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Reinhard Luhrmann - One of the best experts on this subject based on the ideXlab platform.
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cryo em structure of a pre catalytic human Spliceosome primed for activation
Cell, 2017Co-Authors: Karl Bertram, Cindy L Will, Reinhard Luhrmann, Henning Urlaub, Olexandr Dybkov, Dmitry E Agafonov, David Haselbach, Majety N Leelaram, Berthold Kastner, Holger StarkAbstract:Summary Little is known about the Spliceosome's structure before its extensive remodeling into a catalytically active complex. Here, we report a 3D cryo-EM structure of a pre-catalytic human spliceosomal B complex. The U2 snRNP-containing head domain is connected to the B complex main body via three main bridges. U4/U6.U5 tri-snRNP proteins, which are located in the main body, undergo significant rearrangements during tri-snRNP integration into the B complex. These include formation of a partially closed Prp8 conformation that creates, together with Dim1, a 5′ splice site (ss) binding pocket, displacement of Sad1, and rearrangement of Brr2 such that it contacts its U4/U6 substrate and is poised for the subsequent Spliceosome activation step. The molecular organization of several B-specific proteins suggests that they are involved in negatively regulating Brr2, positioning the U6/5′ss helix, and stabilizing the B complex structure. Our results indicate significant differences between the early activation phase of human and yeast Spliceosomes.
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regulation of prp43 mediated disassembly of Spliceosomes by its cofactors ntr1 and ntr2
Nucleic Acids Research, 2017Co-Authors: Jeanbaptiste Fourmann, Marcel J Tauchert, Ralf Ficner, Patrizia Fabrizio, Reinhard LuhrmannAbstract:: 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. Furthermore, we show that Ntr1΄s C-terminal domain (CTD) plays a safeguarding role by preventing Prp43 from disrupting wild-type Spliceosomes other than the ILS. Ntr1 and Ntr2 can also discriminate between wild-type and defective Spliceosomes. In both type of Spliceosomes, Ntr1-CTD impedes Prp43-mediated disassembly while the Ntr1GP promotes disassembly. Intriguingly, Ntr2 plays a specific role in defective Spliceosomes, likely by stabilizing Ntr1 and allowing Prp43 to enter a productive interaction with the GP motif of Ntr1. Our data indicate that Ntr1 and Ntr2 act as 'doorkeepers' and suggest that both cofactors inspect the RNP structure of spliceosomal complexes thereby targeting suboptimal Spliceosomes for Prp43-mediated disassembly.
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SnapShot: Spliceosome Dynamics II
Cell, 2015Co-Authors: Markus C. Wahl, Reinhard LuhrmannAbstract:Numerous mechanisms exploit or modulate the conformational/compositional dynamics of Spliceosomes to regulate splicing. The majority of higher eukaryotic protein-coding genes contain more than one intron and the derived pre-mRNAs can be alternatively spliced. Diverse principles ensure the reliable identification of authentic splice sites while concomitantly providing flexibility in splice site choice during alternative splicing. Some species contain a second type of minor (U12-type) Spliceosome.
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the rna helicase aquarius exhibits structural adaptations mediating its recruitment to Spliceosomes
Nature Structural & Molecular Biology, 2015Co-Authors: Inessa De, Sergey Bessonov, Karine Dos Santos, Cindy L Will, Reinhard Luhrmann, Romina V Hofele, Henning Urlaub, Vladimir PenaAbstract:Aquarius is an RNA helicase associated with Spliceosomes. Luhrmann, Pena and colleagues now provide structural insights into how Aquarius is recruited to the Spliceosome, revealing a new spliceosomal building block that aids in Aquarius positioning.
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The G-patch protein Spp2 couples the Spliceosome-stimulated ATPase activity of the DEAH-box protein Prp2 to catalytic activation of the Spliceosome.
Genes & Development, 2015Co-Authors: Zbigniew Warkocki, Patrizia Fabrizio, Cornelius Schneider, Sina Mozaffari-jovin, Jana Schmitzová, Claudia Höbartner, Reinhard LuhrmannAbstract:Structural rearrangement of the activated Spliceosome (B(act)) to yield a catalytically active complex (B*) is mediated by the DEAH-box NTPase Prp2 in cooperation with the G-patch protein Spp2. However, how the energy of ATP hydrolysis by Prp2 is coupled to mechanical work and what role Spp2 plays in this process are unclear. Using a purified splicing system, we demonstrate that Spp2 is not required to recruit Prp2 to its bona fide binding site in the B(act) Spliceosome. In the absence of Spp2, the B(act) Spliceosome efficiently triggers Prp2's NTPase activity, but NTP hydrolysis is not coupled to ribonucleoprotein (RNP) rearrangements leading to catalytic activation of the Spliceosome. Transformation of the B(act) to the B* Spliceosome occurs only when Spp2 is present and is accompanied by dissociation of Prp2 and a reduction in its NTPase activity. In the absence of Spliceosomes, Spp2 enhances Prp2's RNA-dependent ATPase activity without affecting its RNA affinity. Our data suggest that Spp2 plays a major role in coupling Prp2's ATPase activity to remodeling of the Spliceosome into a catalytically active machine.
