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

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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.