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Karin Murakami, Kenji Nakano, Toshiyuki Shimizu, Umeharu Ohto
DEAH-box RNA helicase 15 (DHX15) plays important roles in RNA metabolism, including in splicing and in ribosome biogenesis. In addition, mammalian DHX15 also mediates the innate immune sensing of viral RNA. However, structural information on this protein is not available, although the structure of the fungal orthologue of this protein, Prp43, has been elucidated. Here, the crystal structure of the ADP-bound form of human DHX15 is reported at a resolution of 2.0 Å. This is the first structure to be revealed of a member of the mammalian DEAH-box RNA helicase (DEAH/RHA) family in a nearly complete form, including the catalytic core consisting of the two N-terminal RecA domains and the C-terminal regulatory domains (CTD)...
June 1, 2017: Acta Crystallographica. Section F, Structural Biology Communications
Lauren T Neves, Stephen Douglass, Roberto Spreafico, Srivats Venkataramanan, Tracy L Kress, Tracy L Johnson
In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from premessenger RNA (pre-mRNA). Recent studies show the processes of RNA synthesis and RNA processing to be spatio-temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In Saccharomyces cerevisiae, H2A.Z is deposited into chromatin by the SWR-C complex, is found near the 5' ends of protein-coding genes, and has been implicated in transcription regulation...
April 1, 2017: Genes & Development
Julien Robert-Paganin, Maral Halladjian, Magali Blaud, Simon Lebaron, Lila Delbos, Florian Chardon, Régine Capeyrou, Odile Humbert, Yves Henry, Anthony K Henras, Stéphane Réty, Nicolas Leulliot
No abstract text is available yet for this article.
February 17, 2017: Nucleic Acids Research
Marcel J Tauchert, Jean-Baptiste Fourmann, Reinhard Lührmann, Ralf Ficner
The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact modus operandi of Prp43 and of all other spliceosomal DEAH-box RNA helicases is still elusive. Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43•ATP-analog•RNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains...
January 16, 2017: ELife
Julien Robert-Paganin, Maral Halladjian, Magali Blaud, Simon Lebaron, Lila Delbos, Florian Chardon, Régine Capeyrou, Odile Humbert, Yves Henry, Anthony K Henras, Stéphane Réty, Nicolas Leulliot
The DEAH box helicase Prp43 is a bifunctional enzyme from the DEAH/RHA helicase family required both for the maturation of ribosomes and for lariat intron release during splicing. It interacts with G-patch domain containing proteins which activate the enzymatic activity of Prp43 in vitro by an unknown mechanism. In this work, we show that the activation by G-patch domains is linked to the unique nucleotide binding mode of this helicase family. The base of the ATP molecule is stacked between two residues, R159 of the RecA1 domain (R-motif) and F357 of the RecA2 domain (F-motif)...
December 9, 2016: Nucleic Acids Research
Jean-Baptiste Fourmann, Marcel J Tauchert, Ralf Ficner, Patrizia Fabrizio, Reinhard Lührmann
The DEAH-box NTPase Prp43 disassembles spliceosomes in co-operation with the cofactors Ntr1/Spp382 and Ntr2, forming the NTR complex. How Prp43 is regulated by its cofactors to discard selectively only intron-lariat spliceosomes (ILS) and defective spliceosomes and to prevent disassembly of earlier and properly assembled/wild-type spliceosomes remains unclear. First, we show that Ntr1΄s G-patch motif (Ntr1GP) can be replaced by the GP motif of Pfa1/Sqs1, a Prp43΄s cofactor in ribosome biogenesis, demonstrating that the specific function of Ntr1GP is to activate Prp43 for spliceosome disassembly and not to guide Prp43 to its binding site in the spliceosome...
April 20, 2017: Nucleic Acids Research
Jean-Baptiste Fourmann, Olexandr Dybkov, Dmitry E Agafonov, Marcel J Tauchert, Henning Urlaub, Ralf Ficner, Patrizia Fabrizio, Reinhard Lührmann
The DEAH-box NTPase Prp43 and its cofactors Ntr1 and Ntr2 form the NTR complex and are required for disassembling intron-lariat spliceosomes (ILS) and defective earlier spliceosomes. However, the Prp43 binding site in the spliceosome and its target(s) are unknown. We show that Prp43 fused to Ntr1's G-patch motif (Prp43_Ntr1GP) is as efficient as the NTR in ILS disassembly, yielding identical dissociation products and recognizing its natural ILS target even in the absence of Ntr1's C-terminal-domain (CTD) and Ntr2...
