Pre-spliceosome assembly in Saccharomyces pombe
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Pre-mRNA splicing is a key event in the regulation of gene expression. The remarkable precision of splicing is at least partially built into the mechanism of pre-spliceosome formation, which occurs during the early stage of spliceosome assembly and defines intron-exon boundaries. In order to better understand the mechanism of pre-spliceosome assembly, we initiated a new system for biochemical analysis of pre-spliceosome from the fission yeast Schizosaccharomyces pombe, because study of evolutionarily distant systems can provide new insights and perspectives into the mechanism of complex systems.;The S. pombe in vitro system is efficient in the formation of pre-spliceosomes. We found that the protein complex of SF 1-U2AF was crucial for the formation of pre-spliceosomes and the interaction between SF1 and U2AF is more stable than that observed in other systems. We proposed a novel model for the action of SF1 and U2AF, that the SF1-U2AF complex is pre-formed prior to pre-spliceosome formation and recruited as a complex to facilitate recognition of the 3' end of introns.;It is important to know the protein composition of the pre-spliceosome in order to better understand how the splicing machinery correctly selects the rather variable splice sites. Therefore, we devised a scheme for purification of pre-spliceosomes using two powerful affinity tags. One is the TAP tag on a U2 snRNP core protein---U2-A'; the other one is the MS2 binding site on pre-mRNA. 28 proteins of purified pre-spliceosomes were identified by mass spectrometry. Only U1 and U2, but no U4, U5, or U6 snRNP proteins were found.;Purified pre-spliceosomes were mono-dispersed and homogeneous, suitable for cryo-electron microscopy. A future goal will be to build an architectural representation of pre-spliceosomes at higher resolution, which will be invaluable for a better understanding of the inner workings of the spliceosome. Together, the protein composition of highly purified S. pombe pre-spliceosomes and protein-protein interactions gives us a strong base on which to build our understanding of the organization of components during the assembly of pre-spliceosomes. In summary, the S. pombe in vitro system has provided new perspectives on the mechanism of pre-spliceosome formation, which helps elucidate how the splicing machinery targets the correct intron-exon boundaries. (Abstract shortened by UMI.).