The Not4 ubiquitin ligase utilizes its conserved RNA binding domains to regulate global proteostasis and RNA polymerase II-dependent transcription

The conserved Ccr4-Not complex regulates all aspects of the RNA polymerase II (Pol II) gene expression pathway, while it also controls proteasome assembly and function. The Not4 RING domain ubiquitin ligase is a core subunit that also contains an RNA recognition motif (RRM) and C3H1 domain (collectively referred to as the RRM-C) whose function is unknown. Herein, we demonstrate that the Not4 RRM-C contributes to both Not4-dependent regulation of the proteasome and Pol II. Disruption of both Not4 ligase activity and the RRM-C domain simultaneously replicates the proteasome-dependent deubiquitylation and catalytic activity misregulation found in Not4-deficient cells. Transcriptome analysis reveals that the Not4 RRM-C affects a subset of Pol II-dependent genes involved in specific biological functions, including transcription elongation, cyclin-dependent kinase regulated nutrient responses, and ribosomal biogenesis. At these genes, the Not4 RRM-C negatively regulates Pol II binding. While Not4 RRM-C disruption modestly increases between Ccr4-Not and Pol II, a Not4 ligase mutant decreases Ccr4-Not association with Pol II, thus implicating Not4-dependent ubiquitylation in mediating Ccr4-Not interaction with Pol II. Collectively, our results suggests the cellular RNA environment may integrate control of global proteostasis and Pol II transcription through the Not4 ubiquitin ligase. There are two conditions analyzed in this study (cells expressing Not4WT or Not4RRM) with three replicates per condition.