Molecular mechanisms connecting signal transduction and RNA processing: Structural studies of the STAR family of protein

Lead Research Organisation: University of Leicester
Department Name: Biochemistry

Abstract

Post-transcriptional gene regulation is a major regulatory event in cells that allow for protein diversity out of a limited number of genes. These regulations are modulated by extracellular signals through cell signalling. Very little is known about the molecular mechanisms that govern these regulations and modulations. I intend to study at a molecular level a family of proteins, called STAR, which connects cell signalling and post-transcriptional gene regulation. For that I will use a combination of biochemical and biophysical methods, especially NMR spectroscopy. The first main aspect of the project will be to characterize structurally how STAR proteins specifically recognize their target RNAs. The second aspect will involve protein-protein interactions and will be to characterize how cell signalling affects the functions of these proteins in post-transcriptional gene regulation.
Since these proteins play a role in tumour progression and in the life cycle of retroviruses, like HIV-1, these structural studies will provide the molecular basis for developing specific drugs optimized for cancer and retroviral therapies.

Technical Summary

Cellular processes are tightly regulated at different levels by various signaling pathways depending on extracellular stimulations. I aim at better understanding the interplay between signaling pathways and post-transcriptional gene regulation. Post-transcriptional gene regulation consists of major events in cells, such as alternative splicing, RNA export, or translation regulation, that are still not fully understood. The project I intend to develop seeks at unraveling at the molecular level the functional properties of the STAR (Signal Transduction and Activation of RNA) family of protein. These proteins provide a direct connection between cell signaling and post-transcriptional modifications.

The STAR family is involved in many cellular processes such as signal transduction, post-transcriptional gene regulation, tumorigenesis and viral metabolism. All these proteins are involved in the regulation of alternative splicing and RNA export. Importantly, Sam68, the best characterized member of this family, has important roles in post-transcriptional gene regulation that are connected with tumor progression and retroviral life cycle.

STAR proteins contain a STAR domain responsible for RNA binding, several proline and tyrosine rich sequences that promote phosphorylation by diverse kinases, arginine-glycine rich sequences that are methylated by methyltransferase enzymes, and nuclear localization signals allowing the protein to shuttle between the nucleus and the cytoplasm.

There are no structures of a full STAR domain free or in complex with RNA. I therefore plan to solve the NMR structures of STAR domains of different STAR proteins free and in complex with their RNA targets in order to define a general mechanism for RNA recognition by STAR family members. In addition, Sam68 regulate the export of HIV-1 RNAs by binding the viral RNA and the Rev protein. I plan to study biochemically and structurally the trimolecular complex formed between Sam68, Rev and the HIV-1 RNA.

Phosphorylation and arginine methylation of STAR proteins affect their RNA binding capability and therefore their functions in post-transcriptional gene regulation. An aspect of the project will be to study structurally theses domains in complex with signaling molecules to understand how post-translational modifications are achieved in STAR proteins and how these modifications affect the RNA binding capacities.

Summarizing, I will apply my knowledge of molecular biology, biochemistry, NMR, protein/RNA structural biology, and docking to unravel the structural properties of the STAR family. Since STAR proteins play an important role in tumor progression and HIV RNA export, these structural studies will help initiating application for optimized viral and cancer therapies.
 
Description Wellcome Trust Vacation Scholarship
Amount £1,080 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Vacation Scholarship
Sector Charity/Non Profit
Country United Kingdom
Start 07/2013 
End 08/2013
 
Title FOLDeR 
Description RNA G-quadruplex structures are thought to affect biological processes, including translation and pre-mRNA splicing, but it is not possible at present to demonstrate that they form naturally at specific sequences in long functional RNA molecules. We have developed a new strategy, footprinting of long 7-deazaguanine-substituted RNAs (FOLDeR), that allows for the first time to investigate the formation of G4s long functional RNAs and under functional conditions. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact This novel method has allowed my group to investigate the effect of G-quadruplex binders on the alternative splicing of an apoptotic factor. Importantly, we have identified one small molecule that affects alternative splicing and induces cell death (Weldon et al, manuscript in preparation). This method has also been presented at various conferences through oral presentation and many laboratories have contacted us to obtain further practical details of the method suggesting that this method is currently used in other laboratories. 
 
