Identification and characterisation of RNA targets of the RNA binding proteins TRa2a and TRa2B

Abstract

Alternative splicing – the production of multiple messenger RNA isoforms from a single gene – is regulated in part by RNA binding proteins. The overall aim of this study was to identify and characterise novel targets of the RNA binding proteins Tra2α and Tra2β, in order to further understand their biological functions. Tra2β is implicated in male germ cell development and in the initial stages of this project, I utilised data from a previous Tra2β HITS-CLIP experiment to validate and characterise novel RNA targets from the mouse testis using minigenes. These included a large testis-enriched exon from Nasp and a posion exon from Tra2a. The identification of a Tra2β-responsive poison exon within the Tra2a gene suggested that Tra2β may directly regulate Tra2α protein expression. Subsequent experiments in a human breast cancer cell line revealed that following depletion of Tra2β, Tra2α is up-regulated, and could functionally compensate in splicing regulation. Tra2β is also up-regulated in several human cancers and we hypothesised that Tra2β may regulate alternative splicing programmes of functional importance in cancer. Therefore for the majority of this project, I investigated RNA targets of Tra2α and Tra2β in the human invasive breast cancer cell line MDA-MB-231. Two transcriptome-wide approaches were used to identify RNA targets in this study. Firstly, I used iCLIP to map the transcriptome-wide binding sites of Tra2β in MDA-MB-231 cells. Secondly, I used RNA-seq to investigate the functional effect of joint Tra2 protein depletion on the transcriptome. Combining the iCLIP and RNA-seq data facilitated the identification of target exons which were both directly bound by Tra2β and functionally responsive to Tra2 protein depletion. Unexpectedly, Tra2 protein dependent exons included both alternative and constitutively spliced exons. A Gene Ontology enrichment analysis of the experimentally validated exons revealed that Tra2 protein dependent exons were functionally enriched in genes associated with chromosome biology. These included a functionally important exon from CHEK1, which encodes a key DNA damage response protein. Joint depletion of Tra2α and Tra2β led to reduced expression of the full-length CHK1 protein, accumulation of the DNA damage marker γH2AX and decreased cell viability. Together, this data suggests that human Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation which are important for cell viability.