Developing tools to investigate combinatorial control of mRNA metabolism

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

Abstract

Our body consists of more than 200 different cell-types that have different sizes, forms, and functions. For example, skin cells are flat and protect our body, whereas neurons can be very long and transmit signals from distant parts of the body to our brain. Nevertheless, all cells contain the same genetic information, which is organized into genes. What makes the cells unique and different from one to another is which genes are turned on or off. When this switch does not work properly, it can lead to developmental defects or diseases such as cancer.

The genetic information is stored in the form of DNA. The DNA is then copied to a molecule called RNA, which is the template for the synthesis of proteins in a process called translation. The proteins make our cells how they look like and what they do.

RNAs are not naked in a cell but rather covered by several proteins, so-called RNA-binding proteins. These RNA-binding proteins can remove or rearrange parts of the RNA, store or deliver them to particular locations, and ultimately destroy the RNA. They also control how efficiently RNAs are translated into proteins. RNA-binding proteins therefore act as a control tower directing the fate of RNA, being stored, translated or destroyed. As a consequence, if a RNA-binding protein does not work properly, it can lead to diseases.

Besides the RNA-binding proteins, research in the last years revealed that there are certain classes of RNAs - so called non-coding RNAs - that can bind to and regulate other RNAs. RNAs are therefore combinatorially controlled by both RNA-binding proteins and non-coding RNAs.

Therefore, it is of immense interest to know, which proteins and non-coding RNAs interact with RNA and how this may be changed in case of a disease. Nevertheless, accessing this information is challenging as researchers lack simple and robust tools to investigate it.

Our objective is to develop these tools and comprehensively identify the proteins and non-coding RNAs that are bound to RNAs. We first aim to get a general view of all the proteins that interact with RNA in a cell. We will then engineer a handle on a particular RNA to pull it out and look for the proteins and other RNAs that sit on it. We will then have a close look how the composition of these 'trans-acting factors' changes upon conditions that simulate the environment in cancer cells.

One way to establish a new tool for research is to test it in a model which is simpler to handle than human cells. We will therefore first establish the tool in the bakers yeast, which is a single-celled organisms called Saccharomyces cerevisiae. We then go one step ahead and establish it human cells in order to identify the RNA-binding proteins and non-coding RNAs that regulate RNAs with pivotal functions in cancer.

At the end, we expect a better understanding how combinations of RNA-binding proteins and non-coding RNAs affect the fate of RNAs. We hope that this insight will give us important clues about how the production of proteins can go wrong to play a critical role in cancer cells. Ultimately, this may lead to new targets for drug development and the treatment of human disease.

Technical Summary

The fate of mRNAs in the cytosol is determined by RNA-binding proteins and non-coding RNAs such as microRNAs. Despite the major roles of these trans-acting factors for RNA metabolism, we still lack sophisticated tools to comprehensively identify the RNA-binding proteins and RNAs that combinatorially control the fate of mRNAs.

We therefore aim to develop these tools to comprehensively identify the proteins and RNAs that interact with mRNAs in the yeast Saccharomyces cerevisiae and in human cells. Thereby, we will implement global and specific approaches. On the global level, we will define the set of proteins that are bound to native polyadenylated mRNAs and therefore, we will combine RNA-protein crosslinking with an established method for the purification of mRNAs via oligo-dT coupled magnetic beads, which is then followed by in-depth mass spectrometry analysis of bound RNA-binding proteins. We will then identify the trans-acting factors from normal and stress-treated cells, which will provide a comprehensive view of the composition and dynamics of mRNA binding proteins - the mRNA-protein interactome.

For the targeted approach, we will establish a new tandem RNA affinity purification strategy that combines RNA aptamer based purification with an antisense oligonucleotide approach to identify the set of proteins or RNAs that specifically bind to selected messages - or the 3' UTR thereof - with mass spec and sequencing, respectively. We will establish and optimize the method on well-characterized bud-tip localized mRNAs in yeast, and investigate the complement of proteins and microRNAs that are implicated in combinatorial control of cancer-related messages in human cells.

