Abstract
In this work, we show that Not4 and Not5 from the Ccr4-Not complex modulate translation elongation dynamics and change ribosome A-site dwelling occupancy in a codon-dependent fashion. These codon-specific changes in not5Δ cells are very robust and independent of codon position within the mRNA, the overall mRNA codon composition, or changes of mRNA expression levels. They inversely correlate with codon-specific changes in cells depleted for eIF5A and positively correlate with those in cells depleted for ribosome-recycling factor Rli1. Not5 resides in punctate loci, co-purifies with ribosomes and Rli1, but not with eIF5A, and limits mRNA solubility. Overexpression of wild-type or non-complementing Rli1 and loss of Rps7A ubiquitination enable Not4 E3 ligase-dependent translation of polyarginine stretches. We propose that Not4 and Not5 modulate translation elongation dynamics to produce a soluble proteome by Rps7A ubiquitination, dynamic condensates that limit mRNA solubility and exclude eIF5A, and a moonlighting function of Rli1.
Highlights
The Ccr4-Not complex is a global regulator of mRNA metabolism in eukaryotic cells
5,048 transcripts were detected as genuinely translated above the threshold (>1 reads per kilobase per million of sequencing reads [RPKMs]; Table S1), with very good reproducibility between biological replicates (Figure S1A), well-defined three-nucleotide periodicity (Figure S1B), and the majority of ribosome protected fragment (RPF) lengths between 28 and 31 nucleotides (Figure S1C)
In not5D, they exhibited higher amounts of RPFs accumulating within the 50 vicinity of the coding sequences (CDSs) relative to the 30 end of the CDS
Summary
The Ccr4-Not complex is a global regulator of mRNA metabolism in eukaryotic cells (for review see Collart, 2016) It regulates transcription and RNA quality control in the nucleus (Azzouz et al, 2009a; Kruk et al, 2011; Reese, 2013) and represses gene expression in the cytoplasm (Rouya et al, 2014; Sandler et al, 2011). RQC-induced ribosome splitting requires Rli, an ATP-binding protein that directly binds the ribosome in the inter-subunit space, at the GTPase binding center. Rli couples peptide release and ribosome splitting, in collaboration with termination factors or with the ribosome rescue factor Dom (Guydosh and Green, 2014) It promotes pre-initiation complex assembly (Dong et al, 2004)
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