The spliceosome is a highly conserved dynamic macromolecular machine that catalyzes the excision of non-coding introns from pre-messenger RNAs (pre-mRNA) to form mature messages (mRNA), a process called pre-mRNA splicing. The ~3 MDa complex, composed of RNAs and proteins, assembles in a series of regulated steps from four snRNPs (U1, U2, U5, U4/U6) and numerous non-snRNP splicing factors. While the spliceosome is a fundamental cellular machine, its complex and dynamic nature has made obtaining structural information challenging. Single particle cryo-electron microscopy (cryo-EM) is a powerful structural approach for studying the structure of dynamic complexes that are difficult to study using other structural techniques such as X-ray crystallography. We are taking a structure/function approach, using single particle cryo-EM, biochemistry, in vivo functional assays, and yeast genetics, to define the conformational changes required for the transition from a pre- to post-activated Schizosaccharomyces pombe spliceosome. S. pombe is an ideal model organism for studying the structure and function of spliceosomes due to the high conservation of core splicing machinery, the ability to purify spliceosomes amenable for single particle cryo-EM analysis, and the availability of genetic approaches. We have generated DExD/H RNA helicase temperature sensitive alleles to trap spliceosomes at various stages of the splicing reaction. We are now using these complexes to map global conformational changes that occur during the transition from a pre- to post-splicing complex.

Structural analysis of the dynamic Schizosaccharomyces pombe spliceosome using single particle cryo-electron microscopy

251st National Meeting (2016)

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