Time-resolved profiling of RNA binding proteins throughout the mRNA life cycle. Time-resolved profiling of RNA binding proteins throughout the mRNA life cycle

mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNP remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA-binding proteins (RBPs) across ten distinct time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed numerous RBPs with unexpected dynamics: the TRanscription-EXport complex (TREX) recruited posttranscriptionally after NXF1 binding, challenging the current view of transcription-coupled mRNA export mechanism, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with GO annotations. Our data can be explored at chronology.rna.snu.ac.kr. Overall design: Pulse-chased mRNA analysis using 10 min 4sU labeling and MTS-biotin pulldown

信使RNA（mRNA）在其整个生命周期中会持续与其结合的蛋白质伴侣发生动态更替，但目前我们对mRNA-蛋白质复合物（mRNP, messenger ribonucleoprotein）重塑过程的认知，受限于时序数据的缺失。本研究通过光活化核糖核苷脉冲代谢标记、UVA交联、聚腺苷酸化RNA（poly(A)+ RNA）分离以及质谱分析，获取了具备时序分辨率的mRNA互作组数据集。该纵向研究方案可在10个不同时间点对700余种RNA结合蛋白（RNA-binding protein, RBP）完成定量检测。总体而言，mRNA结合的时序顺序与已知的蛋白质功能、亚细胞定位及分子互作模式高度吻合。然而本研究也发现了众多呈现出意外动态变化的RNA结合蛋白：例如转录-输出复合体（Transcription-EXport complex, TREX）会在NXF1结合后以转录后方式被招募至mRNA复合物，这一发现挑战了当前关于转录偶联mRNA输出机制的主流认知；而应激颗粒（stress granule）蛋白在衰老的mRNA-蛋白质复合物中富集，提示其在mRNA生命周期的晚期阶段发挥功能。为系统性地鉴定功能未知的mRNA结合型RNA结合蛋白（mRBPs），本研究采用机器学习方法，将mRNA结合动态数据与基因本体注释（Gene Ontology annotations, GO annotations）进行比对分析。本研究的数据集可通过chronology.rna.snu.ac.kr网站进行检索浏览。实验整体设计：采用10分钟4-硫尿核苷（4sU）标记结合MTS-生物素亲和下拉的方式，开展脉冲追踪mRNA分析。