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Understanding intron removal through systematic analysis of mutations in splicing factors on spliceosome assembly and mRNA production

Principal Supervisor: Olga Makarova - Department of Molecular and Cell Biology

Co-supervisor: Kayoko Tanaka (Leicester), Corinne Smith (Warwick)

PhD project title: Understanding intron removal through systematic analysis of mutations in splicing factors on spliceosome assembly and mRNA production

University of Registration: Leicester

Project outline:

Splicing is a key process in gene expression in which introns are removed from pre-mRNA and exons are joined together. In humans, splicing is responsible for large protein diversity, producing on average six protein isoforms per gene. Thus, the number of protein variants produced from the genome vastly exceeds the number of genes found in the genome. Naturally, the relative expression levels of these isoforms play a critical role in health and disease. The project aims to obtain an integrated view of the splicing process and its regulatory mechanisms through multidisciplinary approaches.

The cellular machinery that removes introns is called a spliceosome. The spliceosome is a highly complex and dynamic machinery, which is composed of five RNA molecules and as many as 200 proteins (1). It has been recently revealed that mutations or changes in expression of splicing factors (SF) are responsible for many pathological conditions including cancer. These findings have identified the spliceosome as a new therapeutic target.

The project focuses on the role of disease-associated splicing factors and/or their mutations on spliceosome assembly and mRNA production. The study will be instrumental in defining new molecular targets for drug design.

In order to analyse systematically various mutations in each SF component, we employ a highly tractable model organism, fission yeast S.pombe. Firstly, wild-type yeast strains expressing a tagged version of a protein will be generated for isolation of spliceosomal complexes (2-3). In parallel, a strain harbouring a mutated version of the same SF will be produced. The isolated complexes will be analysed by mass spectrometry and visualised using electron microscopy to determine protein composition and assembly status, correspondingly (4-6). Functionality of the complexes will be evaluated in vitro in splicing assays (4, 7), as well as in vivo by examining RNA expression profiles across the genome through RNA-seq technology (8). Finally, physiological significance will be explored by observing cellular phenotypes, including cell cycle progression, cell morphology, adaptation to environmental stresses and capability of differentiation.

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Figure 1. A work flow of the project. Splicing complexes will be isolated from wild-type or mutant yeast strains harbouring mutations in the spliceosome components. Their molecular status will be determined by MS and EM analyses. Their function will be studied through in vitro splicing assays and RNA-seq analysis which reflects in vivo function of the splicing complexes. Physiological significance of the mutations will be examined by observing cellular phenotypes such as cell cycle progression, cell morphology, adaptation to environmental stresses and differentiation capabilities.

References: 

  1. Makarova, O. V. (2014) Spliceosome: the unravelling complexity. The Biochemist 36, 46-52.
  2. Yan, C., Hang, J., Wan, R., Huang, M., Wong, C. C. L., and Shi, Y. (2015) Structure of a yeast spliceosome at 3.6-angstrom resolution. Science 349, 1182-1191.
  3. Hang, J., Wan, R., Yan, C., and Shi, Y. (2015) Structural basis of pre-mRNA splicing. Science 349, 1191-1198.
  4. Makarov, E. M., Owen, N., Bottrill, A., and Makarova, O. V. (2012) Functional mammalian spliceosomal complex E contains SMN complex proteins in addition to U1 and U2 snRNPs. Nucleic Acids Res 40, 2639-52.
  5. Hernandez, H., Makarova, O. V., Makarov, E. M., Morgner, N., Muto, Y., Krummel, D. P., and Robinson, C. V. (2009) Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP. PLoS One 4, e7202.
  6. Boehringer, D., Makarov, E. M., Sander, B., Makarova, O. V., Kastner, B., Luhrmann, R., and Stark. H (2004) Three-dimensional structure of a pre-catalytic human spliceosomal complex B. Nat Sruct Biol 11, 463-468.
  7. Makarov, E. M., Makarova, O. V., Urlaub, H., Gentzel, M., Will, C. L., Wilm, M., and Luhrmann, R. (2002) Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome. Science 298, 2205-2208.
  8. Bitton, D. A., Rallis, C., Jeffares, D. C., Smith, G. C., Chen, Y. Y. C., Codlin, S., Marguerat, S., and Bahler, J. (2014) LaSSO, a strategy for genome-wide mapping of intronic lariats and branch points using RNA-seq. Genome Res. 24, 1169-1179.

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

Design of the construct to create a S. pombe strain with SFs tagged for affinity purification of splicing complexes. Gene deletion and mutagenesis in fission yeast cells. PCR amplification of the construct and transformation of S. pombe. Verification of the strain by PCR and Western blotting. Purification of the spliceosomal complexes using affinity tag and chromatography. Determination of protein composition by mass spectrometry. EM visualization of complexes and structure determination. In vitro splicing assays. Analysis of transcriptome using targeted and genome wide approaches.

Contact: Dr Olga Makarova, University of Leicester