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The role of initiation factor complex assembly and phosphorylation in controlling mRNA recruitment to ribosomes during differentiation.

Reference	BB/E014399/1
Principal Investigator / Supervisor	Professor Simon Morley
Co-Investigators / Co-Supervisors
Institution	University of Sussex
Department	Sch of Life Sciences
Funding type	Research
Value (£)	289,850
Status	Completed
Type	Research Grant
Start date	07/05/2007
End date	06/05/2010
Duration	36 months

Abstract

The regulation of translation initiation is a key point in gene expression, and can rapidly alter the temporal and spatial expression of either the whole transcriptome or specific mRNAs, without requiring new transcription. Central to translation initiation are eIF4GI/II, which act as a scaffold for the assembly of the initiation complex via contacts with the mRNA cap-binding protein eIF4E, eIF4A, eIF3, and the poly(A)-binding protein (PABP). The eIF4G proteins exist as a number of isoforms and we aim to identify their particular roles in the selective de novo recruitment of mRNAs for translation during muscle cell differentiation. This is particularly timely as recent proteomic work has shown a number of proteins to be expressed up to 5-fold higher during differentiation without any corresponding change in mRNA levels. To address the role for translational control in this process, we have begun to investigate initiation complex formation in differentiating C2C12 myoblasts, showing that the selective mRNA recruitment for translation is associated with enhanced recovery of eIF4GI and eIF4GII with eIF4E. As interference with specific signalling pathways accelerates the rate of differentiation and the recruitment of eIF4GI and eIF4GII into the eIF4F complex, we will use a combination of cell-permeable inhibitors, over-expression of eIF4E kinase (Mnk1) and RNAi to evaluate the biochemical role of Mnk1 in this physiological response. We also propose to use over-expression and RNAi to determine how the selective recruitment of eIF4GI and eIF4GII into the eIF4F complex influences the selection of target skeletal muscle-specific mRNAs for translation. This will also allow us to identify novel regulatory sites of phosphorylation of eIF4GI and eIF4GII stimulated during cellular differentiation, providing a platform for future work. Together, these studies will substantially increase our general understanding of the control of protein synthesis higher eukaryotes.

Summary

Critical information stored in the gene sequences of the genetic material (DNA) has to be decoded by the cell to produce a wide variety of essential proteins, as and when they are required. The sequence of each gene specifies the sequence of a given protein, with many proteins themselves in turn responsible for the synthesis of other types of structures in the cell. The general transfer of information from DNA to protein is carried out by the messenger RNA (mRNA), which is a copy of the DNA sequence and has to be decoded by a complex, highly regulated machine termed a ribosome, in a process known as translation. To work efficiently, accurately, and to allow the ribosome to function in the best interests of the cell, this machinery requires other proteins (translation initiation factors; eIF) that interact with each other, the mRNA and the ribosome in a highly regulated manner. One of these, eIF4G, acts as a scaffold protein, onto which the ribosome and several other initiation factors assemble. In mammalian cells, two slightly different genes contain the sequence for eIF4G, and the two genes also express different variants of the final protein. However, at this time we do not know whether these different forms of eIF4G protein perform different or overlapping functions in the cell. Using a model cell system that recapitulates control systems shown to function in the body, we wish to investigate the role of assembly of these components of the translational machinery in selecting specific mRNAs for translation to allow cells to make the correct types and amounts of proteins required for muscle cell regeneration. This study will improve our understanding of the different stages of gene expression, and may also lead to the specific control of certain mRNAs that are deregulated during diseases.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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