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Chris Smith

Research Focus

Our main efforts have been focused at understanding the molecular mechanisms of regulated alternative splicing decisions in model systems (a-tropomyosin, a-actinin and PTB). Tropomyosin (TM) and actinin are spliced with smooth muscle specificity, while alternative splicing of PTB pre-mRNA occurs via the influence of PTB protein itself in an autoregulatory feedback loop involving alternative splicing leading to Nonsense Mediated Decay (NMD).

A number of interesting themes have emerged from investigation of these systems including: the role and mechanism of PTB as a splicing repressor, the involvement of co-regulators with PTB, the existence of exons with unsually distant branch points, the occurrence of regulated splicing events that lead to NMD.

We are also interested in applying the quantitative proteomic techniques to define functional targets of splicing regulators by proteomic analysis after RNAi knockdown of splicing regulators. Quantitative proteomics approaches have the promise to complement other global approaches (for example, alternative splicing microarrays) in the attempt to define the “circuitry” of alternative splicing programmes.

In our initial application of this approach we analysed the effects of PTB knockdown in HeLa cells. As a result, we characterised a network of cross-regulation between PTB and its tissue restricted paralogs nPTB and ROD1 involving non-productive splicing events. PTB targets could only be identified upon knockdown of both PTB and nPTB, which was upregulated in a compensatory response to PTB knockdown.

Publications

  1. Llorian, M., Schwartz, S., Clark, T.A., Hollander, D., Tan, L-Y., Spellman, R., Gordon, A., Schweitzer, A.C., de la Grange, P., Ast, G., Smith, C.W.J. (2010). Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB. Nature Structural & Molecular Biology, [Epub ahead of print].
  2. Cherny, D., Gooding, C., Eperon, G.E., Coelho, M.B., Bagshaw, C.R., Smith, C.W.J. and Eperon, I.C. (2010). Stoichiometry of a regulatory splicing complex revealed by single molecule analyses. EMBO Journal 29, 2161-2172.
  3. Spellman, R.H., Llorian, M. and Smith, C.W.J. (2007). Functional redundancy and cross-regulation between the splicing regulator PTB and its paralogs nPTB and ROD1. Molecular and Cellular Biology 27, 420-434.
  4. Matlin, A.J., Southby, J., Gooding, C. and Smith, C.W.J. (2007). Repression of a-actinin SM exon splicing by assisted binding of PTB to the polypyrimidine tract. RNA 13, 1214-1223.
  5. Rideau, A.P., Gooding, C., Simpson, P.J., Monie, T.P., Lorenz, M., Hüttelmaier, S., Singer, R.H., Matthews, S., Curry, S. and Smith, C.W.J. (2006). A peptide motif in Raver1 mediates splicing repression by interaction with the PTB RRM2 domain. Nature Structural and Molecular Biology 13, 839-848.
  6. Nagaraja-Grellscheid, S. and Smith, C.W.J. (2006). An apparent pseudo-exon acts both as an alternative exon that leads to NMD and as a zero-length exon. Molecular and Cellular Biology 26, 2237-2246.
  7. Gooding, C., Clark, F., Wollerton, M.C., Nagaraj-Grellscheid, S., Groom, H. and Smith, C.W.J. (2006) A class of human exons with predicted distant branch points revealed by analysis of AG dinucleotide exclusion zones. Genome Biology 7, R1.
  8. Matlin, A.J., Clark, F. and Smith, C.W.J. (2005). Understanding alternative splicing; towards a cellular code. Nature Reviews Molecular and Cellular Biology 6, 386-398.

Key lab techniques: minigene construction, transfection and analysis in cultured cells. RNA interference, 2D-Difference Gel Electrophoresis (Cambridge Centre for Proteomics).

Key lab reagents: various splicing regulator expression constructs and siRNAs (PTB, nPTB, raver1), PTB antibodies.

Lab contact: Chris Smith: cwjs1@cam.ac.uk Clare Gooding: cg126@mole.bio.cam.ac.uk

Lab website: www.bioc.cam.ac.uk/uto/smithc.html

Proteomics website: www.bio.cam.ac.uk/proteomics