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.
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.
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.
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.
People |
ORCID iD |
| Andre Gerber (Principal Investigator) |
Publications
Albihlal WS
(2018)
Unconventional RNA-binding proteins: an uncharted zone in RNA biology.
in FEBS letters
Gerber AP
(2021)
RNA-Centric Approaches to Profile the RNA-Protein Interaction Landscape on Selected RNAs.
in Non-coding RNA
Iadevaia V
(2020)
Tandem RNA isolation reveals functional rearrangement of RNA-binding proteins on CDKN1B/p27Kip1 3'UTRs in cisplatin treated cells.
in RNA biology
Martino F
(2022)
The mechanical regulation of RNA binding protein hnRNPC in the failing heart.
in Science translational medicine
Matia-González AM
(2021)
Biochemical approach for isolation of polyadenylated RNAs with bound proteins from yeast.
in STAR protocols
Matia-González AM
(2021)
Oxidative stress induces coordinated remodeling of RNA-enzyme interactions.
in iScience
Zavolan M
(2018)
Mirroring the multifaceted role of RNA and its partners in gene expression.
in FEBS letters
| Description | We addressed 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 focused 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 focused our investigations towards yeast 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) has also been under investigation. In the following, I summarise some of our main findings: 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 that shared 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, and we recently confirmed the interactions with proteins in vitro (see below). Finally, we 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 performed in vitro RNA-protein interaction assays. These assays confirmed interactions of Pfk enzymes with particular RNA fragments identified in aim 1. We have also produced and purified the recombinant Pfk's 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. Nevertheless, we were able to obtain consistent RNA-binding data, showing that yeast Pfk protein can directly interacts with RNA. On this line, we recently tested the related human phosphofructokinase enzymes and found that they also interact with RNA specifically bind RNA in RNA electrophoretic mobility shift assays (REMSA). An investigation of the RNA properties and RNA targets of human proteins shall be the subject for further investigation in the future. To test for potential associations between enzymatic activity and RNA binding, we are 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. Testing a series of mutant enzymes, our results further suggest that RNA-binding is not directly dependent on enzymatic activity. Aim 3: We pursued work to understand the physiological impact of RNA-binding properties of metabolic enzymes. First, we confirmed that RNA-interaction of certain glycolytic enzymes is dependent on the metabolic state of the cells in yeast as well as in human cells. Secondly, we found that Pfk proteins are associated with polysomes, and we have obtained additional data suggesting that yeast Pfk can act as a translational regulator. Thirdly, systematic analysis of proteome changes in pfk mutants corroborated changes related to experimentally determined mRNA targets (with help of Alexander Schmidt, Biozentrum, Basel, Switzerland). Finally, in collaboration with Dr. Matteo Barberis, a Reader of Systems Biology at University of Surrey and an international recognised expert in yeast cell-cycle control, we have monitored a specific cell-cycle defect in one of the yeast Pfk paralog. This is interesting as it suggests that the phenotype is likely caused by aberrant regulation of the mRNA targets for this enzyme, providing a directly link between metabolic status of cells and post-transcriptional control of cell-cycle via a glycolytic enzyme. As noted above, along the proposed yeast studies, we have obtained preliminary data that strongly suggests evolutionary conservation of enzyme-RNA interactions (yeast-human) in general, and of Pfk-RNA interaction in particular (Albihlal & Gerber 2018, FEBS Lett.). In the future, we wish to further investigate the role of human PFK proteins in RNA metabolisms and its potential implications in disease (i.e. cancer biology). We wish to note, that the main body of work of this grant is in final stages for submission in a high-impact journal. We thought that it will be more valuable to the community to have all information in one larger paper than publishing individual smaller pieces separately. |
| 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). The PDRA working on the project (WA) presented the work at Translation UK 2019, RNA UK 2020, RNA society meeting 2020 and at invited seminars. We also submitted collaborative grant proposals to HFSP and the ERC. Of note, the primary results are not published yet (manuscript in preparation). We assume that our results will have a significant impact and attracts the attention in the field. |
| Sectors | Healthcare Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