Description Elliot 
Organisation Newcastle University
Country United Kingdom 
Sector Academic/University 
PI Contribution In this collaboration, we investigate the structural basis of RNA recognition by the RNA binding protein TSTAR. Our structural work is used by the Elliott's lab to design mutants and test them in functional splicing assays.
Collaborator Contribution The functional splicing assays on mutant RNAs performed by the Elliot's lab are used to validate our structural studies
Impact This collaboration will results in a common publication as research article in the near future.
Start Year 2011
 
Description Eperon 
Organisation University of Leicester
Country United Kingdom 
Sector Academic/University 
PI Contribution In this collaboration, we investigate the structural basis of RNA recognition by the RNA binding protein TSTAR. Our structural work is used by the Eperon's lab to design mutants and test them in functional splicing assays.
Collaborator Contribution The functional splicing assays on mutant proteins performed by the Eperon's lab are used to validate our structural studies
Impact This collaboration will results in a common publication as research article in the near future.
Start Year 2011
 
Description SMSDrug.net 
Organisation University of Strathclyde
Department SMSDrug.net
Country United Kingdom 
Sector Academic/University 
PI Contribution In 2012, the UK RNA splicing community has decided to create a network entitled "Therapeutic Strategies Targeting the Spliceosome" sponsored by SMSDrug.net. The network is composed of a core of around 20 leading PIs from the UK and aims at developing collaborations to propose new ways of treating diseases such as cancer through modulation of alternative splicing. Through this network, I have, in collaboration with scientist at the Beatson Institute in Glasgow and at the University of Durham, set up a screening assay to target the RNA binding proteins Sam68 and TSTAR. The assay is developed by the European Screening Centre Newhouse (http://www.lifesci.dundee.ac.uk/research/esc). My lab provided the proteins and RNAs necessary for the assay, the structural knowledge of the interaction and the protocol for the initial binding assay.
Collaborator Contribution The European Screening Centre Newhouse is developing the assay in a suitable format for application to the European Lead Factory (ELF) program and is testing 3000 small molecules. Other members of our network will contribute through functional assays.
Impact This aim of this partnership is to develop new treatments by targeting aberrant alternative splicing in human diseases
Start Year 2012
 
Description Sattler 
Organisation Helmholtz Zentrum München
Country Germany 
Sector Academic/University 
PI Contribution We have obtained numerous structures of the RNA binding protein TSTAR in complex with RNA. In this collaboration, one of my PhD student joined the Sattler's lab to perform SAXS experiments.
Collaborator Contribution The Sattler's lab has measured SAXS data on our protein-RNA complexes in order to confirm that the X-ray structure is consistent with the structure observed in solution.
Impact This collaboration will results in a common publication as research article in the near future.
Start Year 2014
 
Description G4 and Horizon 2020 
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 Public/other audiences
Results and Impact Based on our article published in Nature Chemical Biology, an interview of myself was published in Pan European Networks, the magazine of Horizon 2020.
Year(s) Of Engagement Activity 2017
URL http://www.horizon2020publications.com/H13/#160
 
Description G4 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 Public/other audiences
Results and Impact We published a press release describing our article in Nature Chemical biology and describing a novel method to identify G-quadruplexes in RNAs.This release was used in more than 10 articles in scientific websites.
Year(s) Of Engagement Activity 2016
URL https://www2.le.ac.uk/offices/press/press-releases/2016/november/new-advance-in-rna-studies-holds-ou...
 
Description Open days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact around 120 pupils (Year 13) from different regional schools visited our department. I was in charge of the coordination of the guided tour of our building that included 5 short activities (10 minutes each) such as fluorescence microscopy, structural biology, what is cancer, ...
Year(s) Of Engagement Activity 2017
URL https://www2.le.ac.uk/departments/molcellbiol/file-store/open-day-2016
 
Description Sam68 structure 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 Public/other audiences
Results and Impact We created a press release describing our article in Nature Communications and describing the structure of the oncogenic Sam68 splicing factor.This press release has been picked up by more than 10 external scientific websites.
Year(s) Of Engagement Activity 2016
URL https://www2.le.ac.uk/offices/press/press-releases/2016/january/new-study-creates-first-3d-vision-of...