RNA Binding and Metabolism: Elucidating the Role of Glycolytic Enzymes in Posttranscriptional Gene Regulation

A cell is the smallest unit of an organism. All cells of every organism contain a copy of the same genetic information, which is organised into genes in the form of DNA. During the process of gene expression, DNA is copied to an intermediate molecule called RNA, which can then serve as a template for the synthesis of proteins. Proteins define the shape and function of each cell of the organism.

Physically, RNAs are covered by proteins, so-called RNA-binding proteins. These proteins can remove or rearrange parts of the RNA, store, or deliver it to particular locations within the cell, and ultimately degrade it. They also control when and how messenger RNAs (mRNAs; refers to the class of RNAs that encode proteins) are translated into proteins. If an RNA-binding protein does not work properly, it can lead to malfunction of the cell and ultimately to disease.

Due to their tremendous importance, we and other researchers have used a new experimental approach to catalogue all of the RNA-binding proteins that interact with mRNAs in cells. Besides detecting previously validated RNA-binding proteins, we found that many proteins with other well-established functions, such as enzymes, are also able to interact with RNA. Enzymes are proteins that perform essential chemical reactions; for instance, they provide and control the flow of energy required to keep cells alive. Whilst individual examples of enzymes binding to RNA have been characterised previously, the finding that most or even all enzymes of an important energy-generating pathway, termed glycolysis, interact with RNAs in yeast intrigued us.

During glycolysis, glucose is transformed via a pathway involving several sequential steps, into another chemical. During this process, energy is generated. This important pathway is highly conserved in all organisms and is tightly controlled. In yeast, glycolysis is essential for the generation of ethanol from glucose, a feature of yeast that has been used for centuries for the production of wine, beer and other goods. In humans, the pathway is highly active in cancer cells and thus, provides a target for the development of new strategies for cancer treatment.

Intrigued by our findings that all of the enzymes in this essential pathway bind to RNA, we wish to understand both the basis and the function of these interactions. Therefore, we propose to comprehensively identify the RNA targets for the enzymes of this pathway. We will then investigate where and how they bind to the RNA, and specifically abrogate mRNA-binding sites in cells, to see whether it affects the fate of particular mRNAs. Likewise, we will measure whether abolishing this binding has an effect on the activity of the protein encoded by the mRNAs and the associated pathway.

With our research, we hope to discover previously unrecognised connections between RNA regulation and the chemical reactions that fuel cells. This knowledge is likely to have impact on diverse important aspects of our society, from food-production, to the development of new cancer treatments.

RNA-binding proteins (RBPs) mediate the post-transcriptional control of gene expression. They bind to distinct elements in mRNAs, thereby regulating the processing, localisation, translation and decay of mRNAs. Hundreds of "classical" RBPs bearing one or several characteristic RNA-binding domains (RBDs) have been annotated; however, recent experimental evidence suggests the existence of many additional "unorthodox" RBPs that lack such domains. Interestingly, this novel category of RBPs includes numerous metabolic enzymes. Evidence for a physiological role for enzyme-mRNA interactions has been obtained for individual examples; however, it remains to be determined whether metabolic enzymes play a more general role in post-transcriptional regulation and/or whether it may be relevant for cellular metabolism.

With our proposed research, we wish to fill this knowledge gap by studying RNA-binding functions of metabolic enzymes in budding yeast Saccharomyces cerevisiae, which is the best-annotated eukaryotic organism. Thereby, we will focus on the glycolytic enzymes, for which we have obtained recent data strongly suggesting specific mRNA-binding activity.

To achieve this goal, we will comprehensively profile the RNA targets, and the binding sites therein, for several glycolytic proteins at a transcriptome-wide scale. Enzyme-mRNA interactions will be validated with biochemical assays in vitro and by mutation of the binding sites in vivo. Finally, the generation of mutants will enable us to monitor the implications of specific enzyme-mRNA interactions on post-transcriptional gene regulation and/or intermediary metabolism.

Our studies will reveal previously unrecognised links between post-transcriptional regulation and metabolism. In the longer term, it will generate impact in biotechnological applications and medicine, i.e. in the development of new strategies for the treatment of cancer.

Since our project deals with two fundamental aspects of cell biology, namely RNA regulation and metabolism, it will capture the interest of several groups of beneficiaries outside of the academic research community (already identified in the academic beneficiaries section). In the following, we outline some of these groups, and define how they will benefit from our research (further information is also given in the "Pathways to Impact" attached to this proposal).

i) Industrial biotechnology: Baker's yeast is the organism most commonly used to produce ethanol via the fermentation process, for beer, wine and alcoholic drink production, and in addition it is also used in both food and bioethanol production. Since our research is focused on the regulatory elements of the key pathway in the fermentation process (glycolysis), any new knowledge about the properties and regulation of this pathway will be of significant interest for these industries. In the long term, our research may lead to new strategies to increase the efficiency of the fermentation process, which would deliver substantial economic impact. Of note, members of our department have established contacts to companies working in this field (see pathways to impact for details).

ii) Pharmaceutical companies: In view of the current limitations of cancer chemotherapy, there has been a resurgence of interest in glycolysis, to determine whether tumours could be killed by energy deprivation (Warburg effect). Thus, the understanding of additional activities of glycolytic enzymes could be of great interest when devising new strategies for cancer treatment. Moreover, several drugs targeting glycolytic enzymes are already in clinical trials, and knowledge of additional regulatory devices for these enzymes may allow for further reiteration on these, as well as to the development of new drugs. In the long term, our research may lead to substantial economical and societal impact. AG has established contacts to Pfizer and Novartis, two pharmaceutical companies developing cancer treatments.

iii) UK trained workforce: This proposal includes the training of a PDRA researcher who will acquire new skills and knowledge in RNA biology, enzymes, and metabolism as well as in bioinformatics (e.g. next-generation sequencing data analysis). The PDRA will thus mature into a highly trained researcher who will be able to pursue a career in academic or industrial research. In addition, the PDRA will be in a position to teach high-level techniques to postgraduate students. This will impact in the area of training and delivery of highly skilled researchers.

iv) Undergraduate and postgraduate students: The proposed research will contribute to fundamental theories and concepts underpinning the regulation of gene expression and metabolism. We will impart this new knowledge to undergraduate students, via teaching activities and research project supervision.
v) The general public: Since our research will have such widespread implications, ranging from medicine/health to the food/beverage/biofuel industry by connection two previously separated fields of research, (namely RNA biology and metabolism), we expect that our result will attract substantial interest from the media and the general public. Hence, our research will have impact in the broader areas of public engagement, public health and societal issues.

Finally, by completing this project, we will reinforce the UK's position in the field of RNA research and metabolism, contributing to the attraction of talented undergraduate students and postgraduate researchers to UK universities. It also enhances our collaborations with international leading scientists and thus, it will also impact in the area of international development.