Degradation factor 1, Def1, regulates mRNA translation and decay through Ccr4-Not-dependent ubiquitylation of the ribosome.

Yeast Def1 is well known for its role in regulating RNA polymerase II elongation and degrading the large subunit of polymerase during transcriptional stress. It is an abundant cytoplasmic protein that undergoes stress-induced processing and is then transported to the nucleus. Previous research from our lab has shown that Def1 interacts with various proteins involved in mRNA decay and translation control, and that it regulates mRNA half-lives, suggesting an important role in the cytoplasm. In this study, we report that Def1 binds polyribosomes and that its null mutant strain exhibits phenotypes indicating a role in translation. Ribo-seq analysis revealed that deleting DEF1 altered ribosome footprints on mRNAs and increased the dwell time of ribosomes at non-optimal codons in the A-site. Additionally, results from a codon-optimality reporter assay suggest that Def1 facilitates the degradation of mRNAs containing non-optimal codons. The Ccr4-Not complex links codon optimality to mRNA decay, and Def1s binding to ribosomes depends on its ubiquitin-binding domain, as well as the ubiquitylation of eS7a in the small ribosomal subunit by the Ccr4-Not complex. Moreover, the polyglutamine-rich, unstructured C-terminus of Def1 is crucial for its interaction with RNA decay and translation factors. This indicates that Def1 functions as a ubiquitin-dependent scaffold, connecting translation status to mRNA decay. In summary, we have identified a cytoplasmic function for Def1 in translation and established it as a regulator of gene expression, spanning both transcription and translation processes.