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Shiheng Liu, Xueni Li, Lingdi Zhang, Jiansen Jiang, Ryan C Hill, Yanxiang Cui, Kirk C Hansen, Z Hong Zhou, Rui Zhao
The spliceosome undergoes dramatic changes in a splicing cycle. Structures of B, Bact , C, C*, and intron lariat spliceosome complexes revealed mechanisms of 5'-splice site (ss) recognition, branching, and intron release, but lacked information on 3'-ss recognition, exon ligation, and exon release. Here we report a cryo-electron microscopy structure of the postcatalytic P complex at 3.3-angstrom resolution, revealing that the 3' ss is mainly recognized through non-Watson-Crick base pairing with the 5' ss and branch point...
December 8, 2017: Science
Xiaofeng Zhang, Chuangye Yan, Jing Hang, Lorenzo I Finci, Jianlin Lei, Yigong Shi
Mechanistic understanding of pre-mRNA splicing requires detailed structural information on various states of the spliceosome. Here we report the cryo electron microscopy (cryo-EM) structure of the human spliceosome just before exon ligation (the C∗ complex) at an average resolution of 3.76 Å. The splicing factor Prp17 stabilizes the active site conformation. The step II factor Slu7 adopts an extended conformation, binds Prp8 and Cwc22, and is poised for selection of the 3'-splice site. Remarkably, the intron lariat traverses through a positively charged central channel of RBM22; this unusual organization suggests mechanisms of intron recruitment, confinement, and release...
May 18, 2017: Cell
Karl Bertram, Dmitry E Agafonov, Wen-Ti Liu, Olexandr Dybkov, Cindy L Will, Klaus Hartmuth, Henning Urlaub, Berthold Kastner, Holger Stark, Reinhard Lührmann
Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2-U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 Å, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains...
February 16, 2017: Nature
Sebastian M Fica, Chris Oubridge, Wojciech P Galej, Max E Wilkinson, Xiao-Chen Bai, Andrew J Newman, Kiyoshi Nagai
The spliceosome excises introns from pre-mRNAs in two sequential transesterifications-branching and exon ligation-catalysed at a single catalytic metal site in U6 small nuclear RNA (snRNA). Recently reported structures of the spliceosomal C complex with the cleaved 5' exon and lariat-3'-exon bound to the catalytic centre revealed that branching-specific factors such as Cwc25 lock the branch helix into position for nucleophilic attack of the branch adenosine at the 5' splice site. Furthermore, the ATPase Prp16 is positioned to bind and translocate the intron downstream of the branch point to destabilize branching-specific factors and release the branch helix from the active site...
February 16, 2017: Nature
Paul A Howles, Leigh K Gebbie, David A Collings, Arvind Varsani, Ronan C Broad, Stephen Ohms, Rosemary J Birch, Ann H Cork, Tony Arioli, Richard E Williamson
The putative RNA helicase encoded by the Arabidopsis gene At1g32490 is a homolog of the yeast splicing RNA helicases Prp2 and Prp22. We isolated a temperature-sensitive allele (rsw12) of the gene in a screen for root radial swelling mutants. Plants containing this allele grown at the restrictive temperature showed weak radial swelling, were stunted with reduced root elongation, and contained reduced levels of cellulose. The role of the protein was further explored by microarray analysis. By using both fold change cutoffs and a weighted gene coexpression network analysis (WGCNA) to investigate coexpression of genes, we found that the radial swelling phenotype was not linked to genes usually associated with primary cell wall biosynthesis...
May 2016: Plant Molecular Biology
Daniel R Semlow, Mario R Blanco, Nils G Walter, Jonathan P Staley
During pre-mRNA splicing, a central step in the expression and regulation of eukaryotic genes, the spliceosome selects splice sites for intron excision and exon ligation. In doing so, the spliceosome must distinguish optimal from suboptimal splice sites. At the catalytic stage of splicing, suboptimal splice sites are repressed by the DEAH-box ATPases Prp16 and Prp22. Here, using budding yeast, we show that these ATPases function further by enabling the spliceosome to search for and utilize alternative branch sites and 3' splice sites...
February 25, 2016: Cell
Stephen Klusza, Amanda Novak, Shirelle Figueroa, William Palmer, Wu-Min Deng
During Drosophila oogenesis, the endopolyploid nuclei of germ-line nurse cells undergo a dramatic shift in morphology as oogenesis progresses; the easily-visible chromosomes are initially polytenic during the early stages of oogenesis before they transiently condense into a distinct '5-blob' configuration, with subsequent dispersal into a diffuse state. Mutations in many genes, with diverse cellular functions, can affect the ability of nurse cells to fully decondense their chromatin, resulting in a '5-blob arrest' phenotype that is maintained throughout the later stages of oogenesis...
2013: PloS One
Thomas Ohrt, Peter Odenwälder, Julia Dannenberg, Mira Prior, Zbigniew Warkocki, Jana Schmitzová, Ramazan Karaduman, Ingo Gregor, Jörg Enderlein, Patrizia Fabrizio, Reinhard Lührmann
Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5' and 3' exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3' splice site (3'SS) to the active site requires only Slu7/Prp18 but not Prp22...
July 2013: RNA
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
Janine O Ilagan, Robert J Chalkley, A L Burlingame, Melissa S Jurica
In spliceosomes, dynamic RNA/RNA and RNA/protein interactions position the pre-mRNA substrate for the two chemical steps of splicing. Not all of these interactions have been characterized, in part because it has not been possible to arrest the complex at clearly defined states relative to chemistry. Previously, it was shown in yeast that the DEAD/H-box protein Prp22 requires an extended 3' exon to promote mRNA release from the spliceosome following second-step chemistry. In line with that observation, we find that shortening the 3' exon blocks cleaved lariat intron and mRNA release in human splicing extracts, which allowed us to stall human spliceosomes in a new post-catalytic complex (P complex)...
