Characterization of the CCR4-NOT complex subunits CNOT1 and CNOT4 using rapid auxin-induced degradation in cultured cells

ABSTRACT: CCR4-NOT regulates multiple steps in gene regulation, including transcription, mRNA decay, protein ubiquitylation and translation. Originally discovered in yeast, this complex is highly conserved across eukaryotes, although its composition and functions differ between mammals and yeast. For example, unlike yeast Not4, human CNOT4 (E3 Ubiquitin Ligase) does not form a stable complex with CCR4-NOT, and its functions are less clear compared to its yeast counterpart. To investigate the roles of CNOT1 (the central scaffold subunit) and CNOT4, we developed a rapid auxin-induced degron cell culture system that allows for the acute depletion of these proteins. We studied the effects of their absence on complex integrity, cell growth, and mRNA expression and turnover. Our transcriptome-wide analysis revealed that depleting CNOT1 altered the expression of thousands of transcripts, with the majority showing increased abundance and a general decrease in mRNA decay. Although CNOT4 does not associate with the complex through standard biochemical methods, BioID proximity labelling confirmed its association with the complex in cells. However, depleting CNOT4 did not affect the integrity of CCR4-NOT. In contrast to the effects observed with CNOT1 depletion, reducing CNOT4 levels led to only modest changes in RNA steady-state levels and unexpectedly accelerated global mRNA decay. Finally, we show that the changes in mRNA stability in CCR4-NOT depleted cells correlated with the codon optimality of the transcripts. Our data suggest that CNOT4 exerts opposite effects on mRNA metabolism compared to CNOT1, and that CNOT4 may have functions distinct from those of complex subunits that promote mRNA degradation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE277237 | GEO | 2025/10/23

REPOSITORIES: GEO