The DNA damage response and RNA Polymerase II regulator Def1 has posttranscriptional functions in the cytoplasm

Yeast Def1 promotes transcription elongation and RNA polymerase II degradation during genotoxic stress. Def1 is cytoplasmic, and DNA damage signals cause its proteolytic processing, liberating its N-terminus to enter the nucleus. Cytoplasmic functions for this abundant protein have not been identified. Proximity-labeling (BioID) experiments indicate that Def1 binds many proteins involved in posttranscriptional control and translation of mRNAs. Deleting DEF1 reduces both mRNA synthesis and decay rates and directly tethering Def1 to a reporter mRNA suppressed expression, suggesting that Def1 directly regulates mRNAs post-transcriptionally. Surprisingly, we found that Def1 interacts with polyribosomes, which requires its N-terminal ubiquitin-binding domain and ubiquitylation of eS7a (Rsp7A) in the small subunit of the ribosome by the Not4 protein in the Ccr4-Not complex. Ribo-seq analysis revealed that deleting DEF1 affected the translation efficiency of a specific subset of transcripts, a genome-wide shift in ribosome footprints on mRNAs and an increase in the dwelling of ribosomes at non-optimal codons. This, together with the high sensitivity of def1Δ to translation inhibitors further suggest impaired translation. The polyglutamine-rich unstructured C-terminus of Def1 is required for its interaction with decay and translation factors, suggesting that Def1 acts as a ubiquitin-dependent scaffold to link translation status to mRNA decay. Thus, we have identified a novel function for this transcription and DNA damage response factor in post-transcriptional regulation in the cytoplasm and establish Def1 as a master regulator of gene expression, functioning from transcription to translation.