Investigating the role of 4E-T, an eIF4E-binding protein resident in P-bodies, in gene expression control

Lead Research Organisation: University of Cambridge
Department Name: Biochemistry

Abstract

DNA is copied into messenger RNA in the nucleus, and following transfer to the cytoplasm, mRNA is translated into protein. mRNAs are translated into protein at different rates, leading to vastly different protein levels, which in part explains why muscle and red blood cells, for example, are so distinct though sharing the same genes. Regulation of mRNA translation is principally controlled by eIF4E, which binds the unusual cap structure at the 5' end of mRNA. eIF4E is important because it interacts with eIF4G which recruits ribosomes, the translational machinery, to mRNAs. Alternative eIF4E-binding proteins exist, such as 4E-BPs and these prevent eIF4G binding to eIF4E and reduce translation.

I propose to investigate a more recently discovered eIF4E-binding protein called 4E-T, which also competes with eIF4G for eIF4E-binding. Interestingly, 4E-T and a fraction of eIF4E is concentrated in punctate cytoplasmic "bodies" visible in the microscope, called P-bodies, which lack ribosomes, and are thought to be sites where mRNAs are stored and returned to translation, or where mRNAs are degraded. Despite considerable interest, the role of these P-bodies remains enigmatic, though they influence for example virus replication.

Published and unpublished work from us and other investigators show that 4E-T regulates a sub-set of mRNAs, but it is not known what sort of proteins they encode, nor is it known whether 4E-T has to be in P-bodies to exert this control, what proteins it binds there, or what its modification by phosphorylation means for this control. These are the questions we want to answer, using human cells in culture. We believe this work is highly timely since aberrant levels of eIF4E (and its phosphorylation) has been implicated in cell growth, proliferaion and in cancer development, and therefore investigation of a factor that interacts with eIF4E is important. Moreover, the role of P-bodies is still relatively unexplored, and our work will make a significant contribution to our understanding of how mRNAs are regulated in these foci. Last, we're fortunate to collaborate with Professor Anne Willis from the MRC Toxicology Unit, Leicester, to undertake a part of the analysis of 4E-T mRNA targets, and to extend and benefit from a Translational Resource database there, containing data from our and similar experiments from other investigators.

The proposed work is novel, focused and timely, and the outcomes will contribute to our understanding and possible exploitation of the regulation of translation initiation by eIF4E and its interacting factors.

Technical Summary

Translational regulation plays a critical role in the control of cell growth and proliferation. A key player in translational control is eIF4E, the mRNA 5' cap-binding protein. Translation initiation, the major control level, begins with the rate-limiting binding of the eIF4F (eIF4E, eIF4G and the RNA helicase eIF4A) complex to the 5' cap and is completed upon start codon recognition by the preinitiation complex. eIF4E sandwiches the cap via conserved tyrosines and binds the YXXXXLphi sequence in eIF4G on its convex side.

Inhibitors of translation initiation known as eIF4E-binding proteins (4E-BPs) contain similar YXXXXLphi motifs, and act as molecular mimics of eIF4G. New eIF4E-binding proteins have been reported that tether it to specific mRNAs, the best characterised of these being the mammalian 4E-T(ransporter) protein, found enriched in distinct cytoplasmic foci, P-(rocessing) bodies. P-bodies concentrate enzymes that are involved in mRNA repression and turnover and sequester mRNAs away from the translational machinery.

Our hypothesis is that 4E-T plays a critical role in the transition of mRNAs from active translation to being committed for silencing in P-bodies. In particular, we propose that 4E-T silences, at the level of translation and/or mRNA decay, the expression of a subset of mRNAs in P-bodies, until its phosphorylation in mitosis, when eIF4E is released from 4E-T.

To investigate this novel and fundamental aspect of gene expression regulation, we propose to identify the subset of mRNAs whose expression is regulated by 4E-T, to determine whether the regulation by 4E-T at the level of translation or mRNA decay; to identify the protein factors that interact with 4E-T in P-bodies and assess if the regulation by 4E-T is dependent on its residence in P-bodies; and to identify and determine the functional importance of the site(s) of phosphorylation in 4E-T in dividing cells.

Planned Impact

This is a basic bioscience proposal, and our main pathways to impact include dissemination of our data to our academic colleagues via publication and meeting involvement, the training of undergraduate, postgraduate and post-doctoral associates, collaboration with centres of excellence, and participation in outreach activities such as Cambridge Science Week.

Our results will be efficiently communicated to academics in our own and related fields via the scientific literature, attendance at conferences and invited presentations (see data sharing). Publication in internationally recognised scientific journals, invited presentations at institutes and the preparation of accessible review articles can efficiently disseminate the results of research to those in fields other than our own, including clinicians and those working in pharma or other areas of industry. Local dissemination of results to researchers and students is aided by seminar programs and local interest groups including the Cambridge RNA Club and OnCamiR.

My laboratory has a history of training others (from school placements to undergraduates to experienced researchers) in a variety of methodologies, with some of those trained now working in non-academic scientific roles.

My lab also organised/s (2010, 2011) a successful summer research experience program for US undergraduates from Madison Wisconsin (SCORE; director Professor Marvin Wickens), where we organise student placements in labs of the Cambridge RNA community as well as hosting students ourselves. NSF funding is currently beinght sought to extend the programme for a further three years.

My lab participates in Cambridge Science Week activities, including designing (PhD students) and judging (PI) posters that challenge postgraduate students to communicate their research to a non-expert audience. The goal is to convey an important scientific message that they have an interest in, clearly and concisely to the general public.

