The role of exon sequences in C complex spliceosome structure.
Pre-mRNA splicing is catalyzed by a large ribonucleoprotein complex called the spliceosome. Previous electron microscopy reconstruction of C complex spliceosomes arrested between the two chemical steps of splicing revealed an averaged core structure consisting of three primary domains surrounding a central cavity. Here we characterize the involvement of pre-mRNA in this structured core of C complex by protection mapping. We find that the 3' end of the cleaved 5' exon and intron sequences flanking the branched lariat are buried in the complex. Upstream regions of the 5' exon and the entire 3' exon, including the mutant 3' splice site, are accessible and can be removed by nucleolytic cleavage. Furthermore, we show that the second-step active site of the complex, which is arrested by a 3' splice site mutation, can accept a normal 3' splice site in trans to catalyze exon ligation. Removal of the accessible exon regions alters the protein composition of the complex, but the core spliceosome proteins associated with the uridine-rich small nuclear ribonucleoproteins U2, U5, and U6 and the Prp19 complex as well as several other proteins remain intact. Two-dimensional averaged images of an exon-trimmed complex closely resemble C complex assembled on full-length pre-mRNA, supporting the hypothesis that the electron microscopy model of C complex reflects the core structure of a catalytically competent particle. Trimming the 3' exon does, however, alter the distribution of particles that appear to be missing some density, suggesting that the exon plays a role in stabilizing C complex.
Ilagan
Janine
J
Department of Molecular, Cell and Developmental Biology, University of California, 1156 High Street, Santa Cruz, CA 95064, USA.
Yuh
Patrick
P
Chalkley
Robert J
RJ
Burlingame
A L
AL
Jurica
Melissa S
MS
D001483:Base Sequence; D005091:Exons; D009154:Mutation; D011487:Protein Conformation; D012322:RNA Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing; D017381:Spliceosomes / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*
D012322:RNA Precursors; D022821:RNA Splice Sites