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

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description During this project we have made significant findings.

mRNA translation and its control allows for rapid changes in protein synthesis that permits cells and organisms to maintain cellular homeostasis and respond to various stimuli, including perturbations such as temperature. Here we investigate the response of mammalian cells to cooling. Cooling of mammalian cells to sub-physiological temperatures (typically around 32oC) elicits translational reprogramming and we have recently demonstrated in this work that this is mediated via translation elongation rate control. Cooling is relevant to transplant medicine, treatment of heart and brain damage, brain plasticity and ageing, mammalian hibernation and is widely utilised in industrial biotechnology applications for the production of biotherapeutic recombinant proteins destined for use in the clinic. We have shown that this control of protein synthesis can be manipulated using reagents that modify the lipid content of the cells that results in changes to the calcium ion flux in the cell and confirmed this is the key mechanism controlling protein synthesis in mammalian cells upon cooling. We have also shown that one of the translation initiation factors that helps assembly the ribosome and mRNA for protein synthesis called eIF4G, binds to a cold induced protein and mapped where and how this occurs. Work is on-going to define how the calcium related cool phenotypes can be manipulated such that these could be applied in a medical or biotechnological setting.

We have also shown that a protein called p58 can be important in controlling translation and that manipulation of this can enhance the amount of therapeutic antibody produced from a CHO cell at both normal and at cooling temperatures.

Finally, and most importantly, we have recently identified new cold-shock protein, RTN3 and established that expression of this protein, and elongation control of mRNA translation play a key role in the mammalian cell response to cold-shock but also in protection against neurodegeneration and tumourigenesis.
Exploitation Route As stated above, this project is on-going. However, the results to date could be translated into application in the fields of transplant medicine, treatment of heart and brain damage, brain plasticity and ageing, mammalian hibernation and industrial biotechnology applications for the production of biotherapeutic recombinant proteins. In the medical field we envisage the control of cooling responses without the need for cooling using a more controlled delivery of drugs that elect these responses to potentially be developed. These findings can also be used to replicate the effect of cooling in the manufacture of therapeutic recombinant proteins without the need for cooling or to further enhance the effects.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The findings from this project have relevance to transplant medicine, treatment of heart and brain damage, brain plasticity and ageing, mammalian hibernation and is widely utilised in industrial biotechnology applications for the production of recombinant proteins manufactured from cultured mammalian cells. To date the cooling effect has been used and manipulated to help address issues with the production of a particular recombinant biotherapeutic protein manufactured for use in the clinic. Recent work has shown that cold-shock responses can help offer protection against neurodegeneration and has resulted in the lead to the development of programmes to investigate this effect in further detail with the aim of developing clinical treatments.
First Year Of Impact 2015
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description MC Training Network EU - Horizon 2020 MSCA
Amount € 819,863 (EUR)
Funding ID ITN 642663 
Organisation European Commission 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 03/2015 
End 03/2019
 
Description Wellcome Trust Collaborative Award
Amount £1,877,553 (GBP)
Funding ID UNS16981 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2016 
End 08/2021
 
Description Lonza Biologics Mass Spec Fingerprinting Project 
Organisation Lonza Group
Country Global 
Sector Private 
PI Contribution Development of a mass spectrometry fingerprinting technology for the identification of highly productive recombinant CHO cell lines.
Collaborator Contribution Cell line development, fermentation and bioreactor runs and validation, provision of reagents and expertise.
Impact IP/Patents, publications.
Start Year 2008
 
Description 7 open days at University 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Describing the research we undertake in the laboratory, covering all aspects of the research and the impact this has/can have. Particular questions around genetic modification of cells to produce recombinant proteins in all sessions and discussions around both the ethical aspects of this and the potential applications of such technology.
Year(s) Of Engagement Activity 2015,2016
 
Description Open Days At University 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This activity is take take parents and secondary students around my research laboratories to explain the research undertaken and to demonstrate some of the research that we do in the laboratory.

Parents and students asked about engineering of cell lines and therapeutic recombinant protein drugs and how these are made, cost implications and on-going research.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011
 
Description School visit/outreach for several days at Simon Langton Boys Grammar - MBP2 project showing students how to clone, express and purify recombinant proteins in the laboratory and discuss science behind this. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Helping with research activity in school - cloning and expression of recombinant proteins
Year(s) Of Engagement Activity 2015,2016
 
Description Turkey Public Biotechnology Talk 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Presentation to public audience in Turkey on behalf of the British Council around biotechnology and its application. Event was filmed and followed by a question and answer session, answering questions sent in before the talk by social media and then from the audience. The event was filmed and shown on national TV in Turkey. Large range of topics discussed around the application of biotechnology to every day life and issues with long discussion/debate.
Year(s) Of Engagement Activity 2016
URL http://www.britishcouncil.org.tr/en/programmes/education/science-innovation-talks/biotechnology