March 2013: RNA
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
Louis Düring, Michael Thorsen, Darima Sophia Njama Petersen, Brian Køster, Torben Heick Jensen, Steen Holmberg
A functional relationship between chromatin structure and mRNA processing events has been suggested, however, so far only a few involved factors have been characterized. Here we show that rsc nhp6ΔΔ mutants, deficient for the function of the chromatin remodeling factor RSC and the chromatin architectural proteins Nhp6A/Nhp6B, accumulate intron-containing pre-mRNA at the restrictive temperature. In addition, we demonstrate that rsc8-ts16 nhp6ΔΔ cells contain low levels of U6 snRNA and U4/U6 di-snRNA that is further exacerbated after two hours growth at the restrictive temperature...
2012: PloS One
Milton A English, Lin Lei, Trevor Blake, Stephen M Wincovitch, Raman Sood, Mizuki Azuma, Dennis Hickstein, P Paul Liu
BACKGROUND: Vertebrate hematopoiesis is a complex developmental process that is controlled by genes in diverse pathways. To identify novel genes involved in early hematopoiesis, we conducted an ENU (N-ethyl-N-nitrosourea) mutagenesis screen in zebrafish. The mummy (mmy) line was investigated because of its multiple hematopoietic defects. RESULTS: Homozygous mmy embryos lacked circulating blood cell types and were dead by 30 hr post-fertilization (hpf). The mmy mutants did not express myeloid markers and had significantly decreased expression of progenitor and erythroid markers in primitive hematopoiesis...
May 2012: Developmental Dynamics: An Official Publication of the American Association of Anatomists
Chi-Kang Tseng, Hsueh-Lien Liu, Soo-Chen Cheng
The assembly of the spliceosome involves dynamic rearrangements of interactions between snRNAs, protein components, and the pre-mRNA substrate. DExD/H-box ATPases are required to mediate structural changes of the spliceosome, utilizing the energy of ATP hydrolysis. Two DExD/H-box ATPases are required for the catalytic steps of the splicing pathway, Prp2 for the first step and Prp16 for the second step, both belonging to the DEAH subgroup of the protein family. The detailed mechanism of their action was not well understood until recently, when Prp2 was shown to be required for the release of U2 components SF3a and SF3b, presumably to allow the binding of Cwc25 to promote the first transesterification reaction...
January 2011: RNA
Zbigniew Warkocki, Peter Odenwälder, Jana Schmitzová, Florian Platzmann, Holger Stark, Henning Urlaub, Ralf Ficner, Patrizia Fabrizio, Reinhard Lührmann
The spliceosome is a ribonucleoprotein machine that removes introns from pre-mRNA in a two-step reaction. To investigate the catalytic steps of splicing, we established an in vitro splicing complementation system. Spliceosomes stalled before step 1 of this process were purified to near-homogeneity from a temperature-sensitive mutant of the RNA helicase Prp2, compositionally defined, and shown to catalyze efficient step 1 when supplemented with recombinant Prp2, Spp2 and Cwc25, thereby demonstrating that Cwc25 has a previously unknown role in promoting step 1...
December 2009: Nature Structural & Molecular Biology
Tadashi Kawashima, Matteo Pellegrini, Guillaume F Chanfreau
The role of many splicing factors in pre-mRNA splicing and the involvement of these factors in the processing of specific transcripts have often been defined through the analysis of loss-of-function mutants in vivo. Here we show that inactivating the nonsense-mediated mRNA decay (NMD) results in an enhancement of splicing phenotypes associated with several S. cerevisiae splicing factor mutations. Tiling microarrays showed that inactivation of the NMD factor Upf1p in the prp17Delta and prp18Delta mutant strains results in a larger spectrum of splicing defects than what is observed in the single mutants, including new transcripts previously shown unaffected by Prp17p or Prp18p inactivation...
December 2009: RNA
Denis Kudlinzki, Christian Nagel, Ralf Ficner
The Homo sapiens DExD/H-box protein hPrp22 is a crucial component of the eukaryotic pre-mRNA splicing machinery. Within the splicing cycle, it is involved in the ligation of exons and generation of the lariat and it additionally catalyzes the release of mature mRNA from the spliceosomal U5 snRNP. The yeast homologue of this protein, yPrp22, shows ATP-dependent RNA-helicase activity and is capable of unwinding RNA/RNA duplex molecules. A truncated construct coding for residues 950-1183 of human Prp22, comprising the structurally and functionally uncharacterized C-terminal domain, was cloned into an Escherichia coli expression vector...
September 1, 2009: Acta Crystallographica. Section F, Structural Biology and Crystallization Communications
Ondrej Gahura, Katerina Abrhámová, Michal Skruzný, Anna Valentová, Vanda Munzarová, Petr Folk, Frantisek Půta
Human transcription co-regulator SNW1/SKIP is implicated in the regulation of both transcription elongation and alternative splicing. Prp45, the SNW/SKIP ortholog in yeast, is assumed to be essential for pre-mRNA processing. Here, we characterize prp45(1-169), a temperature sensitive allele of PRP45, which at permissive temperature elicits cell division defects and hypersensitivity to microtubule inhibitors. Using a synthetic lethality screen, we found that prp45(1-169) genetically interacts with alleles of NTC members SYF1, CLF1/SYF3, NTC20, and CEF1, and 2nd step splicing factors SLU7, PRP17, PRP18, and PRP22...
January 1, 2009: Journal of Cellular Biochemistry
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