The role of the CCR4-NOT complex and mRNA regulatory elements in determining protein synthesis, destination and complex formation.

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Recent evidence indicates that CCR4-NOT is of central importance in regulation of the spatial proteome, but the underlying mechanisms by which it responds to information within mRNA sequence to regulate protein expression and location are poorly understood. This proposal will address these questions by combining global interrogation of the regulation of the subcellular RNAome and proteome by CCR4-NOT with detailed mechanistic analysis of its regulation of cellular and viral mRNA targets at the ER, and how this relates to miRNA function and detection of ribosome pausing.

Using Huh7 liver cells as a model for ER-associated translation, high expression of miR-122, and HCV replication, we will couple rapid CNOT1 depletion by the auxin-inducible degron system with the LOPIT and LoRNA methods that were recently developed in the Lilley lab. These methods will give an unprecedented overview of the subcellular distribution of proteins and RNAs, respectively, and how they are regulated by CCR4-NOT. We will also apply the LoRNA method to obtain the first global characterisation of miRNA localisation across the cell. Immunofluorescence and in situ hybridisation will be used to confirm important results.

In parallel, we will directly investigate how CCR4-NOT functions at the ER, using membrane-cytoplasmic fractionation and ribosome profiling to characterise how CNOT1 depletion regulates ribosome pausing and ER targeting. Detailed mechanistic analysis of the effects of CCR4-NOT and miRNAs on translation and stability of cellular targets and HCV RNA will use approaches including siRNA-mediated knockdown, 4-thio-uridine labelling of nascent RNA and poly(A) tail analysis in membrane-cytoplasmic fractions. Reporter assays will determine the role of ribosome pause sites and UTR elements in dictating miRNA and CCR4-NOT function at the ER, and in mRNA and miRNA subcellular localisation.