title: Insights into the activation of the helicase Prp43 by biochemical studies and structural mass spectrometry.
abstract: Splicing of precursor messenger RNA is a hallmark of eukaryotic cells, which is carried out by the spliceosome, a multi-megadalton ribonucleoprotein machinery. The splicing reaction removes non-coding regions (introns) and ligates coding regions (exons). The spliceosome is a highly dynamic ribonucleoprotein complex that undergoes dramatic structural changes during its assembly, the catalysis and its disassembly. The transitions between the different steps during the splicing cycle are promoted by eight conserved DExD/H box ATPases. The DEAH-box protein Prp43 is responsible for the disassembly of the intron-lariat spliceosome and its helicase activity is activated by the G-patch protein Ntr1. Here, we investigate the activation of Prp43 by Ntr1 in the presence and absence of RNA substrate by functional assays and structural proteomics. Residues 51-110 of Ntr1 were identified to be the minimal fragment that induces full activation. We found protein-protein cross-links that indicate that Prp43 interacts with the G-patch motif of Ntr1 through its C-terminal domains. Additionally, we report on functionally important RNA binding residues in both proteins and propose a model for the activation of the helicase.
authors.affiliation: Department for Molecular Structural Biology, Institute for Microbiology and Genetics, Georg-August-University Göttingen, D-37077 Göttingen, Germany, Bioanalytical Mass Spectrometry Group, Max-Planck-Institute of Biophysical Chemistry, D-37077 Göttingen, Germany and Bioanalytics, Department of Clinical Chemistry, University Medical Center Göttingen, D-37075 Göttingen, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Christian
authors.forename: Romina V
authors.initials: RV
authors.lastname: Hofele
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.forename: Ralf
authors.initials: R
authors.lastname: Ficner
mesh_terms: D020816:Amino Acid Motifs; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D064267:Intrinsically Disordered Proteins / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D054730:Protein Interaction Domains and Motifs; D012313:RNA / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D064267:Intrinsically Disordered Proteins; D029701:Saccharomyces cerevisiae Proteins; C507652:Spp382 protein, S cerevisiae; D012313:RNA; C109034:PRP43 protein, S cerevisiae; D053487:DEAD-box RNA Helicases