| Description | During the last years, the interest in unconventional RNA-binding proteins has been increasing, though it has been recognised that working with those can be challenging and need experimental adaptations. Along our research interests supported with this grant, the RNA-binding functions for several metabolic enzyme have been further characterised by well-renowned laboratories. Overall, these studies including our own suggest that enzyme RNA-interaction have great impact for controlling cell homeostasis, proliferation and differentiation. Their implications in disease are not well explored but our own data suggests new links between glucose metabolism and cell proliferation. As such, the results from out study have been discussed with industrial stakeholder at various occasions (incl. smaller conferences organized by Proventa and Oxford Global which bring together academics and industrial partners). We are also considering a patent on recent findings. We recently made a very 'unexpected' discovery in regard of the function for phosphofructokinase (PFK, a glycolytic enzyme) in RNA metabolisms (which was a main subject of this funded research) and which was obtained with new international collaborators. Due to its importance, we have undertaken further research funded by other means to integrate those findings in final manuscript which is currently being prepared. As such, the latest outcomes are surprising and should be further investigated over the next years, possibly with additional collaborators. Of our findings are transferrable to human (which is currently tested), we believe it could become highly relevant as a new device for controlling cell proliferation in cancer cells; and being of interest to embark into drug screening which is feasible to simple assay set-up to monitor the RNA-related activity of PFK. Overall, the results of our study are currently used by various collaborator groups for further investigations, ranging from biochemical characterisations, structural investigations etc. Contacts with industrial partners are taken on board. Upon publication of the final study expected, our study may attract significant interest from academic but also from industrial stakeholders |
| First Year Of Impact | 2020 |
| Sector | Agriculture, Food and Drink,Creative Economy,Education,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 | 08/2017 |
| End | 08/2022 |
| Title | GSE151522: Transcriptome-wide Profiling of Yeast Phosphofructokinase 1 and 2 RNA targets with fRIP-seq |
| Description | The RNA targets of yeast Saccharomyces cerevisiae Pfk1 and Pfk2 were mapped on a transcriptome scale using formaldehyde-assisted crosslinking and RNA-protein immunoprecipitation followed by high throuput sequencing (fRIP-seq). Tandem affinity purification (TAP) tagged Pfk1 and Pfk2 were usesd to capture the transcritps bound by Pfk1 and Pfk2. We also included a mock IP control i.e. wild type treated exactly the same way as the fRIP-seq samples to account for non-specific RNA interactions with the beads. Moreover, we ran total RNA-seq on wild type control for data normalization. We found that Pfk1 and Pfk2 bind mainly to mRNAs that code for proteins invovled in distinct functions . Our data suggests that, besides their well-characterized functions as glycolytic enzymes, they perfom additional functions as RNA-binding involved in posttranscriptional regulation of gene expression. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2023 |
| Provided To Others? | Yes |
| Impact | Raw RNA seq data that is accessible to community via GEO151522. |
| URL | https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/geo/query/acc.cgi?acc=GSE151522 |
| Title | PXD005943: Saccharomyces cerevisiae mRBPome in oxidative stress conditions |
| Description | Yeast S. cerevisiae proteomics data have been deposited in the ProteomeXchange Consortium database under accession code PXD005943. This data is associated with the following publication: Ana M Matia-González, Ibtissam Jabre, Emma E Laing, André P Gerber (2021)Oxidative stress induces coordinated remodeling of RNA-enzyme interactions. iScience 24(7), 102753 |
| Type Of Material | Database/Collection of data |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | Data has been deposited and allows researchers to extract raw data for analysis of yeast mRNA binding proteins under mild oxidative stress conditions. |
| URL | http://www.ebi.ac.uk/pride/archive/projects/PXD005943 |
| Title | PXD008498: MS data - cisplatin induces the rearrangement of RBPs on CDKN1B/p27 mRNA |
| Description | The mass spectrometry proteomics data deposited to ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD008498. Associated with the manuscript: Iadevaia, V., Wouters, M.D., Kanitz, A., Matia-González, A.M., Laing, E.E., Gerber, A.P. (2020) Tandem RNA isolation reveals functional rearrangement of RNA-binding proteins on CDKN1B/p27Kip1 3'UTRs in cisplatin treated cells. RNA Biol. 17(1), 33-46. https://doi.org/10.1080/15476286.2019.1662268 |
| Type Of Material | Database/Collection of data |
| Year Produced | 2019 |
| Provided To Others? | Yes |
| Impact | Raw data deposited to ProteomeXchange Consortium to make it accessible to other researchers. |
| URL | https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD008498 |
| Title | Tandem RNA isolation reveals functional rearrangement of RNA-binding proteins on CDKN1B/p27Kip1 3'UTRs in cisplatin treated cells |
