Deciphering the functions of the RNA binding protein T-STAR in mouse development

RNA binding proteins play a key role in organising gene expression in the cell, and control the important process of alternative splicing through which a single gene can produce multiple different mRNAs and proteins. Most genes produce alternatively spliced mRNAs, and some individual genes also produce many splice isoforms. In the most common kind of alternative splicing in mammals entire exons are either spliced in or left out of the mRNA. The insertion of alternative exons can be very important for downstream protein function.

Alternative splicing is controlled by nuclear RNA binding proteins. Many nuclear RNA binding proteins exist as families of closely related proteins, but whether family members have different or overlapping functions is usually a mystery. A good example of this is the T-STAR RNA binding protein, which is in the same family as Sam68. T-STAR and Sam68 are important for normal health. The T-STAR gene becomes amplified in some cases of medulloblastoma, and Sam68 becomes sequestered in the severe neurodegenerative disorder Fragile X Tremor Associated Ataxia. T-STAR and Sam68 function similarly in transfected cells to regulate splicing of the same target exons. We want to establish the roles of endogenous T-STAR protein and if it interacts with Sam68 to control splicing in vivo.

Three aspects of this project are timely:
(1) We have recently made a T-STAR null mouse to use in the current project. There is a window of opportunity here where we are the only group worldwide with a T-STAR null mouse (we have submitted our mouse to the European Mouse Mutant Archive for release next August).
(2) In preliminary data using a powerful high throughput approach we have identified splicing defects in the knockout mice. The four already identified alternative exons which we have found to be controlled by T-STAR in the mouse brain include alternative exons in the Neurexin genes which encode proteins essential for wiring up the nervous system. The Neurexin genes have regional splicing patterns in the wild type brain, but this regional control totally breaks down in the absence of T-STAR protein. This indicates that T-STAR is the master regulator controlling this regional splicing pattern.
(3) Through international collaboration with the Sette group in Rome we have access a Sam68 null allele to cross in to our T-STAR null mouse. Hence we will be able to analyse changes in phenotype and identify splicing defects in double knockout mice (missing Sam68 and T-STAR).

Aims and objectives.
In this current project we will comprehensively identify T-STAR target RNAs to discover if T-STAR predominantly regulates synaptic proteins or is responsible for establishing regional patterns of splicing in the brain (or both). We will establish if T-STAR and Sam68 function redundantly in the control of some target exons, and to what extent they overlap in expression in the brain. We will map where T-STAR binds RNA and work out the important features through which this binding regulates alternative splicing. We will examine the phenotypic consequences for the mouse when alternative splicing events are blocked by removing T-STAR. Finally we will address whether T-STAR and Sam68 work redundantly to control important developmental steps, particularly in the testis where both proteins are expressed at high levels as well as the brain.

We expect that the results of this project will be significant in understanding how splicing factors interact to enable flexible use of information in the genome and the development of complex tissues like the brain and specialised cell types. Our project will discover new regulated targets of splicing control, interrogate mechanisms of regulation and phenotypic consequences when this is blocked. The main beneficiaries from this work will be scientists interested in gene expression, scientists and students who will be trained and members of the public that we will engage.

This project will decipher the role of the RNA binding protein T-STAR and the extent of functional overlap with the related Sam68 protein. We will firstly comprehensively identify target exons under the control of T-STAR and T-STAR/Sam68 in brain RNA from different genotype mice using RNAseq analysis. We will use immunohistochemistry to identify which brain cell types express T-STAR and Sam68, and correlate this with the endogenous splicing profile of target exons when T-STAR is removed.

We will use minigenes to test if T-STAR and Sam68 regulation of alternative events is through direct RNA binding, by co-transfecting versions of T-STAR and Sam68 which can and cannot bind RNA into cells and monitoring splicing by RT-PCR. We will further use these minigenes to dissect mechanisms of splicing control. We will scan regulated events for potential T-STAR binding sites, and then use EMSAs (Electrophoretic Mobility Shift Assays) to confirm sites of RNA-protein binding. We will dissect physiological T-STAR response elements using site directed mutagenesis of minigenes, and test the effect on splicing after transfection into cells.

Our preliminary data identify strong splicing defects in important nervous system proteins without T-STAR. These splicing defects are particularly strong in the forebrain, and suggest there will be abnormalities in synapse function, and in behaviour including perception, memory, spatial localisation and navigation. We will test for these abnormalities using thin slice physiology, and behavioural analyses in our T-STAR null mice.

Finally we will determine whether T-STAR and Sam68 proteins interact with each other in development using mice with compound mutations in the genes encoding T-STAR and Sam68. In each case we will carefully monitor mouse development and anatomy in collaboration with our colleagues in the Sette lab in Rome, and correlate this with use of particular alternative splicing events by molecular analysis in Newcastle.

Who will benefit from this research? Expected beneficiaries will include students at Newcastle University, professional scientists working on the project, and the general public in terms of engagement and training of personnel in the healthcare professions, medical charities and the scientific profile and local economy in Newcastle.

How will they benefit from this research? An important impact of our work will be in science education. Newcastle University is a research led university, and work in the lab feeds through into taught classes as well as projects carried out by undergraduate and postgraduate students. In the case of lab based projects students get the opportunity to become directly involved for a time in research projects, and both the PI and coPI on this grant are involved in project student supervision. The impact in science education from this grant will be immediate (Years 1-3).

This project will be enhance the professional research skills of Dr. Ingrid Ehrmann who is the Research Co-investigator on this project and will interact with local bioinformatics support for the RNAseq analysis. Expanding expertise in the application of post-genomic technology to basic research such as we plan in this project can make valuable contributions industrially. Ingrid will also be involved in the mouse behaviour analysis which is a new area for her (Years 1-3).

Although our basic research is "blue skies" in nature, it does have significant translational impacts in normal health and medicine and we have collaborated with clinicians and other research groups to realise these. In particular we have trained a Clinical Training Fellow within the group to PhD level who is now a urological surgeon with a CRUK Clinician Scientist Award at the Beatson Institute in Glasgow; and an NHS permanent scientist to PhD level who is involved in diagnostic work. We also supply reagents that we make to the general community (we have made antisera available through Cancer Research Technology, and we have made our targeted T-STAR allele available through the European Mutant Mouse Archive). We also interact with medical charities, particularly those involved in breast and prostate cancer, and our work will be included in newsletters and fundraising activities of these charities.

Our project will have impact in terms of public engagement.The work we do broadly in the area of genetics and its role in development is of interest to the general public. We will contribute to public talks (Cafe Scientifique and the British Science Festival which is in Newcastle in Year 1 of the grant). We will host sixth form students who are interested in a career in science or medicine, and give talks at a local schools Science and Engineering weeks (Years 1-3 of grant).

A longer term social and economic impact of our study will be in the continued development of the local science infrastructure. Newcastle is a post-industrial city in which the University is a major local employer, and is developing itself as a Science City in which the economy is based on afoundation of research and technology. Within the North East of England over 140,000 people are employed in biotechnology, life sciences, NHS and associated organisations. The Institute of Genetic Medicine (IGM) is part of the city
centre-located International Centre for Life, which includes both aspects of Science education and start up companies, and links with local patient care under the NHS. Key to this vision is Research Excellence. The creation of an excellent basic research base has acted as a magnet to attract both high technology companies and further local investment in healthcare. The excellent reputation of Newcastle University and other local universities in both basic and applied scientific research is critical for the success of Newcastle as a Science city, and continued Research Council funding of excellent science is crucial for this.