The project should provide a valuable platform to study post-transcriptional control of mRNAs. We expect to discover new RNA-binding proteins and principles of combinatorial control. At the end, it could even provide new targets for the development of drugs for cancer treatment.

Planned Impact

Who will benefit?

This project is primarily a basic research project, which nevertheless is likely to lead to new insights of interest to several groups of users and beneficiaries outside of the academic research community (already identified in the previous section). In the medium term a wider group of beneficiaries are expected:

1) Pharmaceutical/Biotechnological companies involved in drug discovery. Several small molecules that target the post-transcriptional control of aberrantly expressed messages are currently developed and/or in clinical trials (e.g. PTC Therapeutics). Our RNA affinity purification method could identify the targets of these drugs. Therefore, the method may become of considerable interest to the biotech industry that develops small-molecules to target gene expression regulatory circuits or that work in the broader field of gene therapy.

2) The UK trained workforce will benefit from this proposal through the training of a PDRA researcher who will acquire new skills in RNA biology, systems biology, proteomics and two model systems (yeast, human cell culture) from the combined expertise of the applicants.

3) As the applicants teach in a research-led environment, undergraduate and postgraduate students will benefit from hearing about this work.

4) The general public in terms of gaining a better understanding of RNA regulation and its implications in human disease.

How will they benefit?

1) Our results will be of significant interest to clinicians and/or pharmaceutical industries who are seeking to develop new therapies for cancer treatment. VEGF is currently the major target for cancer therapy and therefore, information about its post-transcriptional control could provide new entry points for the development of new drugs for treatment. Moreover, our general ambition to explore the interactive space of RNA-binding proteins will probably lead to the discovery of unexpected proteins that interact with RNA. In this regard, we recently identified several metabolic enzymes that also bind to RNA and thus, they could are likely to have dual functions. Many drugs are derived against enzymes and so, unraveling additional functions for them is important to understand drug action.

2) By developing skills in RNA biology/ proteomics/ systems biology, the PDRA will 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 delivering highly skilled people.

3) The knowledge obtained though this research will contribute to fundamental theories and concepts underlying gene expression regulation. We will impart this new knowledge to undergraduate students, via teaching activity and research project supervision. School pupils will also benefit though the numerous engagement activities we undertake to promote RNA biology.

4) It is anticipated that the current application will be of great interest to the media and the public. This work not only provides an exciting scientific story, but the research also relates to public health and could have long lasting implications for disease treatment, therefore impacting in the area of Public engagement, public health and societal issues.

Finally, by completing this novel project, we will reinforce the UK's position in the field of RNA research, contributing to attract more talented undergraduate students and postgraduate researchers to UK universities and stimulate research. Thus, this project will also impact in the International development area.

Publications

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Description The aims of this project were: (1) to develop methodology to define the set of proteins that are bound to native polyadenylated mRNAs under specific cellular conditions - referring to the mRNA-binding proteome (mRBPome) and 2) to identify the proteins/RNAs bound to selected mRNAs or more specifically, the 3-'UTR thereof.

Aim 1: We have completed the 1st aim of the proposed research and published the results in a high-impact peer-reviewed journal (Matia-Gonzalez et al. 2015, NSMB). Therein, we combined RNA-protein crosslinking with an established method for the purification of mRNAs via oligo-dT coupled magnetic beads, which was followed by in-depth mass spectrometry (MS) analysis of bound proteins (supported by the MS facility at University of Bristol). We defined a repertoire of 765 and 594 proteins that reproducibly interacted with polyadenylated RNAs in the unicellular eukaryote yeast Saccharomyces cerevisiae and in multicellular nematodes (Caenorhabditis elegans; with grant support from the Swiss National Science Foundation). We also reported the differential association of mRNA-binding proteins (mRPBs) upon induction of apoptosis in C. elegans L4-stage larvae, suggesting stress- specific remodelling of proteins bound to mRNAs on a global-scale. Intriguingly, we found that most proteins composing mRBPome's, including components of early metabolic pathways and the proteasome, were evolutionarily conserved between yeast and C. elegans. We speculated - on the basis of our evidence that glycolytic enzymes could bind distinct glycolytic mRNAs - that enzyme-mRNA interactions relate to an ancient mechanism for post-transcriptional coordination of metabolic pathways that was established during the transition from the early 'RNA world' to the 'protein world'. The results from this study were used to frame a successful BBSRC grant application (BB/N008820/1).

