title: Recognition of the 3' splice site RNA by the U2AF heterodimer involves a dynamic population shift.
abstract: An essential early step in the assembly of human spliceosomes onto pre-mRNA involves the recognition of regulatory RNA cis elements in the 3' splice site by the U2 auxiliary factor (U2AF). The large (U2AF65) and small (U2AF35) subunits of the U2AF heterodimer contact the polypyrimidine tract (Py-tract) and the AG-dinucleotide, respectively. The tandem RNA recognition motif domains (RRM1,2) of U2AF65 adopt closed/inactive and open/active conformations in the free form and when bound to bona fide Py-tract RNA ligands. To investigate the molecular mechanism and dynamics of 3' splice site recognition by U2AF65 and the role of U2AF35 in the U2AF heterodimer, we have combined single-pair FRET and NMR experiments. In the absence of RNA, the RRM1,2 domain arrangement is highly dynamic on a submillisecond time scale, switching between closed and open conformations. The addition of Py-tract RNA ligands with increasing binding affinity (strength) gradually shifts the equilibrium toward an open conformation. Notably, the protein-RNA complex is rigid in the presence of a strong Py-tract but exhibits internal motion with weak Py-tracts. Surprisingly, the presence of U2AF35, whose UHM domain interacts with U2AF65 RRM1, increases the population of the open arrangement of U2AF65 RRM1,2 in the absence and presence of a weak Py-tract. These data indicate that the U2AF heterodimer promotes spliceosome assembly by a dynamic population shift toward the open conformation of U2AF65 to facilitate the recognition of weak Py-tracts at the 3' splice site. The structure and RNA binding of the heterodimer was unaffected by cancer-linked myelodysplastic syndrome mutants.
authors: Department of Chemistry, Ludwig-Maximilians University, Munich, D-81377 Munich, Germany.|NanoSystems Initiative Munich, Ludwig-Maximilians University, Munich, D-81377 Munich, Germany.|Center for Integrated Protein Science Munich, Ludwig-Maximilians University, Munich, D-81377 Munich, Germany.|Center for Nanoscience, Ludwig-Maximilians University, Munich, D-81377 Munich, Germany.
authors: Lena
authors: L
authors: Voith von Voithenberg
authors: Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany.|Biomolecular NMR, Technische Universität München, 85748 Garching, Germany.|Center for Integrated Protein Science Munich at Fakultät für Chemie, Technische Universität München, 85748 Garching, Germany.
authors: Carolina
authors: C
authors: Sánchez-Rico
authors: Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany.|Biomolecular NMR, Technische Universität München, 85748 Garching, Germany.|Center for Integrated Protein Science Munich at Fakultät für Chemie, Technische Universität München, 85748 Garching, Germany.
authors: Hyun-Seo
authors: HS
authors: Kang
authors: Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany.|Biomolecular NMR, Technische Universität München, 85748 Garching, Germany.|Center for Integrated Protein Science Munich at Fakultät für Chemie, Technische Universität München, 85748 Garching, Germany.|Institut für Molekularbiologie und Biochemie, Medizinische Universität Graz, 8010 Graz, Austria.
authors: Tobias
authors: T
authors: Madl
authors: Biotechnologie et Signalisation Cellulaire, UMR 7242, Ecole Superieure de Biotechnologie de Strasbourg, 67412 Illkirch, France.
authors: Katia
authors: K
authors: Zanier
authors: Department of Chemistry, Ludwig-Maximilians University, Munich, D-81377 Munich, Germany.|NanoSystems Initiative Munich, Ludwig-Maximilians University, Munich, D-81377 Munich, Germany.|Center for Integrated Protein Science Munich, Ludwig-Maximilians University, Munich, D-81377 Munich, Germany.|Center for Nanoscience, Ludwig-Maximilians University, Munich, D-81377 Munich, Germany.
authors: Anders
authors: A
authors: Barth
authors: Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany.|Biomolecular NMR, Technische Universität München, 85748 Garching, Germany.|Center for Integrated Protein Science Munich at Fakultät für Chemie, Technische Universität München, 85748 Garching, Germany.
authors: Lisa R
authors: LR
authors: Warner
authors: Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany; d.lamb@lmu.de sattler@helmholtz-muenchen.de.|Biomolecular NMR, Technische Universität München, 85748 Garching, Germany.|Center for Integrated Protein Science Munich at Fakultät für Chemie, Technische Universität München, 85748 Garching, Germany.
authors: Michael
authors: M
authors: Sattler
authors: Department of Chemistry, Ludwig-Maximilians University, Munich, D-81377 Munich, Germany; d.lamb@lmu.de sattler@helmholtz-muenchen.de.|NanoSystems Initiative Munich, Ludwig-Maximilians University, Munich, D-81377 Munich, Germany.|Center for Integrated Protein Science Munich, Ludwig-Maximilians University, Munich, D-81377 Munich, Germany.|Center for Nanoscience, Ludwig-Maximilians University, Munich, D-81377 Munich, Germany.
authors: Don C
authors: DC
authors: Lamb
mesh_terms: D019281:Dimerization; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D022821:RNA Splice Sites; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism; D000072668:Splicing Factor U2AF / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D022821:RNA Splice Sites; D011994:Recombinant Proteins; D000072668:Splicing Factor U2AF; C076753:U2AF1 protein, human; C000607494:U2AF2 protein, human; D012313:RNA
keywords: NMR; U2AF; dynamics; spFRET; splicing