Developing tools to investigate combinatorial control of mRNA metabolism

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.

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.

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.