2016: ELife
Marcel J Tauchert, Jean-Baptiste Fourmann, Henning Christian, Reinhard Lührmann, Ralf Ficner
RNA helicases are indispensable for all organisms in each domain of life and have implications in numerous cellular processes. The DEAH-box RNA helicase Prp43 is involved in pre-mRNA splicing as well as rRNA maturation. Here, the crystal structure of Chaetomium thermophilum Prp43 at 2.9 Å resolution is revealed. Furthermore, it is demonstrated that Prp43 from C. thermophilum is capable of functionally replacing its orthologue from Saccharomyces cerevisiae in spliceosomal disassembly assays.
February 2016: Acta Crystallographica. Section F, Structural Biology Communications
Annika U Heininger, Philipp Hackert, Alexandra Z Andreou, Kum-Loong Boon, Indira Memet, Mira Prior, Anne Clancy, Bernhard Schmidt, Henning Urlaub, Enrico Schleiff, Katherine E Sloan, Markus Deckers, Reinhard Lührmann, Jörg Enderlein, Dagmar Klostermeier, Peter Rehling, Markus T Bohnsack
A rapidly increasing number of RNA helicases are implicated in several distinct cellular processes, however, the modes of regulation of multifunctional RNA helicases and their recruitment to different target complexes have remained unknown. Here, we show that the distribution of the multifunctional DEAH-box RNA helicase Prp43 between its diverse cellular functions can be regulated by the interplay of its G-patch protein cofactors. We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase...
2016: RNA Biology
Jason K K Low, Hogune Im, Melissa A Erce, Gene Hart-Smith, Michael P Snyder, Marc R Wilkins
Arginine methylation on nonhistone proteins is associated with a number of cellular processes including RNA splicing, protein localization, and the formation of protein complexes. In this manuscript, Saccharomyces cerevisiae proteome arrays carrying 4228 proteins were used with an antimethylarginine antibody to first identify 88 putatively arginine-methylated proteins. By treating the arrays with recombinant arginine methyltransferase Hmt1, 42 proteins were found to be possible substrates of this enzyme. Analysis of the putative arginine-methylated proteins revealed that they were predominantly nuclear or nucleolar in localization, consistent with the localization of Hmt1...
February 2016: Proteomics
Daipayan Banerjee, Peter M McDaniel, Brian C Rymond
The Prp43 DExD/H-box protein is required for progression of the biochemically distinct pre-messenger RNA and ribosomal RNA (rRNA) maturation pathways. In Saccharomyces cerevisiae, the Spp382/Ntr1, Sqs1/Pfa1, and Pxr1/Gno1 proteins are implicated as cofactors necessary for Prp43 helicase activation during spliceosome dissociation (Spp382) and rRNA processing (Sqs1 and Pxr1). While otherwise dissimilar in primary sequence, these Prp43-binding proteins each contain a short glycine-rich G-patch motif required for function and thought to act in protein or nucleic acid recognition...
May 2015: Genetics
Julien Robert-Paganin, Stéphane Réty, Nicolas Leulliot
RNA helicases from the DEAH/RHA family are present in all the processes of RNA metabolism. The function of two helicases from this family, Prp2 and Prp43, is regulated by protein partners containing a G-patch domain. The G-patch is a glycine-rich domain discovered by sequence alignment, involved in protein-protein and protein-nucleic acid interaction. Although it has been shown to stimulate the helicase's enzymatic activities, the precise role of the G-patch domain remains unclear. The role of G-patch proteins in the regulation of Prp43 activity has been studied in the two biological processes in which it is involved: splicing and ribosome biogenesis...
2015: BioMed Research International
Yan-Ling Chen, Régine Capeyrou, Odile Humbert, Saïda Mouffok, Yasmine Al Kadri, Simon Lebaron, Anthony K Henras, Yves Henry
We provide evidence that a central player in ribosome synthesis, the ribonucleic acid helicase Prp43p, can be activated by yeast Gno1p and its human ortholog, the telomerase inhibitor PINX1. Gno1p and PINX1 expressed in yeast interact with Prp43p and the integrity of their G-patch domain is required for this interaction. Moreover, PINX1 interacts with human PRP43 (DHX15) in HeLa cells. PINX1 directly binds to yeast Prp43p and stimulates its adenosine triphosphatase activity, while alterations of the G patch abolish formation of the PINX1/Prp43p complex and the stimulation of Prp43p...
