Investigating the regulation of alternative splicing by Tra2 proteins and RBMX in triple negative breast cancer cells

Abstract

Alternative splicing is a key process that enables the expression of a large number of proteins from a limited number of genes. Regulation of alternative splicing has been directly linked with the hallmarks of cancer. Human Transformer 2-α and -β are two homologous RNA binding proteins involved in the regulation of alternative splicing that are overexpressed in many different cancers. Previous work suggests Tra2β interacts closely with RBMX, encoded by the gene RBMX, to regulate the splicing of key pathologically related genes such as SMN2. The objective of this study is to investigate the role of alternative splicing regulators in breast cancer in order to further understand their biological function. Firstly, I utilise previously obtained RNAseq data from the double knockdown of Tra2α and Tra2β in the MDA-MB-231 breast cancer cell line to identify an extended number of genes regulated by Tra2 proteins using recently developed bioinformatics pipelines. The analysis revealed a range of alternative events including intron retentions, alternative 3’ and 5’ splice sites and exon repression events, suggesting different mechanisms of splice site regulation by Tra2 proteins. I experimentally validated the results of the RNAseq analysis by PCR. Subsequent Gene Ontology enrichment analysis of Tra2-regulated targets reveals several genes involved in the cellular response to DNA damage. I employed western immunoblotting to investigate the protein expression changes in these genes and was able to detect short protein isoforms expressed for MBD4 and XPA. Subsequently, nuclear fractionation and immunofluorescence microscopy of Tra2 depleted cells showed cytoplasmic localisation of the short isoforms for both MBD4 and XPA. In order to determine the role of the alternative isoforms regulated by Tra2 proteins, antisense oligonucleotide targeting exon 5 of BRIP1, exon 3 of CHEK1, and exon 5 of XPA were transfected into MDA-MB-231 cells. Expression of the short XPA isoform using antisense oligonucleotides results in an increase of γH2AX and reduced cell viability. This data suggests Tra2 proteins sustain the DNA integrity and protect the cells from death by ensuring the expression of correctly spliced DNA repair genes. In order to compare the role of Tra2 proteins with the commonly associated splicing regulator RBMX, I knocked down the expression of RBMX in MDA-MB-231 cells and obtained RNAseq data. Bioinformatics analysis was able to identify many commonly regulated alternative events, but also some novel isoforms regulated by RBMX alone. RNAseq data also unveiled an interesting autoregulatory mechanism for the RBMX gene, whereby the expression of RBMX is regulated by alternative splicing of the 3’ UTR. Subsequent examination of Upf1 knockdown cells showed stabilisation of the alternating isoform of RBMX indicating it is targeted by nonsense mediated decay. Future work into the role of RBMX regulated targets similar to the work done for Tra2-regulated genes could reveal a complete and clear picture of the function of RBMX in breast cancer cells.