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

While nearly all cells in an organism contain identical DNA, enormous differences in the behaviour of cells occur over the course of development and in different tissues. These differences are achieved by the regulation of gene expression, the process by which the DNA of a gene is copied to mRNA (transcription), and the mRNA is used as a template to make the encoded protein (translation). The network of proteins produced within a cell then dictates its function.

Control of the production of the correct proteins in the right place at the right time is crucial in allowing cells to carry out their function and to respond rapidly to changes in environment. A central hub in this regulation is the CCR4-NOT protein complex. CCR4-NOT induces mRNA degradation, both globally and through targeted recruitment to specific mRNAs, and has additional functions in detecting ribosome pausing during the elongation stage of translation and modulating cotranslational assembly of protein complexes.

microRNAs (miRNA) are a biologically and medically important family of small RNA molecules that induce mRNA degradation and translation repression by binding to sequence-specific sites in the 3'untranslated region (UTR) of mRNAs and directly recruiting CCR4-NOT. In addition, we have shown that liver-specific miR-122 has an unusual role in promoting hepatitis C virus (HCV) replication. CCR4-NOT also promotes HCV replication, while we have shown that the proteins eIF4A2 and DDX6 are directly recruited by CCR4-NOT and involved in miRNA regulation of both cellular targets and HCV. It remains unclear how CCR4-NOT integrates signals from miRNAs and ribosome pausing to control the production, location and assembly of proteins. This will be determined in this proposal.

The endoplasmic reticulum (ER) is a network of membranes within eukaryotic cells where secreted and membrane proteins are synthesised. mRNAs that encode these proteins are directed to the ER where translation occurs. We have recently found that CCR4-NOT specifically regulates ribosome pausing on ER-targeted mRNAs, and that there are differences in miRNA repression at the ER and in the cytoplasm. HCV replication occurs in a membranous web derived from the ER, but it is not known whether miR-122 or CCR4-NOT regulation of HCV occurs in this location.

In this proposal, we will use state-of-the-art approaches to determine how CCR4-NOT controls the level and location of proteins, mRNAs and miRNAs throughout the cell. This will be followed by detailed interrogation of the mechanisms by which CCR4-NOT and miRNAs regulate cellular and viral targets in the ER and cytoplasm.

This study will provide an unprecedented global overview of the effects of CCR4-NOT and miRNAs on the whole cell and on a detailed molecular level. This will make an important contribution to the fundamental understanding the control of gene expression, while also providing new avenues to manipulate mRNA metabolism with considerable relevance to the emerging field of RNA therapeutics.

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.