title: Dynamic Contacts of U2, RES, Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes.
abstract: Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast B(act) spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the B(act) crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25's step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome's protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of Cellular Biochemistry, Göttingen, Germany.
authors.forename: Cornelius
authors.initials: C
authors.lastname: Schneider
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of Cellular Biochemistry, Göttingen, Germany.
authors.forename: Dmitry E
authors.initials: DE
authors.lastname: Agafonov
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of Cellular Biochemistry, Göttingen, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Schmitzová
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of Cellular Biochemistry, Göttingen, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Hartmuth
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of Cellular Biochemistry, Göttingen, Germany.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Fabrizio
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of Cellular Biochemistry, Göttingen, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
mesh_terms: D002352:Carrier Proteins / Q000235:genetics*; D002384:Catalysis; D007438:Introns / Q000235:genetics; D009711:Nucleotides / Q000235:genetics; D012322:RNA Precursors / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics; D000072260:RNA Splicing Factors; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000235:genetics; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics* / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D017381:Spliceosomes / Q000235:genetics*
chemical_list: C534389:Bud13 protein, S cerevisiae; D002352:Carrier Proteins; C544745:Cwc25 protein, S cerevisiae; D009711:Nucleotides; C488197:PRP45 protein, S cerevisiae; C077018:PRP8 protein, S cerevisiae; D012322:RNA Precursors; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D017413:Ribonucleoprotein, U2 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins