An integrated systems approach to posttranscriptional gene expression

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

As with many of the essential processes in living cells, a great deal of qualitative information about posttranscriptional gene expression has been accumulated, yet our quantitative understanding of the pathways of translation and mRNA turnover as complete processes (systems) is minimal. This means that we are frequently unable to model adequately either the processes themselves or their regulation, thus limiting potential progress in our understanding of the molecular basis of function and control in the translation and mRNA turnover machineries. This is particularly evident in relation to the control of the switching of mRNA molecules from translatable templates to targets of degradation, which remains completely uncharacterised in quantitative terms. I would like to dedicate five years of my research career to the application of a novel combination of advanced techniques drawn from a number of disciplines in order to place our understanding of these steps of posttranscriptional gene expression in a quantitative framework that can be used as the basis for testable and predictive modelling. This will enable a step-change in our fundamental understanding of these key processes in the cell because all structural and functional data will be built into one coherent framework. The techniques to be applied will include process control analysis, fast reaction kinetic analysis, studies of molecular structure and dynamics, and mathematical modelling. The overall strategy was originally embodied in my review of 1998 (McCarthy, J.E.G. Posttranscriptional control of gene expression in yeast. Microbiol. Molec. Biol. Rev. 62, 1492-1553), but only now has the field reached a sufficiently advanced stage of experimental/theoretical progress where an intensive approach of this scale can be truly effective. In the wider context of the BBSRC's commitment to supporting members of the scientific community who can promote the strategic aims of the research council, this ties in with my more general commitment to promoting interdisciplinary research and training in the UK, as evident from my leadership of the MIB project and of the RSC Chemical Biology Interface Forum, as well as from my managerial involvement with the Manchester Centre for Integrated Systems Biology (MCISB) and its associated Doctoral Training Centre.

Technical Summary

This will be a coherent research programme combining several complementary threads of investigation focused on eukaryotic posttranscriptional gene expression, using yeast as a model system. The first component will be the development of a comprehensive systems model for rate control in the eukaryotic translation pathway and in the route that leads mRNA molecules into pathways of degradation. This work will be enhanced to incorporate spatial and temporal resolution into the analysis via an approach termed Spatially (and Temporally) Resolved Molecular Systems Analysis [SReMSA (STReMSA)]. This will provide a detailed, quantitative platform for understanding the dynamic control and regulation of eukaryotic posttranscriptional gene expression. The above systems approach will be complemented by a series of structural, allied to functional, studies at the molecular level that will provide the mechanistic detail that is needed for an understanding of component steps in the pathways. X-ray crystallography, NMR and cryo-EM will all be used to enhance our structural understanding of the ribosome and its complexes with translation factors, and of key components of the mRNA decay pathway. Mechanistic studies will involve the use of FRET, EPR, fast reaction spectroscopy and single molecule studies (particularly AFM and TIRFM). Various forms of modelling will be used to analyse the data generated in this research programme, including rate control modelling, local dynamics and kinetic modelling, intermolecular interactions modelling, and structural modelling.

Publications

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Tait S (2010) Local control of a disorder-order transition in 4E-BP1 underpins regulation of translation via eIF4E. in Proceedings of the National Academy of Sciences of the United States of America

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Dixon N (2010) Reengineering orthogonally selective riboswitches. in Proceedings of the National Academy of Sciences of the United States of America

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Malys N (2011) Translation initiation: variations in the mechanism can be anticipated. in Cellular and molecular life sciences : CMLS

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Firczuk H (2013) An in vivo control map for the eukaryotic mRNA translation machinery. in Molecular systems biology

 
Description This Fellowship grant enabled my group to achieve multiple objectives, including the following:

We developed a new approach to study how proteins involved in the translation and degradation of messenger RNA in living cells are localised, and how this localisation changes when the cells are placed under stress. A novel tetracysteine motif (TCM), combined with biarsenical dyes, was used to tag key regulatory proteins in the mRNA degradation machinery.

We performed a comprehensive in vivo rate control study of yeast translation that involved the construction of more than 20 regulatable genpmic expression constructs, 'genetic 'titrations' of the encoded protein factors, protein synthesis assays, quantitative mass spectrometry and advanced systems modelling. This work revealed that rate control is distributed across multiple steps in the pathway and provided unique insight into the roles of duplicated genes.

We used isothermal calorimetry, NMR, surface plasmon resonance, and other methods, to elucidate the mechanism underpinning phosphorylation-dependent regulation of the human cap-binding protein eIF4E by the 4E-binding protein (4E-BP1). The data generated are consistent with a model in which phosphorylation changes the stability of the alpha-helical structure in the 4E-BP1 binding motif, thus modulating binding affinity.

In a challenging and time-consuming part of the research programme, we generated a unique set of dual-tagged 40S ribosomal subunits that can be used to monitor the conformational rearrangements that occur when translation factors bind to the subunit. These important reagents are a valuable resource but have yet to be fully exploited. Individual ribosomal proteins were tagged with either GFP or a tetracysteine motif (TCM)-biarsenical dye combination, thus allowing FRET to occur between tagged pairs.

In collaboration with Robert Gilbert at Oxford, we generated two crystal structures of the S.pombe m7GpppX pyrophosphatase Nhm1. These structures will help us elucidate the mechanism by which this protein distinguishes between capped mRNA molecules of different lengths.
Exploitation Route Our observations on Dhh1 and Pat1 could be followed up by more detailed analysis of the dynamics of ribonucleoprotein complexes in yeast.

The dual-tagged ribosomes represent a potentially powerful set of tools for studying conformational rearrangements in the 40S ribosomal subunit.

The Nhm1 crystal structures represent the platform for elucidating capped-mRNA length selectivity, a fascinating problem.

The in vivo rate control study provides the basis for further in-depth analyses of rate control in the yeast translation machinery.
Sectors Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
Description Some of the work related to rate control has fed into a new project now underway with Ingenza, in which we utilise robotic methods to optimise expression of cellulose degrading enzymes in yeast.
First Year Of Impact 2014
Sector Manufacturing, including Industrial Biotechology