Gene expression analysis and proximity labeling reveal posttranscriptional functions of the yeast RNA Polymerase II regulator Def1

Def1 is a yeast protein that promotes transcription elongation and regulates the degradation of RNA polymerase II during transcription stress. Although Def1 is localized in the cytoplasm, its functions in this cellular compartment are not yet understood. Despite its well-established roles in transcription, a comprehensive genome-wide analysis of its impact on gene expression has not been conducted. Here, we performed RNA sequencing (RNA-seq) analysis on cells lacking DEF1 and surprisingly found that only a few hundred genes exhibited altered expression, both upregulated and downregulated. To evaluate mRNA synthesis and decay rates in these DEF1-deficient cells, we used a nascent transcription metabolic labeling technique called RATE-seq. As expected, we observed reduced synthesis rates across the genome in these cells. Additionally, a global decrease in mRNA decay rates was observed, suggesting that Def1 plays a role in the post-transcriptional regulation of mRNAs. The changes in synthesis and decay rates showed a strong correlation, indicating that this compensation helps buffer steady-state mRNA levels. To gain further insight into Def1's functions, we conducted proximity labeling experiments to identify its protein binding partners within the cells. Our findings revealed that Def1 primarily interacts with cytoplasmic regulators involved in post-transcriptional processes, including proteins responsible for deadenylation, decapping, and translation regulation. Using an mRNA decay reporter assay, we demonstrated that recruiting Def1 to mRNA reduces its expression and accelerates its turnover. In summary, we have identified a novel cytoplasmic function for Def1, establishing it as a key regulator of gene expression in both transcription and mRNA decay. Overall design: The role of Def1 in translation and in the regulation of RNA was studied by using RNA-seq, RNA metabolic labeling, and Ribo-seq Wild type (W303) and def1? strains were grown to log phase (OD~0.8-1) and then treated with 5mM 4-thiouracil For metabolic labeling, cells were labeled for different times, then harvested, followed by RNA isolation and pull-down. For Ribo-seq, RNA was isolated from monosomes For steady-state RNA-seq, RNA was isolated at log phase Libraries were prepared and sequenced. Analysis was carried out.