Localization of the Fission Yeast U5.U2/U6 Spliceosome Subunits

Abstract

The spliceosome is a dynamic macromolecular machine that catalyzes the excision of introns from pre-mRNA to generate protein-coding transcripts. The megadalton-sized spliceosome is composed of four small nuclear RNPs (U1, U2, U5, and U4/U6) and numerous pre-mRNA splicing factors. The formation of an active spliceosome is hypothesized to occur in a stepwise manner requiring the assembly and disassembly of large multiprotein/RNA complexes. A promising structural approach to obtain information about spliceosome complexes is single-particle cryo-electron microscopy (cryo-EM), a powerful technique that is ideal for determining the structures of large dynamic complexes at protein concentrations too low for crystallization. Formerly, our group determined structure of the fission yeast U5.U2/U6 spliceosome complex by cryo-EM. This U5.U2/U6 spliceosome complex contains the U2, U5, and U6 snRNAs, pre-mRNA splicing intermediates, U2 and U5 snRNP proteins, the Nineteen Complex (NTC), and second-step splicing factors. However, the location of these subunits in the complex was not determined. Using antibody labeling and single particle EM we are now localizing these individual subunits within the density map of the U5.U2/U6 spliceosome complex. This work now enables us to propose a structural model for U5.U2/U6 organization.