Departement of Microbiology and Molecular Medicine, Institute of Genetics and Genomics Geneva, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
Present address: Department of Biomolecular Sciences, The Weizmann Institute of Science, 76100, Rehovot, Israel.
Genome Biol. 2023 Feb 20;24(1):30. doi: 10.1186/s13059-023-02871-7.
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.
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.
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.
Ccr4-Not 复合物主要作为主要的真核脱腺苷酶而广为人知。然而，有几项研究揭示了该复合物的作用，特别是与脱腺苷化无关但与翻译有关的 Not 亚基的作用。特别是，已经报道了调节翻译延伸动力学的 Not 凝聚物的存在。评估翻译效率的典型研究依赖于在细胞破裂后获得的可溶性提取物和核糖体分析。然而，凝聚体中的细胞 mRNA 可以被主动翻译，并且可能不存在于此类提取物中。
在这项工作中，通过分析酵母中可溶性和不可溶性 mRNA 衰变中间体，我们确定与可溶性 mRNA 相比，不可溶性 mRNA 富含居住在非最佳密码子上的核糖体。可溶性 RNA 的 mRNA 衰变率较高，但相对于总体 mRNA 衰变，不可溶性 mRNA 的共翻译降解比例较高。我们表明，Not1 和 Not4 的缺失会反向影响 mRNA 的可溶性，并且对于可溶性 mRNA，核糖体根据密码子最优性居住。Not4 的缺失使具有较低非最佳密码子含量和更高表达的 mRNA 变得可溶，而这些 mRNA 由于 Not1 的缺失而变得不可溶。相比之下，Not1 的缺失使线粒体 mRNA 变得可溶，而这些 mRNA 在 Not4 缺失时变得不可溶。
我们的结果表明，mRNA 的可溶性决定了共翻译事件的动力学，并且由 Not1 和 Not4 反向调节，我们还确定这种机制可能已经由 Not1 启动子在核内的关联预先设定。