| Description | Post-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. While recent global RNA interactome capture experiments expanded the repertoire of cellular RBPs quiet dramatically, little is known about the assembly of RBPs on particular mRNAs; and how these associations change and control the fate of the mRNA in drug-treatment conditions. Here we introduce a novel biochemical approach, termed tobramycin-based tandem RNA isolation procedure (tobTRIP), to quantify proteins associated with the 3'UTRs of cyclin-dependent kinase inhibitor 1B (CDKN1B/p27Kip1) mRNAs in vivo. P27Kip1 plays an important role in mediating a cell's response to cisplatin (CP), a widely used chemotherapeutic cancer drug that induces DNA damage and cell cycle arrest. We found that p27Kip1 mRNA is stabilized upon CP treatment of HEK293 cells through elements in its 3'UTR. Applying tobTRIP, we further compared the associated proteins in CP and non-treated cells, and identified more than 50 interacting RBPs, many functionally related and evoking a coordinated response. Knock-downs of several of the identified RBPs in HEK293 cells confirmed their involvement in CP-induced p27 mRNA regulation; while knock-down of the KH-type splicing regulatory protein (KHSRP) further enhanced the sensitivity of MCF7 adenocarcinoma cancer cells to CP treatment. Our results highlight the benefit of specific in vivo mRNA-protein interactome capture to reveal post-transcriptional regulatory networks implicated in cellular drug response and adaptation. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2022 |
| Provided To Others? | Yes |
| Impact | Annotated dataset for research use. |
| URL | https://tandf.figshare.com/articles/dataset/Tandem_RNA_isolation_reveals_functional_rearrangement_of... |
| Title | Tandem RNA isolation reveals functional rearrangement of RNA-binding proteins on CDKN1B/p27Kip1 3'UTRs in cisplatin treated cells |
| Description | Post-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. While recent global RNA interactome capture experiments expanded the repertoire of cellular RBPs quiet dramatically, little is known about the assembly of RBPs on particular mRNAs; and how these associations change and control the fate of the mRNA in drug-treatment conditions. Here we introduce a novel biochemical approach, termed tobramycin-based tandem RNA isolation procedure (tobTRIP), to quantify proteins associated with the 3'UTRs of cyclin-dependent kinase inhibitor 1B (CDKN1B/p27Kip1) mRNAs in vivo. P27Kip1 plays an important role in mediating a cell's response to cisplatin (CP), a widely used chemotherapeutic cancer drug that induces DNA damage and cell cycle arrest. We found that p27Kip1 mRNA is stabilized upon CP treatment of HEK293 cells through elements in its 3'UTR. Applying tobTRIP, we further compared the associated proteins in CP and non-treated cells, and identified more than 50 interacting RBPs, many functionally related and evoking a coordinated response. Knock-downs of several of the identified RBPs in HEK293 cells confirmed their involvement in CP-induced p27 mRNA regulation; while knock-down of the KH-type splicing regulatory protein (KHSRP) further enhanced the sensitivity of MCF7 adenocarcinoma cancer cells to CP treatment. Our results highlight the benefit of specific in vivo mRNA-protein interactome capture to reveal post-transcriptional regulatory networks implicated in cellular drug response and adaptation. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2019 |
| Provided To Others? | Yes |
| Impact | Dataset made accessible to research community |
| URL | https://tandf.figshare.com/articles/dataset/Tandem_RNA_isolation_reveals_functional_rearrangement_of... |
| Title | Tandem RNA isolation reveals functional rearrangement of RNA-binding proteins on CDKN1B/p27Kip1 3'UTRs in cisplatin treated cells |
| Description | Post-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. While recent global RNA interactome capture experiments expanded the repertoire of cellular RBPs quiet dramatically, little is known about the assembly of RBPs on particular mRNAs; and how these associations change and control the fate of the mRNA in drug-treatment conditions. Here we introduce a novel biochemical approach, termed tobramycin-based tandem RNA isolation procedure (tobTRIP), to quantify proteins associated with the 3'UTRs of cyclin-dependent kinase inhibitor 1B (CDKN1B/p27Kip1) mRNAs in vivo. P27Kip1 plays an important role in mediating a cell's response to cisplatin (CP), a widely used chemotherapeutic cancer drug that induces DNA damage and cell cycle arrest. We found that p27Kip1 mRNA is stabilized upon CP treatment of HEK293 cells through elements in its 3'UTR. Applying tobTRIP, we further compared the associated proteins in CP and non-treated cells, and identified more than 50 interacting RBPs, many functionally related and evoking a coordinated response. Knock-downs of several of the identified RBPs in HEK293 cells confirmed their involvement in CP-induced p27 mRNA regulation; while knock-down of the KH-type splicing regulatory protein (KHSRP) further enhanced the sensitivity of MCF7 adenocarcinoma cancer cells to CP treatment. Our results highlight the benefit of specific in vivo mRNA-protein interactome capture to reveal post-transcriptional regulatory networks implicated in cellular drug response and adaptation. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2022 |
| Provided To Others? | Yes |
| Impact | Dataset made accessible to research community. |
