Regulation of MET Alternative Splicing

Abstract

The way in which a gene transcript is processed has a wide impact on protein translation. Alternative splicing, in particular, is under tight regulation and deviations from this can result in disease. The thesis presented here illustrates this concept in the tyrosine kinase receptor MET of which the gene’s 14th exon is alternatively spliced. In recent years, disruption of alternative splice modulation has been highlighted as an important causative factor in disease. Aberrant MET exon 14 splicing has been indicated as a driver in particular forms of lung cancer as well as in osteofibrous dysplasia (OFD), a disease of the bone. I sought to reveal and better understand the regulatory mechanisms underlying this differential splicing event. In order to gain a better understanding of MET exon 14’s splice patterns in normal physiology, I characterised patterns of skipped and unskipped MET transcripts in a range of undiseased model organisms and samples, revealing finely-tuned spatio-temporal splicing patterns. MET exon 14 comprises very weak splice site sequences, which would usually result in high skipping rates. Despite this, exon 14 has a remarkably high inclusion in normal adult tissues. This indicates that there are auxiliary regulatory elements, other than the splice site sequencess, which support exon 14 inclusion. Based on this premise, I carried out a number of experiments in an attempt to identify different cis and trans factors which may have a role in regulation. Firstly, a comprehensive search of the literature for reported variants associated with exon 14 skipping resulted in the identification of a putative cis regulatory region. This 36 base pair area was subsequently confirmed as a crucial enhancer element for MET exon 14 splice inclusion by using hybridising antisense oligonucleotides which competively bound the mRNA reigon and thus inhibited cis-mediated regulation at this specific area. Secondly, I searched for factors which bind at the mRNA as trans-acting splicing enhancers or suppressor and identified a primary candidate: TRA2β. In vitro knockdown experiments to assess effects on exon 14 skipping gave no conclusive evidence, however further tests should be carried out to fully assess its effect. Thirdly, analyses were carried out to assess effect of local and global methylation on exon 14 splicing. Publicly available data revealed differential local methylation profiles at specific CpG methylation sites within and adjacent to MET exon 14 and I was able to correlate these to observed skipping patterns. No correlation was identified between global hypomethylation and skipping in this study, however further investigation is warranted. Overall, results from this research show that the alternative splicing at MET exon 14 is regulated according to a complex spatio-temporal pattern and disruption of this results in disease. Importantly, the previous discovery of germ line mutations that result in constitutive MET exon 14 skipping in OFD has highlighted an important role for splice event in bone development, maintenance and repair. Better understanding of this will develop as further research in this alternative splice regulation continues.