The proposed work involves collaboration with the laboratory of Professor Anne Willis (BBSCR Professorial Fellow, director MRC Toxicology Unit, Leicester, to be trained in and to perform polysomal profiling and other arrays, and in the analysis of the data. The value of these collaborations is in the import and further co-development of a cutting-edge technique to our Department, and in the free exchange of ideas, reagents and additional technologies as appropriate. Staff working on this project will acquire important skills in cutting edge technology that can be applied widely in academic research and in industry. They will also learn how to integrate this information into a wider picture.

Translational control (both mRNA-specific and global) is central to regulating protein synthesis rates and plays a critical role in the control of cell growth and proliferation. A key factor in translational control is eIF4E, the mRNA 5' cap-binding protein. Aberrant expression of eIF4E promotes tumorigenesis and has been implicated in cancer development. Our proposal is to investigate translation regulation by 4E-T, a protein that sequesters eIF4E from the translational machinery. Understanding these regulatory mechanisms can also impact a variety of industrial applications including the production of recombinant proteins, and as a result people working within industry keep abreast of developments in the field of translational control through the scientific literature and attendance at international conferences to which the PI and members of her research group regularly contribute. Cambridge Enterprise exists to help University of Cambridge inventors, innovators and entrepreneurs make their ideas and concepts more commercially successful for the benefit of society, UK economy, the inventors and the University. If applicable, clinical or commercial collaborations arising from this work would be coordinated by Cambridge Enterprise.
 
Description We have shown how an important translation factor called 4E-T which interacts tightly with eIF4E (also known as the cap-binding protein) mediates translational repression when bound to mRNA (Kamenska et al., 2014). Moreover, more recently we have shown that its binding to the DDX6 helicase is important for translational repression, including that mediated by micro RNAs (Kamenska et al., 2016). Moreover, in an important collaboration with a structural group we reported the ternary complex between CNOT1 deadenylase, DDX6 and 4E-T (Ozgur et al., 2015). In contrast, CNOT1-DDX6 cannot interact with Pat1b or Edc3 decapping activators. This suggests a possible temporal change in mRNP formation during translational repression and mRNA decay
Exploitation Route The field of microRNA-mediated silencing and indeed more generally how specific mRNAs are translationally repressed and decayed is receiving considerable interest currently. Other groups will build on our findings to further elucidate the mechanism of action of mcroRNAs, whose levels are regulated in differentiation and disease
Sectors Education,Healthcare,Other

 
Description To date there have been no economic or societal benefits. The funding has provided extensive training for the post-doctoral associate and research assistant. The former is applying for fellowship funding currently
First Year Of Impact 2012
Sector Education,Healthcare
Impact Types Policy & public services

 
Description P-bodies and RNA-binding proteins 
Organisation Pierre and Marie Curie University - Paris 6
Country France 
Sector Academic/University 
PI Contribution Exchange of reagents and training in experimental methods. Continued active collaborations in projects related to BBSRC funding
Collaborator Contribution Exchange of reagents and training in experimental methods. Continued active collaborations in projects related to BBSRC funding
Impact . The DDX6-4E-T interaction mediates translational repression and P-body assembly. Kamenska A, Simpson C, Vindry C, Broomhead H, Bénard M, Ernoult-Lange M, Lee BP, Harries LW, Weil D, Standart N. Nucleic Acids Res. 2016 Jul 27;44(13):6318-34. doi: 10.1093/nar/gkw565. P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes. Ayache J, Bénard M, Ernoult-Lange M, Minshall N, Standart N, Kress M, Weil D. Mol Biol Cell. 2015 Jul 15;26(14):2579-95. doi: 10.1091/mbc.E15-03-0136. Multiple binding of repressed mRNAs by the P-body protein Rck/p54. Ernoult-Lange M, Baconnais S, Harper M, Minshall N, Souquere S, Boudier T, Bénard M, Andrey P, Pierron G, Kress M, Standart N, le Cam E, Weil D. RNA. 2012 Sep;18(9):1702-15. doi: 10.1261/rna.034314.112. RNA-related nuclear functions of human Pat1b, the P-body mRNA decay factor. Marnef A, Weil D, Standart N. Mol Biol Cell. 2012 Jan;23(1):213-24. doi: 10.1091/mbc.E11-05-0415. Distinct functions of maternal and somatic Pat1 protein paralogs. Marnef A, Maldonado M, Bugaut A, Balasubramanian S, Kress M, Weil D, Standart N. RNA. 2010 Nov;16(11):2094-107. doi: 10.1261/rna.2295410. Role of p54 RNA helicase activity and its C-terminal domain in translational repression, P-body localization and assembly. Minshall N, Kress M, Weil D, Standart N. Mol Biol Cell. 2009 May;20(9):2464-72. doi: 10.1091/mbc.E09-01-0035. CPEB interacts with an ovary-specific eIF4E and 4E-T in early Xenopus oocytes. Minshall N, Reiter MH, Weil D, Standart N. J Biol Chem. 2007 Dec 28;282(52):37389-401.
 
Description Biochemical Society Panel membership 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact I have served on a Biochemical Society Theme Panel I:Genes (currently as vice-chair). Our remit is to mainly select proposal for meetings for funding by the Society, and to select Medal and Award winners annually. We also have a remit to policy making in the Biological Sciences, and to extend links between academia and biotech/pharma, to list a couple. We potentially reach several hundred practitioners annually
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description SCORE Program Madison University of Wisconsin 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I help coordinate summer research lab internships (8 week long) in Departments here for penultimate undergraduates from University of Wisconsin, USA.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014
URL https://biochem.wisc.edu/score
 
Description Science Week 
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 The activity was a series of visual demonstrations on DNA and how to decode it, and included one simple experiment. The workshop was of great interest to children and adults and sparked discussion afterwards

As the target audience were children around 10-12 it was too early to have specific outcomes, but no doubt seeing scientists - incidentally many female ones - discuss their work would have had an impact
Year(s) Of Engagement Activity 2013