| URL | https://tandf.figshare.com/articles/dataset/Tandem_RNA_isolation_reveals_functional_rearrangement_of... |
| Description | Collaborator - Dierk Niessing (University of Ulm) |
| Organisation | University of Ulm |
| Country | Germany |
| Sector | Academic/University |
| PI Contribution | AG has a long-standing collaboration with Dierk Niessing. In the frame of the allocated grant projects, AG has contacted Dierk on technical issues for measureing RNA-protein interactions and whether there is an interest in solving the struture of enzyme-RNA complexes. AG has also advice Dierk in a related project on RIP-chip experiments and data interpretation. Both group are keen to embark into a long term collaboration to study biophysical properties of RNA-enzyme interactions. |
| Collaborator Contribution | Dierk's lab has long-standing experties in protein cristallography and solved numerous RNA-protein complexes. His lab has also further optimized and produced good amounts of recombinant enzymes that are used in our lab for RNA-protein interaction and enzymatic assays. We hope to engage in a long term collaboration based on the results obtaiined from associated project grants. |
| Impact | Output are to come. We though have initiated some collaborative work years ago and a paper describing the outcome was submitted during the last year. However, since the manuscript was rejected, further studies are planned to improve the impact and for resubmisssion soon. A manuscript on a glycolytic enzyme is currently in preparation. Continued support with in kind contributions have been confirmed. |
| Start Year | 2020 |
| Description | Collaborator - Dr. Alexander Schmidt, Biozentrum, Basel (Yeast Proteomics) |
| Organisation | University of Basel |
| Department | Biozentrum Basel |
| Country | Switzerland |
| Sector | Academic/University |
| PI Contribution | We got in contact with Alex in the frame of a common grant application with Mihaela Zavolan, University of Basel. Alex heads the MS facility at the University of Basel and conducted several yeast proteomics analysis. |
| Collaborator Contribution | Alex performed some yeast proteomics TMT analysis for pkf mutants for us. Data was processed by Alex and sent to us for further analysis. |
| Impact | We submitted common grant applications (with Mihaela Zavolan) to HFSP and ERC. Alex adviced on and performed yeast proteomics analysis. He will be a co-author on upcoming publications that include MS analysis from his facility. |
| Start Year | 2020 |
| Description | Collaborator - Giancarlo Forte, ICRC, Brno, Czech Republic |
| Organisation | International Clinical Research Center of St. Anne's University Hospital Brno |
| Country | Czech Republic |
| Sector | Public |
| PI Contribution | We have been advising a PhD student (Fabiana Martino) in Prof. Fortes laboratory to establish the formaldehyde-based RNA-binding protein immunoprecipitation (fRIP) in mouse heart tissue samples. We further adviced on RNA-seq and general data analysis. A manuscript describing our collaborative research is currently under revision for publication in high-profile journal. The results from this study and the optimised protocol are key for likewise determination of RNA-protein interactions in tissues. |
| Collaborator Contribution | The partner conducted the experiments after our input for modification. Our advice in embedded in a larger study that revealed the function of a mechano-regulated RNA-binding protein the bridges ECM remodelling and mRNA homeostasis in heart failure. |
| Impact | A common mansucript describing the results has been published in Science Transl. Medicine (Martino F. et al. 2023, Science Transl. Med). |
| Start Year | 2019 |
| 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 collaborated with us over the past years and provided useful materials and reagents. AG joined his lab in 2017 to perform enzymatic assays. |
| 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 results from this collaboration has been presented at several scientific conferences and is currently finalised for publication in a high-quality journal. |
| 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). |
| 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. |
| Impact | We have published several collaborative papers in the past. We have also co-edited a special issue in FEBS Letters in 2018: Zavolan, M., Gerber, A.P. (2018) Mirroring the multifaceted role of RNA and its partner in gene expression. FEBS Lett. 592(17), 2825-2827. We submitted a common grant appication to HFSP and ERC Sinergia grant. Unfortunately, the applications were not successful. |
| 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 | Moderator at SMI workshops |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | I moderated a round-table discussion on timely research topics (e.g. RNA Therapeutics). The session was attended by researchers and key personal from industry and academia. It involved brainstorming of current and future developments in the field, liasing industrial and academic stakeholder. In 2020, I presented our research at Oxford Global, Biological Series (28. August 2020, Online) - talk. Proventa International, 20. Oct. 2020, Zurich - virtual with a round-table discussion about RNA Therapeutics. |
| Year(s) Of Engagement Activity | 2019,2020 |
| 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 |