Defining novel mechanisms of mRNA translational control upon cold-shock in mammalian cells

The synthesis of polypeptides that are ultimately folded and decorated with various modifications in the cell to yield a functional protein is the result of a process called mRNA translation. Protein synthesis is therefore the process by which the information in the genetic material in the cell, DNA is converted via an intermediary substrate mRNA, into proteins. Translational control allows for rapid changes in protein synthesis that permits cells and organisms to maintain cellular homeostasis and respond to various stimuli, including environmental perturbations such as temperature. Surprisingly, the control of mRNA translation and subsequent protein synthesis in mammalian cells at subphysiological temperatures (cold-shock, <37degC) and upon recovery is poorly described even though cold-shock is used in transplant medicine, heart and brain surgery, implicated in mammalian hibernation, brain plasticity and ageing, and is utilised in the biotechnology sector as a method to improve recombinant protein production. Further, the two mammalian cold-shock proteins, cold inducible RNA binding protein (CIRP) and RNA binding motif protein 3 (Rbm3) are implicated in translational control and various cancers. To our knowledge we are the only group in the UK investigating translational control in mammalian cells upon cold-shock. In earlier studies we have generated data that suggests cold specific mechanisms control mRNA translation and protein synthesis in mammalian cells upon cold-shock at 27-32degC. We intend to further our earlier studies by maintaining and extending the link between our two internationally known groups to utilise a combination of interrelated approaches to investigate our over-arching hypothesis that 'upon cold-shock in mammalian cells a coordinated response involving distinct signalling pathways is activated that results in modification of the translational apparatus and its interactions, ribosomal 40S protein subunit turnover, synthesis of mRNAs which contain features recognised by specific trans acting factors, and the synthesis of specific proteins that interact with the translational apparatus to aid mRNA translation'. These studies will further define the control of translation upon cold-shock in mammalian cells, significantly improving our understanding of protein synthesis under such conditions, potentially leading to new approaches to improve protein production from mammalian cells and treatments for heart and brain damage.

The control of mRNA translation and subsequent protein synthesis in mammalian cells at sub-physiological temperatures (cold-shock, <37degC) and upon recovery is poorly described even though cold-shock is used in transplant medicine, heart and brain surgery, mammalian hibernation, brain plasticity and ageing, and is utilised in the biotechnology sector as a method to improve recombinant protein production. The proposed programme of work sets out to investigate the hypothesis that 'upon cold-shock in mammalian cells a coordinated response involving distinct signalling pathways is activated that results in modification of the translational apparatus and its interactions, ribosomal 40S protein subunit turnover, synthesis of mRNAs which contain features recognised by specific trans acting factors, and the synthesis of specific proteins that interact with the translational apparatus to aid mRNA translation'. As such, this proposal builds upon direct evidence of such a link throughout the literature and earlier work from our laboratories suggesting novel and cold specific mechanisms control mRNA translation and protein synthesis in mammalian cells upon cold-shock at 27-32degC. We will utilise a combination of approaches to characterise the links between cold-shock and (i) ribosomal 40S subunit protein turnover; (ii) RNA binding proteins that regulate cold-shock; (iii) the relationship between, eIF2 and eIF2B, elongation, the initiation factor eIF3, protein synthesis, 40S protein turnover and the proteasome; (iv) the signalling pathways activated upon cold-shock; and (v) whether the cold-shock protein CIRP plays a role in globally regulating translation upon cold-shock in mammalian cells. The outcomes will be an increased understanding of physiological processes associated with cooling such as hibernation, brain cooling used in surgery and transplant surgery, and the production of recombinant proteins alongside the potential development of new treatments in these areas.

Impact Summary Who will benefit from this research? In terms of research findings: the primary beneficiaries will be researchers in the academic, medical and biopharmaceutical sectors who are interested in understanding the role of the environment on mRNA translation and protein synthesis. As such, this proposal is relevant to all those academics, medics and industrialists who are interested in the processes controlling the mammalian cell response to cold-shock and wish to understand and/or harness these responses. The outcomes of this research will be an increased understanding of physiological processes associated with cooling such as hibernation, brain cooling used in surgery and transplant surgery, and the production of recombinant proteins alongside the potential development of new approaches and treatments in these areas. The research will also strengthen collaboration between two academic groups with expertise in the cold-shock response in mammalian cells and we believe are the only two groups in the UK presently studying this. What will be done to ensure that they benefit from this research? The investigators are very well placed to inform the activities of academia, clinicians and industry alike, and to exploit their discoveries commercially if opportunities arise. Both PI's have established links with the post-transcriptional regulation academic community within the UK and internationally and as such are well placed to disseminate findings to this group. With regard to clinicians, AEW has direct contact with clinicians active in the area through the MRC unit at Leicester whilst CMS is in contact with those interested in treatment of stroke damage in patients in the NHS in Kent. CMS has established links with industry and is part of the BBSRC/EPSRC BRIC steering group currently involved in developing and delivering research in bioprocessing between industry and academia within the UK. Currently he has been instrumental in the development of a new approach to improve cell line development which is being beta tested with an industrial partner. As such he is well placed to disseminate appropriate information to industry. Results will be published (our targets are a minimum of 3 papers in high quality journals) and presented at conferences (1 national p/a and at least 2 international meetings over the grant period). Locally, results will be presented at departmental meetings and at local special interest groups. In terms of disseminating information to wider audiences, the investigators are active teachers on undergraduate courses and host projects for undergraduate as well as postgraduate projects. All the investigators are active within their institutions in promoting public understanding of science particularly during National Science week. In terms of exploitation: Kent Innovation & Enterprise (KIE), the University of Kent's dedicated business development unit, will provide full support and guidance on any business opportunities that may arise from research findings made and work in collaboration with the MRC unit at Leicester. In terms of staff training; training will be provided for the PDRAs and research technician to develop new skills through the project, but also to develop an understanding of the relevance of science in underpinning post-transcriptional control in the cold and in general and the potential applications of this knowledge.