Not1 and Not4 inversely determine mRNA solubility that sets the dynamics of co-translational events

Abstract

Background The Ccr4-Not complex is mostly known as the major eukaryotic deadenylase. However, several studies have uncovered roles of the complex, in particular of the Not subunits, unrelated to deadenylation and relevant for translation. In particular, the existence of Not condensates that regulate translation elongation dynamics has been reported. Typical studies that evaluate translation efficiency rely on soluble extracts obtained after the disruption of cells and ribosome profiling. Yet cellular mRNAs in condensates can be actively translated and may not be present in such extracts. Results In this work, by analyzing soluble and insoluble mRNA decay intermediates in yeast, we determine that insoluble mRNAs are enriched for ribosomes dwelling at non-optimal codons compared to soluble mRNAs. mRNA decay is higher for soluble RNAs, but the proportion of co-translational degradation relative to the overall mRNA decay is higher for insoluble mRNAs. We show that depletion of Not1 and Not4 inversely impacts mRNA solubilities and, for soluble mRNAs, ribosome dwelling according to codon optimality. Depletion of Not4 solubilizes mRNAs with lower non-optimal codon content and higher expression that are rendered insoluble by Not1 depletion. By contrast, depletion of Not1 solubilizes mitochondrial mRNAs, which are rendered insoluble upon Not4 depletion. Conclusions Our results reveal that mRNA solubility defines the dynamics of co-translation events and is oppositely regulated by Not1 and Not4, a mechanism that we additionally determine may already be set by Not1 promoter association in the nucleus.

Roles of the CCR4‐Not complex in translation and dynamics of co‐translation events

“…This suggests that the structure of ribosomes associated with Not4 and Not5 obtained by Beckmann and collaborators is compatible with the last translating ribosomes that accumulate and limit progression of the Xrn1 exonuclease, in the absence of Not4 and Not5, but also after Not1 depletion, since codon‐specific 5′P‐Seq A‐site RDO changes correlate with those in not4Δ and not5Δ (Allen et al, 2021 ; Allen et al, 2023 ). It may seem contradictory that the Not proteins are contributing to co‐translational assembly if they are associated with decay intermediates whose abundance increase in their absence.…”

“…In budding yeast 5 0 P-Seq has been used to analyze translation elongation dynamics of both the soluble and total RNA pools (including the insoluble RNAs) (Allen et al, 2023). From these studies it was noted that changes in A-site RDOs occur according to codon optimality upon depletion of Not proteins in the soluble RNA pool (by Ribo-Seq and 5 0 P-Seq) and that this is mediated by a specific group of mRNAs whose solubility (distribution between soluble and total RNA pools) changes.…”

“…Ribosomes at this pause site can be expected to be targets for Not5 binding, and in turn Not5 binding might slow translation enough to promote co-translational targeting (Figure 6). Mitochondrial mRNAs are enriched amongst mRNAs whose solubility is inversely modulated by Not1 and Not4 (Allen et al, 2023), F I G U R E 6 Ribosome pausing during translation is important for Not-dependent co-translational mRNA targeting. During translation the ribosome can slow down, for instance when it encounters non-optimal codons, and upon prolonged pausing the E-site can become empty such that the Ccr4-Not complex via Not5 can interact with the ribosome.…”

Structural determinants of co-translational protein complex assembly

“…Considering heteromers, mRNA co-localization is a prerequisite for co-translational assembly. Such co-localization could be achieved actively through zipcode sequences 63 or through partitioning into specific condensates 64 . Alternatively, random diffusion could suffice to promote nascent chain recognition 65 .…”

Phase-separated ribosome-nascent chain complexes in genotoxic stress response

“…What we know so far about NCAs is limited to their discovery in the context of co-translational assembly of Rpt1 and Rpt2 (Panasenko et al 2019). A recent study using ribosome profiling revealed the pivotal role of Not1 in regulating ribosome pausing on numerous mRNAs, indicating that the NCA regulation might be widespread (Gillen et al 2021;Allen et al 2023). However, maybe not all mRNAs translated in assemblysomes show detectable ribosome pausing by ribosome profiling, because assemblysomes are RNase-resistant (Panasenko et al 2019).…”