As suggested in our original application, we next analysed changes in the mRNA-binding proteome (mRBPome) upon application of oxidative stress (implemented through hydrogen peroxide) to yeast cells. We found changes in the mRNA associations of subsets of RBPs, some of them known to have stress-related functions. Possibly more interesting, we found substantial rearrangement of particular classes of metabolic enzymes upon exposure to oxidative stress. Our recent reanalysis of the data showed also substantial paralogue-specific RBP associations with mRNAs upon stress. We validated these findings for a subset of identified proteins with classical tools, such as immunoblot analysis. These results provided key preliminary data for another BBSRC grant BB/S017747/1. We are are currently at final stages to submit this work to a high-quality peer-reviewed journal.
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Aim 2: We have established a variety of RNA affinity purification strategies that combine RNA aptamer-based purification and/or antisense oligonucleotides to identify the set of proteins that specifically bind to selected messages - or the 3' UTR thereof - with mass-spectrometry (MS).

We established a Tandem RNA Isolation Procedure (TRIP) that enables rapid purification of in vivo formed messenger ribonucleoprotein (mRNP) complexes from cells or entire organisms without the need for genetic modification (Matia-Gonzalez et al. 2017, Methods, Iadevaia V. et al. 2018, JoVE). TRIP relies on the purification of polyadenylated mRNAs with oligo(dT) beads from cellular extracts, followed by the capture of specific mRNAs with 3'-biotinylated 2'-O-methylated antisense RNA oligonucleotides, which are recovered with streptavidin beads. TRIP was applied to isolate in vivo crosslinked mRNP complexes from yeast, nematodes and human cells for subsequent analysis of RNAs and bound proteins. The method provides a basis for adaptation to other types of polyadenylated RNAs, enabling the comprehensive identification of bound proteins/RNAs, and the investigation of dynamic rearrangement of mRNPs imposed by cellular or environmental cues. After publication of the original TRIP method (Matia-Gonzalez et al. 2017, Methods), we published a detailed description of TRIP in a prominent Video Journal (Iadevaia, V, et al. 2018, JoVE, open access) to make it accessible to the wider research community.

We next focused on the human cyclin-dependent kinase inhibitor 1B (CDKN1B/p27kip) mRNA, which bears several well characterised binding sites for regulatory mRNA-binding proteins in the 3'UTR. CDKN1B, also termed p27/kip1, codes for an important tumour suppressor that is extensively regulated at the transcriptional, post-transcriptional and protein level. Increased levels of p27 have been reported to correlate with cisplatin resistance (CP; a chemo-therapeutic agent that binds DNA and induces DNA strand crosslinks and is widely for treatment of cancer). In HEK293 cells (an embryonic kidney cell line), which are sensitive to CP treatment, we observed that CDKN1B mRNAs levels increased whereas protein levels decreased upon CP treatment. Furthermore, we found that the mRNA is stabilised up CP treatment through elements in its 3'UTR. In contrast, likewise CP treatment of breast cancer cell lines (MCF7) had no significant effect on p27 expression, which correlated with better survival of these cells upon CP treatment. To explore the underlying mechanisms, we developed an alternative TRIP protocol (termed tobramycin-based TRIP, tobTRIP) combined with mass-spectrometry to systematically identify the RNA-binding proteins (RBPs) bound to the 3'UTR of CDKN1B mRNAs in CP-treated versus non-treated HEK293 cells in vivo (Iadevaia V. et al. 2020, RNA Biol.). Of note - to our knowledge - this was the first study measuring the dynamics of RNA-binding protein associations on specific mRNAs upon changing conditions (i.e. drug treated vs. untreated cells). At the end, we identified more than 50 interacting RBPs, many of them 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. Overall, these results corroborated the importance of post-transcriptional control in cellular drug responses and suggests KHSRP as potential target to modulate CP resistance of certain cancer cells (Iadevaia et al. 2020, RNA Biol.).
Exploitation Route Aim 1: Studies on the RNA-protein interactomes have become rather popular as evident from numerous papers that preformed likewise analysis in other organisms (including plants, fish, flies, and parasites). Thereby, our work has been well-cited and therefore found its recognition in the academic community. Furthermore, the PI has been invited to present this work at conferences, where it attracted a lot of attention beyond the immediate RNA field. Foremost, the discovery of RNA-binding capacity of glycolytic enzymes attracted the interest from the medical field (i.e. Warburg effect, relation to inflammation). Overall, our results provide a robust basis for the study of new RBPs (including metabolic enzymes), which could lead to unexpected findings with substantial impact in the longer term. On this line, the results from aim 1 were also the basis for successful grant applications to the BBSRC (BB/N008820/1 and BB/S017747/1).

