title: Breaking up the C complex spliceosome shows stable association of proteins with the lariat intron intermediate.
abstract: Spliceosome assembly requires several structural rearrangements to position the components of the catalytic core. Many of these rearrangements involve successive strengthening and weakening of different RNA:RNA and RNA:proteins interactions within the complex. To gain insight into the organization of the catalytic core of the spliceosome arrested between the two steps of splicing chemistry (C complex), we investigated the effects of exposing C complex to low concentrations of urea. We find that in the presence of 3M urea C complex separates into at least three sub-complexes. One sub-complex contains the 5'exon, another contains the intron-lariat intermediate, and U2/U5/U6 snRNAs likely comprise a third sub-complex. We purified the intron-lariat intermediate sub-complex and identified several proteins, including U2 snRNP and PRP19 complex (NTC) components. The data from our study indicate that U2 snRNP proteins in C complex are more stably associated with the lariat-intron intermediate than the U2 snRNA. The results also suggest a set of candidate proteins that hold the lariat-intron intermediate together in C complex. This information is critical for further interpreting the complex architecture of the mammalian spliceosome.
authors.affiliation: Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America.
authors.forename: Patricia
authors.initials: P
authors.lastname: Coltri
authors.forename: Kerstin
authors.initials: K
authors.lastname: Effenberger
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Chalkley
authors.forename: A L
authors.initials: AL
authors.lastname: Burlingame
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Jurica
mesh_terms: D005091:Exons / Q000235:genetics; D005990:Glycerol / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007438:Introns / Q000235:genetics*; D013058:Mass Spectrometry; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000187:drug effects; D012326:RNA Splicing / Q000187:drug effects; D012342:RNA, Small Nuclear / Q000378:metabolism; D017381:Spliceosomes / Q000187:drug effects / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
chemical_list: D046912:Multiprotein Complexes; D012342:RNA, Small Nuclear; D014508:Urea; D005990:Glycerol