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

Abstract

Background: The Ccr4-Not complex is most well 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 have been reported. Typical studies that evaluate translation efficiency rely on soluble extracts obtained after disruption of cells and ribosome profiling. Yet cellular mRNAs in condensates can be actively translated and may not be present in such extracts. 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 impact 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. Our results reveal that mRNA solubility defines 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.

Not4-dependent targeting ofMMF1mRNA to mitochondria limits its expression via ribosome pausing, Egd1 ubiquitination, Caf130, No-Go-Decay and autophagy

“…The Not4 subunit of the Ccr4-Not complex plays an important role in Mito-ENCay by contributing to the targeting of the Mmf1-RNC to the mitochondria, in collaboration with the nascent chain MTS and its bound chaperone Egd1. Such a function for Not4 has not previously been described, and is compatible with our previous observation that MMF1 mRNA solubility increases upon Not4 depletion (33). In this context it is interesting to note that Not4-dependent ubiquitination of Rps7A is important for HAC1 translational up-regulation in response to ER stress, and the presence of the HAC1 mRNA at the ER is necessary for this up-regulation (65).…”

“…It was recently shown that condensates of Tis11, an RNA binding protein that can associate with the Ccr4-Not complex, is important for translation in proximity to the ER (73). This supports a possible role of NCAs in mRNA targeting to vesicles and/or organelles, that would explain the regulation of mRNA solubility by Not1 and Not4 that we have recently identified (33), including MMF1 studied here.…”

“…In this context it is interesting to note that Not4-dependent ubiquitination of Rps7A is important for HAC1 translational up-regulation in response to ER stress, and the presence of the HAC1 mRNA at the ER is necessary for this up-regulation (65). HAC1 mRNA solubility, like the solubility of MMF1 , increases upon Not4 depletion (33). Hence, it could be that Not4 contributes to ER targeting of the HAC1 mRNA.…”

“…This has been proposed to enable the Ccr4-Not complex to monitor the translating ribosome for mRNA turnover according to codon optimality (32). Consistently, depletion of Not5 changes A-site ribosome dwelling occupancies inversely to codon optimality (33). In addition, ubiquitination of Rps7A by Not4 can contribute to degradation of mRNAs by No-Go-Decay (NGD) in conditions where the RQC response is defective (34).…”

“…The data was generated and analyzed in (33). To briefly describe its origin, total and soluble RNA were isolated from cells with Not1 and Not4 degron alleles before and after auxin treatment to deplete Not1 and Not4.…”