title: Preparation of yeast whole cell splicing extract.
abstract: Pre-mRNA splicing, the removal of introns from pre-messenger RNA, is an essential step in eukaryotic gene expression. In humans, it has been estimated that 60 % of noninfectious diseases are caused by errors in splicing, making the study of pre-mRNA splicing a high priority from a health perspective. Pre-mRNA splicing is also complicated: the molecular machine that catalyzes the reaction, the spliceosome, is composed of five small nuclear RNAs, and over 100 proteins, making splicing one of the most complex processes in the cell.An important tool for studying pre-mRNA splicing is the in vitro splicing assay. With an in vitro assay, it is possible to test the function of each splicing component by removing the endogenous version and replacing it (or reconstituting it) with a modified one. This assay relies on the ability to produce an extract-either whole cell or nuclear-that contains all of the activities required to convert pre-mRNA to mRNA. To date, splicing extracts have only been produced from human and S. cerevisiae (yeast) cells. We describe a method to produce whole cell extracts from yeast that support splicing with efficiencies up to 90 %. These extracts have been used to reconstitute snRNAs, screen small molecule libraries for splicing inhibitors, and purify a variety of splicing complexes.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Dunn
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Rader
mesh_terms: D002457:Cell Extracts / Q000302:isolation & purification*; D006801:Humans; D007438:Introns; D008967:Molecular Biology / Q000379:methods*; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017381:Spliceosomes / Q000235:genetics*
chemical_list: D002457:Cell Extracts; D012322:RNA Precursors; D012342:RNA, Small Nuclear