Aim 2: We have swiftly published the TRIP method (Matia-Gonzalez et al. 2017, Methods; Iadevaia V. et al. 2018, JoVE) to make it immediately accessible to the wider research community. Soon after publication, we were approached by several groups that wished to implement the approach in their research. Furthermore, we have introduced an alteration to the method that implements an RNA aptamer sequence (tobramycin aptamer), and we investigated the combinatorial control of a cancer-related mRNAs (Iadevaia et al. 2020, RNA Biol.). We have set-up two direct academic collaborations that incorporate the method to study the repertoire of RBPs on particular mRNAs and viral RNAs. Regarding the latter, the method will be used in a successful BBSRC grant application led by Prof. Gill Elliot (University of Surrey) to study proteins interacting with a viral endonuclease RNA (vhs)(BB/T007923/1).

Based on the presentation of our work at the RNA Therapeutics conference in London in 2019, we also got in contact with leading European Biotech companies that expressed an interest in the technology. We have framed a collaborative research project that started in Feb. 2020. TRIP is a key pillar of the project and shall be further refined an optimized.

Overall, the project aims have been completed and the work has now been published in several peer-reviewed high-quality journals, presented at conferences and upon invitations at various occasions. It also provided substantial preliminary data that led to successful grant applications.
Sectors Aerospace, Defence and Marine,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL http://www.surrey.ac.uk/mediacentre/press/2015/new-research-suggests-novel-route-fight-against-cancer
 
Description Our press release on a publication related to aim 1 was widely taken up by diverse press institution all over the world. We have obtained positive feedback from many people beyond academia that were particularly attracted by the idea that many proteins with well-thought functions may have additional functions in the cells. It shows that "old dogmas" can change and our findings expanded our knowledge of the versatility of biological functions. During the last years, several labs have taken-up the idea and published work on unconventional RNA-binding proteins, such as metabolic enzymes unraveled by us and others. Our findings and technology developed in the frame of this grant found great interest from industrial stakeholders. The PI has been invited to present the findings at the several industry/academic conferences, including the RNA Therapeutics conference, 20-21. Feb. 2019 in London. During the last year, we have framed a collaborative research project with a leading Euripean Biotech company that started in Feb. 2021. The method development with this grant (TRIP) is a key pillar for this project. The project may eventually lead to new IP, thus promoting the connections between the UK and European based Biotech sector.
First Year Of Impact 2015
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural,Societal

 
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
 
Title Tandem RNA Isolation Procedure (TRIP) 
Description TRIP enables purification of in vivo formed messenger ribonucleoprotein (mRNP) complexes. The procedure relies on the purification of polyadenylated mRNAs with oligo(dT) beads from cellular extracts, followed by the capture of specific mRNAs with 30-biotinylated 2'-O-methylated antisense RNA oligonucleotides, which are recovered with streptavidin beads. TRIP was applied to isolate in vivo crosslinked mRNP complexes from yeast, nematodes and human cells for subsequent analysis of RNAs and bound proteins. The method provides a basis for adaptation to other types of polyadenylated RNAs, enabling the comprehensive identification of bound proteins/RNAs, and the investigation of dynamic rearrangement of mRNPs imposed by cellular or environmental cues. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact We have been contacted by several academic groups with interest in the technology. We have shared protocols and provided advice on setting-up the procedure to several research groups. It led to active collaborations with two academic research groups; experiments are currently conducted in our laboratory for one of them. Our method attracted the interest from industrial stakeholders (see Awards/Recognition). We are currently in discussion with a Biotech company to use the technology to improve the development of mRNA vaccines and cancer treatment. 
 
