A time-resolved framework for the recruitment of mRNP processing and assembly factors to a site of transcription

Processing and packaging of messenger ribonucleoprotein (mRNP) particles involve complex, coordinated interactions between nascent transcripts, RNA binding proteins (RBPs), and other associated factors. Despite the critical role of co-transcriptional mRNP assembly in gene expression, the temporal dynamics of this process are not well understood. Here, a live cell imaging assay is reported in S. cerevisiae to quantitatively detect recruitment of endogenous fluorescently tagged proteins to a transcriptionally active locus. Protein recruitment to an inducible integrated gene array composed of twenty-five transcriptional units is detected by colocalization with lacO repeats. Using arrays with two different promoters and the same coding sequence (GFA1), arrival times for a variety of mRNP processing and assembly factors were quantified. These analyses revealed Yra1, Cbp80, and Yhs7 as pioneering assembly factors. Notably, Yra1 recruitment occurs independently of the THO complex, with early localization supported by Cbp80 and the RNA recognition motif (RRM) of Yra1. Altogether, this work establishes the first comprehensive temporal framework for understanding protein recruitment during co-transcriptional mRNP assembly, providing mechanistic insights into the dependencies of Yra1 recruitment.信使核糖核蛋白颗粒（messenger ribonucleoprotein, mRNP）的加工与包装，涉及新生转录本、RNA结合蛋白（RNA binding proteins, RBPs）及其他相关因子间复杂且协同的相互作用。尽管共转录mRNP组装在基因表达中发挥关键作用，但该过程的时间动态特征仍未得到充分阐释。本研究报道了一种酿酒酵母（S. cerevisiae）中的活细胞成像检测方法，可定量检测内源性荧光标记蛋白向转录活性位点的招募情况。该方法通过与lacO重复序列（lacO repeats）共定位，实现对由25个转录单元组成的可诱导整合基因阵列的蛋白招募检测。针对携带两种不同启动子、编码序列均为GFA1的基因阵列，研究量化了多种mRNP加工与组装因子的到达时间。分析结果显示，Yra1、Cbp80与Yhs7为先驱组装因子。值得注意的是，Yra1的招募不依赖THO复合物（THO complex），其早期定位依赖Cbp80及Yra1的RNA识别基序（RNA recognition motif, RRM）。综上，本研究构建了首个用于解析共转录mRNP组装过程中蛋白招募的全面时间框架，为阐明Yra1招募的依赖关系提供了机制层面的新见解。