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

Lead Research Organisation: University of Surrey
Department Name: Microbial & Cellular Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description We address the question whether enzyme-mRNA interactions have implications in the post-transcriptional regulation of mRNA fate and/or whether they are directly or indirectly connected to the control of central intermediary metabolism. Thereby, we particularly focus on the glycolytic enzymes in the yeast S. cerevisiae, for which we have obtained evidence that they all bind to mRNAs (Matia-Gonzalez et al. 2015, NSMB).
We have started our investigations with phosphofructokinase, which is a key regulator of the glycolytic pathway. Yeast Pfk is comprised of two paralogous (termed Pfk1 and Pfk2) that form a hetero-octamer. Another glycolytic enzyme, 3-phosphoglycerate kinase (Pgk1) is also under investigation.
In the following, a brief overview on the status of research on the different aims is given:
Aim 1: To determine the RNA targets of glycolytic enzymes on a global-scale, namely for Pfk1, Pfk2 and Pgk1, we initially applied a novel infrared cross-linking immunoprecipitation protocol (irCLIP; Zarnegar et al. 2017, Nat. Methods). However, even after tedious optimisation processes to adapt the protocol to yeast, the final data from these experiments were sub-optimal and not conclusive. We therefore opted for a more conservative approach, using formaldehyde-assisted crosslinking and RNA-protein immunoprecipitation followed by high-throughput sequencing (fRIP-seq) (Hendrickson et al. 2016). Performing duplicate experiments and including mock control samples, we could identify hundreds of RNA targets for Pfk1, Pfk2 and Pgk1 that substantially overlapped. Importantly, the identified RNA targets share common functional and structural attributes, which is a hallmark of many proteins interacting with RNAs (forming a so-called RNA regulon). Furthermore, we identified particular RNA elements that are enriched among the RNA targets, providing a potential platform for interaction. Besides determining the RNA targets, we also performed mass-spectrometry (MS) analysis to identify interacting proteins in crosslinked, non-crosslinked and RNA-digested samples.
Aim 2: To understand the molecular determinants of enzyme-RNA interactions, we have been performing a series of in vitro RNA-protein interaction assays. These assays confirmed interactions of metabolic enzymes with particular RNA fragments identified in aim 1. We have also been producing and purified the recombinant enzymes expressed in E. coli for use in RNA-binding assays. However, we realised that expression of these proteins is not trivial and that the selected glycolytic enzymes are rather unstable and need special buffers for storage. To further test for potential associations between enzymatic activity and RNA binding, we have been collaborating with Prof. Juergen Heinisch (University of Osnabruck, Germany), who is an expert on yeast Pfk proteins. The PI visited Prof. Heinisch's laboratory in 2017 and established enzymatic activity assays in the laboratory. At this point, we could not observe direct links between RNA-binding and enzymatic activity.
Aim 3: We pursued work to understand the physiological impact of RNA-binding properties of metabolic enzymes. We confirmed that RNA-interaction of certain glycolytic enzymes is dependent on the metabolic state of the cells, while others seem to be less dependent. We also observed that Pfk proteins are associated with polysomes, and we have obtained data suggesting the Pfk proteins could act as translational regulator of certain mRNA targets. In the last year, we have also obtained data that indicates a cell-cycle defect of one Pfk paralog in yeast. In collaboration with Dr. Matteo Barberis, a recently appointed Reader of Systems Biology at University of Surrey and an international recognised expert in yeast cell-cycle control, we are currently further characterizing the phenotype. Finally, we have obtained data that shows evolutionary conservation of enzyme-RNA interactions, which suggests important biological functions. On this line, we have published a review in 2018 to document the evolutionary conservation of RNA-binding of metabolic enzymes (Albihlal & Gerber 2018, FEBS Lett.).

In conclusion, we have obtained data that suggest specific functions of glycolytic enzymes in posttranscriptional gene regulation. While there are still some open question that need to be resolved, we are preparing a manuscript to describe the data obtained from this study for publication in a high-quality open access journal.
Exploitation Route We have been in contact with several collaborators that expressed their interest in studying structural and/or functional aspects of enzyme RNA interactions. Our yeast fRIP protocol has been requested by several laboratories. Although not published yet, we have already shared protocols.

The data obtained from this study has been presented at invited lectures given by the PI (e.g. CEITECH, Brno, Czech; UCL, London, Max-Planck, Muenster, Germany) and was presented at several conferences (EMBO, Translation UK 2018). During the last year, the PDRA working on the project (WA) presented the work at Translation UK 2019 and RNA UK 2020 and obtained a lot of attention and interest. Based on our results, we submitted collaborative grant proposals to HFSP and the ERC.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Royal Society Wolfson Research Merit Award
Amount £50,000 (GBP)
Funding ID WM170036 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2017 
End 08/2022
 
Description Collaborator - Juergen Heinisch 
Organisation University of Osnabrück
Country Germany 
Sector Academic/University 
PI Contribution Juergen Heinisch is biochemist and expert for the enzyme phosphofructokinase (Pfk) in yeast. We contacted him to discuss our recent results indicating a relation between the metabolic state and RNA-binding activity of yeast and human Pfk enzymes. He is glad to collaborate with us and provided useful materials and reagents. Furthermore, he offered our Postdoc to join his laboratory to learn how to perform biochemical assays with said enzyme.
Collaborator Contribution He contributed reagents and materials that are useful for our research (e.g. plasmids). Furthermore, he performed Pfk activity assays with cell extracts prepared from cells grown in different media. He is also testing activity of recombinant proteins and how it may be modified by addition of RNA.
Impact The collaboration has only recently started and is currently ongoing.
Start Year 2016
 
Description Collaborator - Mihaela Zavolan, Biozentrum, University of Basel 
Organisation University of Basel
Country Switzerland 
Sector Academic/University 
PI Contribution Mihaela provides advice on bioinformatics matters for RNA-seq data analysis. We exchange protocols and materials across several project. In particular, we met several times to discuss improvements related to CLIP methods (a method to profile the RNA-binding sites of RNA-binding protein in vivo). On our side, we are currently performing TRIP experiments to identify proteins associated with specific mRNAs. Data shall then be further analysed by the Zavolan group.
Collaborator Contribution As mentioned above, Mihaela provides advice on bioinformatics matters for RNA-seq data analysis. We exchange protocols and materials across several project. In particular, we met several times to discuss improvements related to CLIP methods (a method to profile the RNA-binding sites of RNA-binding protein in vivo). We also submitted a common grant application in the last year. We are currently collaborating in project to implement TRIP for study the translation of different mRNAs.
Impact We have published several collaborative papers in the past. We have also co-edited a special issue in FEBS Letters in 2018.
Start Year 2007
 
Description Innovation of Health Partnership building event, School of Veterinary Medicien, University of Surrey 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact The event was thought to build new relations with SMEs in the UK to propagate collaborations and networks. Discussed business ideas related to one of my patent applications with industry representatives.
Year(s) Of Engagement Activity 2017
 
Description Participation at several Open/Applicant days at my institution 
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 Discussion with many prospective students and their parents about the University studies and my research. This sparked questions and interest and increased the interest in my and related subject areas.
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017,2018,2019
 
Description Translation UK 2016 - organiser 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Translation UK is an yearly held scientific conference to discuss latest topics in protein synthesis and post-transcriptional gene regulation. It gathers an UK but also international audience and is complemented by invitation of renowned international keynote speakers. It was organised in collaboration with the Biochemical Society.
Year(s) Of Engagement Activity 2016
URL https://www.biochemistry.org/Events/tabid/379/MeetingNo/SA183/view/Conference/Default.aspx