June 2014: Nucleic Acids Research
Henning Christian, Romina V Hofele, Henning Urlaub, Ralf Ficner
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...
January 2014: Nucleic Acids Research
Prakash Koodathingal, Jonathan P Staley
The spliceosome discriminates against suboptimal substrates, both during assembly and catalysis, thereby enhancing specificity during pre-mRNA splicing. Central to such fidelity mechanisms are a conserved subset of the DEAD- and DEAH-box ATPases, which belong to a superfamily of proteins that mediate RNP rearrangements in almost all RNA-dependent processes in the cell. Through an investigation of the mechanisms contributing to the specificity of 5' splice site cleavage, two related reports, one from our lab and the other from the Cheng lab, have provided insights into fidelity mechanisms utilized by the spliceosome...
July 2013: RNA Biology
Ram Kannan, Sean Hartnett, Rodger B Voelker, J Andrew Berglund, Jonathan P Staley, Peter Baumann
The fission yeast telomerase RNA (TER1) precursor harbors an intron immediately downstream from its mature 3' end. Unlike most introns, which are removed from precursor RNAs by the spliceosome in two sequential but tightly coupled transesterification reactions, TER1 only undergoes the first cleavage reaction during telomerase RNA maturation. The mechanism underlying spliceosome-mediated 3' end processing has remained unclear. We now demonstrate that a strong branch site (BS), a long distance to the 3' splice site (3' SS), and a weak polypyrimidine (Py) tract act synergistically to attenuate the transition from the first to the second step of splicing...
March 15, 2013: Genes & Development
Jean-Baptiste Fourmann, Jana Schmitzová, Henning Christian, Henning Urlaub, Ralf Ficner, Kum-Loong Boon, Patrizia Fabrizio, Reinhard Lührmann
The spliceosome is a single-turnover enzyme that needs to be dismantled after catalysis to both release the mRNA and recycle small nuclear ribonucleoproteins (snRNPs) for subsequent rounds of pre-mRNA splicing. The RNP remodeling events occurring during spliceosome disassembly are poorly understood, and the composition of the released snRNPs are only roughly known. Using purified components in vitro, we generated post-catalytic spliceosomes that can be dissociated into mRNA and the intron-lariat spliceosome (ILS) by addition of the RNA helicase Prp22 plus ATP and without requiring the step 2 proteins Slu7 and Prp18...
February 15, 2013: Genes & Development
Hsin-Chou Chen, Chi-Kang Tseng, Rong-Tzong Tsai, Che-Sheng Chung, Soo-Chen Cheng
The DEAH-box ATPase Prp43 is required for disassembly of the spliceosome after the completion of splicing or after the discard of the spliceosome due to a splicing defect. Prp43 associates with Ntr1 and Ntr2 to form the NTR complex and is recruited to the spliceosome via the interaction of Ntr2 and U5 component Brr2. Ntr2 alone can bind to U5 and to the spliceosome. To understand how NTR might mediate the disassembly of spliceosome intermediates, we arrested the spliceosome at various stages of the assembly pathway and assessed its susceptibility to disassembly...
February 2013: Molecular and Cellular Biology
Denis Kudlinzki, Andreas Schmitt, Henning Christian, Ralf Ficner
Splicing of pre-mRNA requires the activity of at least eight different DEAD/H-box proteins that are involved in distinct steps of the splicing process. These proteins are driving the spliceosomal machinery by ATP-dependent unwinding of dsRNA and/or disrupting protein-RNA complexes. The spliceosomal DEAH-box proteins Prp2, Prp16, Prp22 and Prp43 share homologous C-terminal domains (CTD). We have determined the crystal structure of the CTD of human Prp22 by means of MAD. The fold of the human Prp22-CTD closely resembles that of the yeast Prp43-CTD...
September 8, 2012: Biological Chemistry
Manja A Behrens, Yangzi He, Cristiano L P Oliveira, Gregers R Andersen, Jan Skov Pedersen, Klaus H Nielsen
Small-angle X-ray scattering (SAXS) is a structural characterization method applicable to biological macromolecules in solution. The great advantage of solution scattering is that the systems can be investigated in near-physiological conditions and their response to external changes can also be easily investigated. In this chapter, we discuss the application of SAXS for studying the conformation of helicases alone and in complex with other biological macromolecules. The DEAD-box helicase eIF4A and the DEAH/RHA helicase Prp43 are investigated for their solution structures, and the analysis of the collected scattering data is presented...
2012: Methods in Enzymology
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