与环状RNA和RNA G-四链体相关的蛋白质的原位邻近标记

In situ proximity labeling of proteins associated with circRNA and RNA G-quadruplexes.

作者信息

Mo Jing, Chen Zonggui, Cui Manman, Fang Xin, Li Ruihan, Qin Shanshan, Zhou Zhenlin, Sun Mei, Wu Jinjun, He Chuan, Wang Fang, Zhang Haojian, Zhou Xiang, Weng Xiaocheng

机构信息

State Key Laboratory of Metabolism and Regulation in Complex Organisms, College of Chemistry and Molecule Sciences, Taikang Center for Life and Medical Sciences, Department of Otorhinolaryngology-Head and Neck Surgery, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China.

Department of Chemistry, Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA.

出版信息

Nat Chem Biol. 2025 Aug 7. doi: 10.1038/s41589-025-01993-2.

DOI:10.1038/s41589-025-01993-2

PMID:40775047

Abstract

Profiling the protein interactome of specific RNA loci aids in understanding the molecular mechanisms of regulatory RNA. However, current RNA-centric methods have sufficient space for improvement in terms of efficiency and biocompatibility. Here we developed TurboID-assisted proximity labeling of targeted RNA-interacting proteins (TAPRIP), in which the proximity labeling enzyme miniTurbo is attached to an RNA-targeting element, CIRTS3, to label the targeted RNA-interacting proteins, which are then analyzed by mass spectrometry. We profiled the interactome of mCherry circular RNA (circRNA) and found that HNRNPK modulates the expression of mCherry circRNA and other endogenous circRNAs by binding their flanking introns. Targeting the BCL2 RNA G-quadruplex structure, we found that RBM12 promotes BCL2 translation by binding RNA G-quadruplexes and recruiting ribosomes. RBM12 knockdown markedly reduced proliferation and clonogenicity in MOLM-13, MV4-11 and THP-1 acute myeloid leukemia cells. These findings established the foundation for a versatile technique for analyzing the protein interactome of specific RNA sequences in live cells.

摘要

分析特定RNA位点的蛋白质相互作用组有助于理解调控RNA的分子机制。然而，目前以RNA为中心的方法在效率和生物相容性方面仍有很大的改进空间。在此，我们开发了TurboID辅助的靶向RNA相互作用蛋白的邻近标记（TAPRIP），其中邻近标记酶miniTurbo连接到一个RNA靶向元件CIRTS3上，以标记靶向RNA相互作用蛋白，然后通过质谱分析这些蛋白。我们分析了mCherry环状RNA（circRNA）的相互作用组，发现HNRNPK通过结合其侧翼内含子来调节mCherry circRNA和其他内源性circRNA的表达。靶向BCL2 RNA G-四链体结构，我们发现RBM12通过结合RNA G-四链体并招募核糖体来促进BCL2的翻译。RBM12的敲低显著降低了MOLM-13、MV4-11和THP-1急性髓系白血病细胞的增殖和克隆形成能力。这些发现为一种用于分析活细胞中特定RNA序列的蛋白质相互作用组的通用技术奠定了基础。