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 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 Saccharomyces cerevisiae to detect recruitment of endogenous fluorescently tagged proteins to a transcriptionally active locus. Protein recruitment to an inducible integrated gene array composed of 25 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 mRNP assembly factors. Notably, Yra1 recruitment occurs independently of the THO complex, with early localization supported by Cbp80 and the RNA recognition motif 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.