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. Overall design: RNA-seq profiling of WT and pCUP1-GFA1 25x array strains over hour time-course following treatment with 500 uM Copper Sulfate. Samples collected at 0, 5, 10, 15, 20, 25, 30, 45, and 60 minutes.