An important goal in my laboratory is to understand the structural underpinnings of gene splicing. This work is carried out in collaboration with the Moore laboratory at HHMI, UMass Worcester to obtain purified, homogeneous splicing complexes that are suitable for single particle EM. The spliceosome removes introns from nascent transcripts, an essential step in eukaryotic gene expression. Most introns interrupt precursors to messenger RNAs (pre-mRNAs), and their precise excision is required to create readable mRNAs. Spliceosomes are ribosome-sized (50 - 60 S) complexes composed of pre-mRNA, four small nuclear ribonucleoprotein (snRNP) particles, and a host of associated protein factors. The snRNPs (U1, U2, U4/6, and U5) are, in turn, multicomponent complexes, each containing at least one small stable RNA molecule (snRNA) and five or more tightly bound polypeptides. In all, it has been estimated that nuclear pre-mRNA splicing requires the action of over 100 different gene products. We have recently obtained images of purified spliceosomes (C complex) that have been used to determine an initial 3D structure of the spliceosome . Our goal is now to improve this structure using cryo-electron microscopy of unstained specimens. The 3D structure of one or more of the spliceosomal complexes, at a resolution of about 20 Angstroms or higher, will be invaluable for a better understanding of the inner workings of this large molecular machine.
The catalytically competent C complex (Figure) stands at the end of an ordered pathway by which the snRNPs assemble to form spliceosomes. To better understand this assembly, and how splice sites are recognized, we are also working on earlier splicing complexes. Finally, together with the Moore laboratory, we study post-splicing complexes that remains on the spliced mRNA substrate, such as the exon junction complex (EJC) . The EJC targets the spliced mRNA for nuclear export and is involved in determining its fate in subsequent processing, such as translation by the ribosome.