The following is a general statement about ongoing research on alternative splicing in the Smith lab. Specific projects will be available in one or more of these areas.
Alternative splicing affects the majority of human genes, in some cases giving rise to many functionally distinct protein isoforms in a cell-specific regulated manner. It is therefore a major form of gene control that is of great importance to understand. Research in our group is primarily concerned with the mechanisms responsible for tissue-specific splicing of pre-mRNAs, focusing on the regulation of alternative splicing in mammalian smooth muscle cells. We take two broad complementary approaches. The first is molecular analysis of individual model systems of alternative splicing. This approach involves characterizing the role of specific cis-acting RNA elements and regulatory proteins in enforcing the cell specific splicing decisions. The second approach is to use global approaches (mRNA-Seq) to define large sets of splicing events that are co-regulated e.g. upon knockdown of a specific splicing regulatory protein, or upon cell differentiation. Computational analysis then allows us to make inferences about the possible mechanisms of regulation, which can be tested using exemplar model alternative splicing events. We have recently identified an RNA binding protein that appears to act as a "master" regulator of alternative splicing in smooth muscle cells. This protein is sufficient to switch alternative splicing patterns when over expressed in cells or when added to cell-free splicing assays. Moreover its activities are regulated by its own alternative splicing and by phosphorylation. In addition to using cell lines and primary cells, we will increasingly be using human stem cell derived smooth muscle cells in collaboration with Dr Sanjay Sinha (Cambridge Stem Cell Institute).
We are also interested in an under-appreciated role of alternative splicing in quantitative regulation of gene expression by the deliberate production of mRNAs that are targeted for degradation by the Nonsense Mediated Decay (NMD) pathway. For example, we have characterized a set of auto- and cross-regulatory interactions between the splicing regulator PTBP1 and its tissue-specific paralogs PTBP2 and PTBP3. We have a collaborative project with Martin Turner (Babraham Institute) in which we are generating lymphoid cell conditional knockouts of PTBP1, 2 and 3 to analyse the unique and redundant roles of this family of RNA binding proteins in B and T cells.
Methods used include standard molecular biology and cloning techniques, protein purification, cell culture, RNA interference, analysis of in vivo splicing patterns by nuclease protection, RT-PCR and RT-qPCR, in vitro transcription and splicing reactions, various assays for RNA-protein interactions, quantitative proteomics, mRNA-Seq.
M. Llorian, C. Gooding, N. Bellora, M. Hallegger, A. Buckroyd, X. Wang, D. Rajgor, M. Kayikci, J. Feltham, J. Ule, E. Eyras, C.W.J. Smith. The alternative splicing program of differentiated smooth muscle cells involves concerted non-productive splicing of post-transcriptional regulators. Nucleic Acids Research (2016) doi: 10.1093/nar/gkw560
M. Coelho, J. Attig, N. Bellora, J. König, M. Hallegger, M. Kayikci, E. Eyras, J. Ule, C.W.J. Smith. Nuclear Matrix Protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB. EMBO Journal 34, 653-668 (2015) doi: 10.15252/embj.201489852
C. Gooding, C. Edge, M.B. Coelho, M. Lorenz, M. Winters, C.F. Kaminski, D. Cherny, I.C. Eperon & C.W.J. Smith. MBNL1 and PTB cooperate to repress splicing of Tpm1 exon 3. Nucleic Acids Res. 41, 4765-4782 (2013). doi:10.1093/nar/gkt168
M. Llorian, S. Schwartz, T. Clark, D. Hollander, L-Y Tan, R. Spellman, A. Gordon, A.C. Schweitzer, P. de la Grange, G. Ast, & C.W.J. Smith. Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB. Nature Structural & Molecular Biology 17, 1114-1123, (2010)
R.H. Spellman, M. Llorian and C.W.J. Smith. Functional redundancy and cross-regulation between the splicing regulator PTB and its paralogs nPTB and ROD1. Mol. Cell. 27, 420-434, (2007)
A.P. Rideau, C. Gooding, P.J. Simpson, T.P. Monie, M. Lorenz, S. Hüttelmaier, R.H. Singer, S. Matthews, S. Curry & C.W.J. Smith. A peptide motif in Raver1 mediates splicing repression by interaction with the PTB RRM2 domain. Nature Structural and Molecular Biology. 13, 839-848 (2006).
A.J. Matlin, F. Clark and C.W.J. Smith. Understanding alternative splicing; towards a cellular code. Nature Reviews Mol. Cell. Biol. 6, 386-398 (2005).