Title Tandem RNA Isolation Procedure (TRIP) 
Description TRIP enables purification of in vivo formed messenger ribonucleoprotein (mRNP) complexes. The procedure relies on the purification of polyadenylated mRNAs with oligo(dT) beads from cellular extracts, followed by the capture of specific mRNAs with 30-biotinylated 2'-O-methylated antisense RNA oligonucleotides, which are recovered with streptavidin beads. TRIP was applied to isolate in vivo crosslinked mRNP complexes from yeast, nematodes and human cells for subsequent analysis of RNAs and bound proteins. The method provides a basis for adaptation to other types of polyadenylated RNAs, enabling the comprehensive identification of bound proteins/RNAs, and the investigation of dynamic rearrangement of mRNPs imposed by cellular or environmental cues. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact We have been contacted by several academic groups with interest in the technology. We have shared protocols and provided advice on setting-up the procedure to several research groups. Furthermore, it led to active collaborations with two academic research groups with experiments conducted in our laboratory for one of them. Our method attracted the interest from industrial stakeholders (see Awards/Recognition). We are currently in discussion with a Biotech company to use the technology to improve the development of mRNA vaccines and cancer treatment. 
 
Title Yeast S. cerevisiae and C. elegans mRBPome dataset 
Description Proteomics data have been deposited in the ProteomeXchange Consortium database under accession codes PXD002293 and PXD002226, corresponding to the S. cerevisiae and C. elegans mRBPomes, respectively. This data is linked to the following publication: Matia-González, A.M., Laing, E.E, Gerber, A.P. (2015) Conserved mRNA-binding proteomes in eukaryotic organisms. Nat. Struct. Mol. Biol. 22(12), 1027-33. (epub Nov 23. doi: 10.1038/nsmb.3128)Data was discussed and published in 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Data has been deposited and allows research to extract raw data of yeast and C.elegans mRNA binding proteins for further analysis. 
URL http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD002293
 
Description Collaborator - Jon Hall, ETH Zurich 
Organisation Swiss Federal Institutes of Technology Domain
Country Switzerland 
Sector Public 
PI Contribution Long collaboration with my previous host at the ETH Zurich. Exchange of materials and meetings for scientific exchange. In the frame of the current BBSRC grant, Prof Hall has provided antisense modified RNA oligonucleotides that have been used to set-up a new method to purify endogenously expressed mRNAs from cells. Prof Hall is a named collaborator on the grant.
Collaborator Contribution Long collaboration with my previous host at the ETH Zurich. Exchange of materials and meetings for scientific exchange. We also co-supervised a Postdoc (Dr. Jochen Imig) in the frame of a project funded by the Swiss National Science Foundation, including regular meetings at the ETH Zurich to discuss progress (2010-2014). In the frame of the current BBSRC grant, Prof Hall provide antisense modified RNA oligonucleotides that have been used to set-up a new method to purify endogenously expressed mRNAs from cells. Prof Hall is a named collaborator on the grant.
Impact We published some papers together: Imig, J., Brunschweiger, A., Brümmer, A., Guennewig, B., Mittal, N., Kishore, S., Tsikrika, P., Gerber, A.P., Zavolan, M., Hall, J. (2015) MiR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction. Nat. Chem. Biol. 11(2), 107-14. Towbin, H., Wenter, P., Guennewig, B., Imig, J., Zagalak, J.A., Gerber, A.P., Hall, J. (2013) Systematic screens of proteins binding to synthetic microRNA precursors. Nucleic Acids Res, 41(3), e47.
Start Year 2009
 
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 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 Press release 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release that was picked-up by many professional newsletters.
Year(s) Of Engagement Activity 2015
URL https://www.surrey.ac.uk/mediacentre/press/2015/new-research-suggests-novel-route-fight-against-canc...
 
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