Award details

Investigating metabolite-RNase communication.

Reference	BB/J016179/1
Principal Investigator / Supervisor	Professor Anastasia Callaghan
Co-Investigators / Co-Supervisors
Institution	University of Portsmouth
Department	Inst of Biomedical and Biomolecular Sc
Funding type	Research
Value (£)	337,376
Status	Completed
Type	Research Grant
Start date	01/03/2013
End date	31/08/2016
Duration	42 months

Abstract

The aim of this project is to expand the understanding of our recently discovered communicative link between central metabolism and ribonucleases (RNases) by investigating the details and level of conservation of the mechanism. Such RNases impact the abundance of many mRNA transcripts and structured RNAs, thereby affecting post-transcriptional gene expression. The control of RNase activities contributes to homeostasis and the response to environmental change and is thus likely to be affected by general metabolic activities. Our recent published data confirms that a key central metabolism intermediate in the Krebs cycle, citrate, affects the activity of the E. coli exoribonuclease, polynucleotide phosphorylase (PNPase) and that conversely, cellular metabolism is distributively affected by the activities of PNPase and the multi-enzyme RNA degradosome complex. With our recent fundamental discovery as a basis, this proposal seeks to investigate the newly identified regulatory interactions in more detail. We will investigate the impact of metabolite-PNPase communication at the transcript level using the standard transcriptomic techniques of microarray analysis and real time RT-PCR. Specifically, we will validate and quantify our earlier microarray analysis and explore the rates of transcript decay. We will then determine whether metabolite-PNPase communication is altered when PNPase is in complex form (i.e. as part of the RNA degradosome) or following mutation of a PNPase-metabolite-binding site. Finally, we will identify whether the metabolite-PNPase communicative link represents a conserved mechanism through the identification and characterisation of homologous RNase-metabolite relationships using computational, biochemical, biophysical and structural techniques. The implications of this work stand to be significant, aiding our appreciation of this recently discovered mechanism and providing us with a comprehensive understanding of metabolite-RNase communication.

Summary

Within living cells a whole series of chemical reactions occur in order to provide the energy the cell needs to sustain life. This series of reactions is collectively known as a cell's metabolism. Understanding how metabolism is controlled within a cell is fundamentally important and is directly applicable to medical, environmental and biotechnological advances. At the present time, some aspects of how metabolism is controlled are understood, but we have recently discovered a whole new control mechanism of key importance. It is already known that messenger molecules (RNA) within a cell play a role in controlling metabolism and that in turn, destroyer molecules (RNA degraders) in the cell keep the number of RNA molecules in check. Our studies have identified that one of the chemicals involved in metabolism, known as a metabolite, interacts with an RNA degrader and affects its ability to destroy RNA. Our work therefore indicates that a full feedback system exists within a cell, with metabolites altering the ability of RNA degraders to destroy RNA, which in turn affects cellular metabolism, which impacts metabolites, which then interact with RNA degraders and so the loop continues. The aim of the proposed work is to investigate the newly identified interactions between metabolism and RNA-degraders in detail. Specifically, our objectives are to answer a number of key questions. What changes occur to the population of messenger molecules within the cell when this mechanism takes place and are some messenger molecules targeted earlier than others? By monitoring the population of messenger molecules can it be seen whether the mechanism changes once the RNA-degraders form larger complex structures with other RNA degraders? If we specifically change the metabolite-recognition site on the RNA-degrader, what happens to the population of messenger molecules and what can this tell us about the mechanism? Is this mechanism of communication between metabolism and RNA-degraders found in all types of cells from simple bacteria to complex animal cells? To answer these questions our research will use a comprehensive state-of-the-art toolset of proven practical and computational biological research techniques. Understanding these additional details about the communication between metabolism and RNA-degraders allows us to take the next step towards realising the full impact of our recent discovery. In the longer term, such knowledge could allow scientists to artificially control metabolism within living cells. For example, simple bacterial cells play an important role in many industrial applications and this artificial metabolic control could optimise their use. This may potentially increase efficiency by reducing energy costs, increasing yields and reducing starting material requirements, all of economic and environmental value. Examples include exploitation within the pharmaceutical industry (e.g. more efficient drug production), the food industry (e.g. improvements in food production) and particularly in relation to environmental concerns (e.g. aiding biofuel production and bioremediation projects). In a similar manner, the artificial control of metabolism within animal cells has the potential to offer far reaching therapeutic benefits.

Impact Summary

The potential impact of the proposed work broadly falls into the three main areas: 1) Impacting the field of systems biology With significant transcriptomic data generated during this proposal, a key immediate impact of research will be within the systems biology field. Our work detailing the specific transcripts altered by the metabolite-RNase communicative link will form the basis for the development of metabolite-RNase-transcript models. It will thus be valuable to computational modelling specialists in universities and research institutes, who will be able to use our transcriptomic data to create models for in silico testing of cellular scenarios, which could subsequently be tested experimentally, potentially by metabolomics specialists. Refinement of such models will ultimately allow it to be possible to predict the effect on the transcriptome of a metabolic change within a cell and hence conduct targeted manipulations of the metabolome with therapeutic and industrial benefits in mind. Overall, this will sustain the knowledge economy by supporting the UK's leading role in Systems biology. 2) Instigation of new research programmes The results of the proposed work will impact research groups in the wider field of post-transcriptional gene regulation control, specifically within academic disciplines such as transcriptomics, metabolomics, biochemistry, biophysics, structural biology, molecular biology, microbiology and cell biology. Such researchers can be found within universities, research institutes, charities and industry. Our proposed work will support the instigation of new research programmes that build on our findings; allowing a further characterisation of the mechanism and a full understanding of its cellular impact to be undertaken. Specifically, should our research identify that the metabolite-RNase mechanism that we have discovered in the bacteria, E. coli, is conserved in the other domains of life i.e. archaea and eukaryotes, then a key investigational element will be to undertake in vivo studies in such organisms. This research will provide new information and scientific advancement thereby enhancing the UK's knowledge economy. Maintaining and expanding research in this manner will contribute towards the skills of researchers involved in such studies. Instigating new research programmes will therefore not only benefit scientific research in knowledge terms but will also provide the UK workforce with trained, highly skilled, researchers. 3) Exploitation within the metabolic engineering domain A major longer term future potential application of this work lies within the metabolic engineering domain. This research to clarify the metabolite-RNase communicative link could provide the basis for manipulation of metabolic processes in bacteria to artificially control RNA decay rates. This metabolic intervention could subsequently be used to optimise cellular processes; increasing efficiency thereby reducing energy costs, increasing yields and reducing starting material requirements - all of which have the potential to offer economic and environmental benefits. This exploitation route would therefore be expected to be highly attractive to the private sector, specifically: - to the pharmaceutical industry, for more efficient synthetic drug production such as insulin - to the food industry, for improved food production - to address environmental challenges, such as improved biofuel production and bioremediation The exploitation of scientific knowledge could support the creation of new processes and products, contributing to the economic prosperity for the companies involved and the economic competitiveness of the UK.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Microbiology, Systems Biology
Research Priority	Systems Approach to Biological research
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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