.issn.1009-976X.2022.01.007

Abstract

Abstract: Objective We purified endogenous U6 snRNA （U6 for short） interaction protein from tumor cells by ChIRP technique, and identified the interaction proteome by high sensitivity liquid chromatography-mass spectrometry, systematically revealing the new biological function and molecular regulation mechanism of U6 in tumor cells. Methods A biotin-labeled U6-specific probe was synthesized, and the complex formed by formaldehyde crosslinking U6 and interacting protein was captured by molecular hybridization, and the U6 interacting proteome was identified by high sensitivity mass spectrometry. Bioinformatics analysis was used to analyze the function of U6 in tumor cells. Results 135 specific endogenous U6 interacting proteins were identified through rigorous screening. GO, KEGG, protein complex enrichment analysis and PPI analysis showed that U6 was involved in a series of biological processes from mRNA production to post-transcriptional regulation in addition to interacting with related proteins such as mRNA precursor splicing processing. U6 was also involved in the transcription regulation of mRNA, vesicle transport, nucleation transport of mature mRNA, mRNA degradation, RNA localization, post-transcriptional regulation and other processes. Conclusion Our study systematically revealed the interaction proteome map of U6 in tumor cells for the first time in the world, and the analysis of the map showed that U6 was multidimensional in the whole process of mRNA biological regulation, providing a new direction and idea for the study of the function and mechanism of U6 in tumor development.

Key words: Tumor, ChIRP-MS, U6 snRNA, Splicing

摘要： 目的采用ChIRP技术纯化肿瘤细胞中内源U6 snRNA（简称U6）互作蛋白,结合高灵敏度液相色谱-质谱联用技术对所获得的互作蛋白组进行鉴定,系统揭示U6参与肿瘤细胞中新的生物学功能和分子调控机制。方法合成含生物素标记的U6特异性探针,利用分子杂交技术特异捕获经甲醛交联的内源U6与互作蛋白形成的复合物,并结合高灵敏度质谱分析技术鉴定出U6互作蛋白组。利用生物信息分析手段分析U6在肿瘤细胞中的功能。结果通过严格的筛选本研究最终鉴定到135个特异的内源U6互作蛋白。GO、KEGG、蛋白复合体富集分析和PPI分析显示U6除了与mRNA前体剪接加工等相关蛋白发生相互作用外,还参与了mRNA从产生到转录后调控等一系列生物学过程,包括参与mRNA转录调控、囊泡运输、成熟mRNA的出核转运、mRNA降解、RNA定位、转录后调控等过程。结论我们的研究在国际上首次系统揭示了肿瘤细胞U6的相互作用蛋白组图谱,通过对该图谱的分析表明U6多维度地参与了mRNA生物学发生的全过程调控,为U6参与肿瘤发生发展的功能和机制研究提供了全新的方向和思路。

关键词: 肿瘤, ChIRP-MS, U6 snRNA, 剪接

CLC Number:

R73-3

YANG Zhi-Zhi, YANG Bing, TIAN Bin, LIAO Jian-you. High-throughput interaction proteome mapping revealed that U6 snRNA multi-dimensionally regulates mRNA processing and maturation in tumor cells[J]. Lingnan Modern Clinics In Surgery, 2022, 22(01): 42-50.

杨芝芝, 杨兵, 田彬, 廖建友. 高通量互作蛋白组图谱研究揭示U6 snRNA在肿瘤细胞中多维度调控mRNA加工成熟过程[J]. 岭南现代临床外科, 2022, 22(01): 42-50.

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