Co-translational regulation of Mmf1 expression depends upon its mitochondrial targeting, ubiquitination of Egd1 by Not4, Caf130 and NGD: MitoENCay

The Ccr4-Not complex is a conserved multi protein complex with diverse roles in the mRNA life cycle. Recently we determined that the Not1 and Not4 subunits of Ccr4-Not inversely regulate mRNA solubility and thereby impact dynamics of co-translation events. One mRNA whose solubility is most extremely inversely regulated by Not1 and Not4 is MMF1 encoding a mitochondrial protein that is translated with apparent ribosome pausing. In this work we uncover a mechanism that limits MMF1 overexpression in fermenting yeast and does not require MMF1 5’ or 3’ untranslated sequences but is mediated solely by the MMF1 coding sequence. This mechanism depends upon co-translational targeting of the MMF1 mRNA to the mitochondria, promoted by its mitochondrial targeting sequence, the Egd1 subunit of the NAC complex, the Om14 outer membrane protein and components of the mitochondrial import machinery as well as by Not4. Thereby, levels and translation of the MMF1 mRNA are limited by a mechanism that we have named Mito-ENCay involving ubiquitination of Egd1 by Not4, the Caf130 subunit of the Ccr4-Not complex and RQC. Our results indicate that this mechanism is used for highly expressed mRNAs that depend upon co-translational interactions for appropriate folding, such as ribosomal protein mRNAs in fermenting yeast.Ccr4-Not复合物（Ccr4-Not complex）是一类保守的多蛋白复合物，在信使RNA（mRNA）生命周期中具有多样的生物学功能。近期本研究明确，Ccr4-Not复合物的Not1与Not4亚基可反向调控mRNA的溶解度，进而影响共翻译事件的动态进程。其中，受Not1与Not4反向调控程度最为极端的mRNA之一为MMF1，其编码的线粒体蛋白在翻译过程中表现出显著的核糖体暂停现象。本研究揭示了一种在发酵酵母中限制MMF1过表达的机制：该机制不依赖MMF1的5'或3'非翻译序列，仅由MMF1的编码区介导。该机制的实现依赖于MMF1 mRNA的共翻译线粒体靶向过程，这一过程由其线粒体靶向序列、新生肽链相关复合物（NAC complex）的Egd1亚基、Om14外膜蛋白以及线粒体导入装置的组分共同促进，同时也需要Not4的参与。据此，本研究将该限制MMF1 mRNA水平与翻译过程的机制命名为Mito-ENCay，其核心涉及Not4对Egd1的泛素化、Ccr4-Not复合物的Caf130亚基以及核糖体相关质量控制（RQC）通路。研究结果表明，该机制可作用于依赖共翻译相互作用以实现正确折叠的高表达mRNA，例如发酵酵母中的核糖体蛋白mRNA。