How do a conserved family of RNA binding proteins protect the transcriptome from aberrant processing?

PURPOSE OF RESEARCH: Gene expression requires correct production of mature protein-coding RNAs (mRNAs) by RNA processing. This includes splicing, which joins parts of the gene called "exons" and removes others called "introns". RNA processing is guided by short RNA sequences. This project seeks identify the mechanism how RBMXL2 and RBMX proteins suppress use of "cryptic" RNA processing sequences that would otherwise corrupt productive gene expression.

This project addresses three current knowledge gaps. Firstly, how long exons are efficiently recognised during mRNA maturation is currently poorly understood, yet critically important for expression of key genes, including mouse Meioc (required for meiotic prophase) and human BRCA2 (a tumour suppressor needed for genome stability). In particular, how cryptic RNA processing events are repressed within long exons is very poorly understood, yet may underpin problems associated with deficiency of RBMX family proteins (male infertility, genome instability and Shashi intellectual disability syndrome). Thirdly, how RBMX proteins repress splice sites is unknown. Since repressed RNA processing sites were the majority of targets we identified in our preliminary screens, this suggests that repression is the primary mechanism of action of RBMXL2 and RBMX proteins.

TIMELINESS AND VALUE FOR MONEY: This project is timely since (1) We have already made and carried out initial characterisation of an RBMXL2 knockout mouse. This mouse is male infertile and has already enabled us to identify some cryptic splice sites repressed by RBMXL2. (2) Through collaboration with two world leading groups based in Edinburgh (Prof Ian Adams and Prof Donal O'Carroll) we will be able to purify the exact cell types that arrest in our RBMXL2 knockout mouse and use these for RNA sequencing analyses. (3) Providing value for money, the O'Carroll group will provide us with a mouse line that expresses GFP during meiosis at no charge other than shipping. This line will enable ~99% purification of cells in meiosis from RBMXL2 knockout mice that we can then characterise. (4) Providing extra value for money, this project will make use of a capillary gel electrophoresis system that was recently purchased by a grant from the Pathological Society and matching funds from Newcastle University. (5) We have already identified novel cryptic RNA processing sites that are repressed by RBMX in somatic cells and carried out experiments to globally identify RBMX binding sites in human cells (currently in the sequencing queue of our Genomics Core Facility).

OUTCOMES: We expect that the results of this project will be significant in understanding how the splicing and polyadenylation machineries enable proper RNA processing of long exons, and avoid including cryptic exons. We expect to identify patterns of gene expression that depend on RBMXL2 during male meiosis, improving understanding of the causes of male infertility. Since our preliminary study has already detected RBMX target genes in cancer cells that are important for how cells respond to genotoxic drugs, our research may lead to new strategies to increase the efficacy of chemotherapy. The main beneficiaries from this work will be scientists and students who will be trained, other scientists interested in gene expression, members of the public that we will engage, and possibly in the longer term patients who are treated with genotoxic drugs.

Cryptic sites resemble nucleotide sequences used for RNA processing yet are usually efficiently repressed. This project will investigate the mechanism through which two ancient RNA binding proteins protect the transcriptome from cryptic RNA processing sites and how important this is for expression of key proteins required for genome stability. We recently discovered that the ancient RNA binding protein RBMXL2 represses cryptic splice sites during meiosis - particularly within long exons that do not fit well into current models of exon definition. However, the limited sensitivity and depth of these analyses prevented us from establishing the mechanisms involved. Aim 1 will thus globally analyse the effect of RBMXL2 deletion on splicing in purified meiotic cells and map RBMXL2 RNA binding sites. We will combine these datasets to predict mechanisms of cryptic splicing control, test these using minigene reporters, and investigate effects on gene and protein expression in meiosis. Our preliminary analyses have identified an additional novel role for the RBMXL2-paralog RBMX in repressing cryptic cleavage and polyadenylation sites (polyA sites), particularly in long exons of genes involved in genome stability and intellectual disability. Aim 2 will use a more specific 3'-end sequencing strategy to comprehensively identify polyadenylation sites repressed by RBMX, and will dissect and contrast the mechanisms by which RBMX represses cryptic splice site and polyA sites. RBMXL2 is only expressed in meiosis, and RBMX is expressed in most cells outside of meiosis. Our final aim will use a knockin approach to test whether RBMXL2 can replace RBMX function in splicing and polyA site repression within a human cell model. This project will help explain how long exons are properly recognised for RNA maturation, reveal pathways of gene expression important for male infertility, and provide insight into how sensitivity to genotoxic drugs in cancer cells could be increased.