title: Isolation and accumulation of spliceosomal assembly intermediates.
abstract: Isolating spliceosomes at a specific assembly stage requires a means to stall or enrich for one of the intermediate splicing complexes. We describe strategies to arrest spliceosomes at different points of complex formation and provide a detailed protocol developed for isolating intact splicing complexes arrested between the first and second chemical steps of splicing. Briefly, spliceosomes are assembled on a radiolabeled in vitro-transcribed splicing substrate from components present in nuclear extract of HeLa cells. Spliceosome progression is arrested after the first step of splicing chemistry by mutating the pre-mRNA substrate at the 3' splice site. The substrate also contains binding sites for the MS2 protein, which serve as an affinity tag. Purification of arrested spliceosomes is carried out in two steps: (1) size exclusion chromatography and (2) affinity selection via a fusion of MS2 and maltose-binding protein (MBP). Complex assembly and purification are analyzed by denaturing polyacrylamide gel electrophoresis.
authors.affiliation: Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, CA, USA.
authors.forename: Janine O
authors.initials: JO
authors.lastname: Ilagan
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Jurica
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D058619:Maltose-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D012322:RNA Precursors / Q000235:genetics / Q000302:isolation & purification*; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing; D017381:Spliceosomes / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D058619:Maltose-Binding Proteins; D012322:RNA Precursors; D022821:RNA Splice Sites