title: ATPγS stalls splicing after B complex formation but prior to spliceosome activation.
abstract: The ATP analog ATPγS inhibits pre-mRNA splicing in vitro, but there have been conflicting reports as to which step of splicing is inhibited by this small molecule and its inhibitory mechanism remains unclear. Here we have dissected the effect of ATPγS on pre-mRNA splicing in vitro. Addition of ATPγS to splicing extracts depleted of ATP inhibited both catalytic steps of splicing. At ATPγS concentrations ≥0.5 mM, precatalytic B complexes accumulate, demonstrating a block prior to or during the spliceosome activation stage. Affinity purification of the ATPγS-stalled B complexes (B(ATPγS)) and subsequent characterization of their abundant protein components by 2D gel electrophoresis revealed that B(ATPγS) complexes are compositionally more homogeneous than B complexes previously isolated in the presence of ATP. In particular, they contain little or no Prp19/CDC5L complex proteins, indicating that these proteins are recruited after assembly of the precatalytic spliceosome. Under the electron microscope, B(ATPγS) complexes exhibit a morphology highly similar to B complexes, indicating that the ATPγS-induced block in the transformation of the B to B(act) complex is not due to a major structural defect. Likely mechanisms whereby ATPγS blocks spliceosome assembly at the activation stage, including inhibition of the RNA helicase Brr2, are discussed. Given their more homogeneous composition, B complexes stalled by ATPγS may prove highly useful for both functional and structural analyses of the precatalytic spliceosome and its conversion into an activated B(act) spliceosomal complex.
authors.affiliation: Department of Cellular Biochemistry, MPI for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Dmitry E
authors.initials: DE
authors.lastname: Agafonov
authors.affiliation: Department of Cellular Biochemistry, MPI for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Marieke
authors.initials: M
authors.lastname: van Santen
authors.affiliation: Department of Cellular Biochemistry, MPI for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Berthold
authors.initials: B
authors.lastname: Kastner
authors.affiliation: 3D Electron Cryomicroscopy Group, MPI for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Prakash
authors.initials: P
authors.lastname: Dube
authors.affiliation: Department of Cellular Biochemistry, MPI for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Cindy L
authors.initials: CL
authors.lastname: Will
authors.affiliation: Bioanalytical Mass Spectrometry Group, MPI for Biophysical Chemistry, D-37077 Göttingen, Germany Bioanalytics Group, Institute for Clinical Chemistry, University Medical Center Göttingen, D-37075 Göttingen, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.affiliation: Department of Cellular Biochemistry, MPI for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism; D017381:Spliceosomes / Q000187:drug effects / Q000378:metabolism*
chemical_list: C508357:CDC5L protein, human; D018797:Cell Cycle Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D017411:Ribonucleoproteins, Small Nuclear; C545711:SNRNP200 protein, human; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate
keywords: ATPγS; Prp19/CDC5L complex; pre-mRNA splicing; small molecule; spliceosome