In human cells, almost every gene contains one or more introns that must be removed to generate mRNA. Alternative splicing refers to removing different combinations of introns, thereby generating multiple mRNAs, coding for proteins with different structures, from a single gene. Alternative splicing can generate different protein isoforms, which vary in function, substrate specificity and/or subcellular localisation. Regulation of alternative splicing can modulate the proportions of protein isoforms expressed in different cell types, or under different growth conditions, while defects in splicing can alter protein expression, leading to disease phenotypes. Aberrant splicing is a common feature of human disease mechanisms, including inherited genetic disorders causing neurodegenerative disease such as Spinal Muscular Atrophy (SMA). Defects in alternative splicing are also a hallmark of cancer, with over 15,000 cancer-specific mis-splicing events identified in 27 different types of cancer.
The splicing machinery has therefore emerged as an attractive source of new drug targets. A novel therapeutic strategy is to identify molecules that can modulate splice site selection artificially. For example, antisense oligonucleotides targeting regulatory sequences in the gene SMN2, have provided a major medical advance for the treatment of Spinal Muscular Atrophy. Small molecules have also been identified that can modulate splice site choices in cells and inhibit splicing in vitro, including spliceostatin A, E7107 and herboxidiene. Angus Lamond is a world leader in studying quantitative proteomics and the biochemistry of RNA processing. David Gray is a world leader in drug discovery and chemical biology, including target validation strategies, assay and screen design, hit validation strategies and lead optimisation. Collaborative work by the Lamond and Gray groups has identified families of new small molecule splice site modulators, showing that different molecules affect different transcripts and alternative splice site choices in cells, while arresting assembly of splicing complexes in vitro at specific splice sites (1,2).
The project: To identify the protein targets of novel, small molecule pre-mRNA splicing modulators in human cells, validate the targets identified and characterise their mechanism of action and impact on cell phenotypes. Work involved: The student will prepare extracts from human cells treated with small molecule splicing modulator compounds and use quantitative, mass spectrometry-based proteomics techniques for screening to identify their protein targets. This will include 2-D Thermal Proteome Profiling (3). Putative target proteins will be validated both by interaction studies, using recombinant proteins expressed in vitro and by depletion & add back experiments in cellulo. They will use multiple molecular and chemical methods to characterise the mechanism of action of the splicing modulator and its impact on cell phenotypes. During this project the student will acquire expertise in a wide range of cutting-edge experimental and computational techniques.
The project will be developed as a CASE partnership in collaboration with Platinum Informatics Ltd.
References:1) Pawellek A, McElroy S, Samatov T, Mitchell L, Woodland A, Ryder U, Gray D, Lührmann R, Lamond AI. (2014) Identification of small molecule inhibitors of pre-mRNA splicing. J Biol Chem. 12;289(50):34683-98. doi: 10.1074/jbc.M114.590976. PMID: 25281741
2) Pawellek A, Ryder U, Tammsalu T, King LJ, Kreinin H, Ly T, Hay RT, Hartley RC, Lamond AI. (2017) Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP. eLife Sep 8;6:e27402. doi: 10.7554/eLife.27402. PMID:28884683
3) Kurzawa, N., Becher, I., Sridharan, S., Franken, H., Mateus, A., Anders, S., Bantscheff, M., Huber, W., and Savitski, M. M. (2020) A computational method for detection of ligand-binding proteins from dose range thermal proteome profiles. Nature Communications 11, 5783.