title: Stable tri-snRNP integration is accompanied by a major structural rearrangement of the spliceosome that is dependent on Prp8 interaction with the 5' splice site.
abstract: Exon definition is the predominant initial spliceosome assembly pathway in higher eukaryotes, but it remains much less well-characterized compared to the intron-defined assembly pathway. Addition in trans of an excess of 5'ss containing RNA to a splicing reaction converts a 37S exon-defined complex, formed on a single exon RNA substrate, into a 45S B-like spliceosomal complex with stably integrated U4/U6.U5 tri-snRNP. This 45S complex is compositonally and structurally highly similar to an intron-defined spliceosomal B complex. Stable tri-snRNP integration during B-like complex formation is accompanied by a major structural change as visualized by electron microscopy. The changes in structure and stability during transition from a 37S to 45S complex can be induced in affinity-purified cross-exon complexes by adding solely the 5'ss RNA oligonucleotide. This conformational change does not require the B-specific proteins, which are recruited during this stabilization process, or site-specific phosphorylation of hPrp31. Instead it is triggered by the interaction of U4/U6.U5 tri-snRNP components with the 5'ss sequence, most importantly between Prp8 and nucleotides at the exon-intron junction. These studies provide novel insights into the conversion of a cross-exon to cross-intron organized spliceosome and also shed light on the requirements for stable tri-snRNP integration during B complex formation.
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Boesler
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Rigo
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Dmitry E
authors.initials: DE
authors.lastname: Agafonov
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Berthold
authors.initials: B
authors.lastname: Kastner
authors.affiliation: Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Cindy L
authors.initials: CL
authors.lastname: Will
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
mesh_terms: D045744:Cell Line, Tumor; D005091:Exons / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007438:Introns / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics*; D017381:Spliceosomes / Q000235:genetics*
chemical_list: D005136:Eye Proteins; C454222:PRPF31 protein, human; C115374:PRPF8 protein, human; D022821:RNA Splice Sites; D016601:RNA-Binding Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear
keywords: Prp8; exon definition; pre-mRNA splicing; spliceosome; tri-snRNP