Tetsuo Hashimoto - One of the best experts on this subject based on the ideXlab platform.
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A novel Spliceosome-mediated trans-splicing can change our view on genome complexity of the divergent eukaryote Giardia intestinalis
Biophysical Reviews, 2011Co-Authors: Ryoma Kamikawa, Yuji Inagaki, Tetsuo HashimotoAbstract:Although spliceosomal introns are an abundant landmark in eukaryotic genomes, the nuclear genome of the divergent eukaryote Giardia intestinalis , the causative agent of giardiasis, has been considered as “intron-poor” with only five canonical ( cis -spliced) introns. However, three research groups (including ours) have independently reported a novel class of spliceosomal introns in the G. intestinalis genome. Three protein-coding genes are split into pieces in the G. intestinalis genome, and each of the partial coding regions was independently transcribed into polyadenylated premature mRNAs (pre-mRNAs). The two pre-mRNAs directly interact with each other by an intermolecular-stem structure formed between their non-coding portions, and are then processed into mature mRNAs by Spliceosome-mediated trans -splicing. Here, we summarize the recently published works on split introns (“splintrons”) in the G. intestinalis genome, and then provide our speculation on the functional property of the Giardia Spliceosomes based on the putative ratio of splintrons to canonical introns. Finally, we discuss a scenario for the transition from typical GT-AG boundaries to non-typical AT-AC boundaries in a particular splintron of Giardia .
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Splintrons in Giardia intestinalis: Spliceosomal introns in a split form.
Communicative & Integrative Biology, 2011Co-Authors: Ryoma Kamikawa, Yuji Inagaki, Andrew J. Roger, Tetsuo HashimotoAbstract:The divergent eukaryotic unicellular organism Giardia intestinalis is an intestinal parasite in humans and various animals. An analysis of a draft genome sequence suggested that G. intestinalis has a much simpler genome organization and gene repertoire than those of other model eukaryotic organisms (e.g., Arabidopsis and human). This general picture of the G. intestinalis genome seemingly agrees with the fact that only four spliceosomal (cis-spliced) introns have been identified in this organism to date. We have recently shown that G. intestinalis possesses a unique gene expression system incorporating Spliceosome-mediated trans-splicing. Some protein-coding genes in G. intestinalis are split into multiple pieces in the genome and each gene fragment is independently transcribed. Two particular pre-mRNAs directly interact with each other by forming an intermolecular-stem structure and are then trans-spliced into a mature mRNA by Spliceosomes. We believe that this trans-splicing secondarily arose from the s...
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split introns in the genome of giardia intestinalis are excised by Spliceosome mediated trans splicing
Current Biology, 2011Co-Authors: Ryoma Kamikawa, Yuji Inagaki, Andrew J. Roger, Masaharu Tokoro, Tetsuo HashimotoAbstract:Summary Spliceosomal introns are hallmarks of most eukaryotic genomes and are excised from premature mRNAs by a Spliceosome that is among the largest, and most complex, molecular machine in cells [1]. The divergent unicellular eukaryote Giardia intestinalis , the causative agent of giardiasis, also possesses Spliceosomes, but only four canonical ( cis -spliced) introns have been identified in its genome to date [2–4]. We demonstrate that this organism has a novel form of Spliceosome-mediated trans -splicing of split introns that is essential for generating mature mRNAs for at least two important genes: one encoding a heat shock protein 90 (HSP90), which controls the conformation of a suite of cellular proteins [5], and the other encoding a dynein molecular motor protein, involved in the motility of eukaryotic flagella [6]. These split introns have properties that distinguish them from other trans- splicing systems known within eukaryotes, suggesting that Giardia independently evolved a unique system to splice split introns.
Henning Urlaub - One of the best experts on this subject based on the ideXlab platform.
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cryo em structure of a pre catalytic human Spliceosome primed for activation
Cell, 2017Co-Authors: Karl Bertram, Cindy L Will, Reinhard Luhrmann, Henning Urlaub, Olexandr Dybkov, Dmitry E Agafonov, David Haselbach, Majety N Leelaram, Berthold Kastner, Holger StarkAbstract:Summary Little is known about the Spliceosome's structure before its extensive remodeling into a catalytically active complex. Here, we report a 3D cryo-EM structure of a pre-catalytic human spliceosomal B complex. The U2 snRNP-containing head domain is connected to the B complex main body via three main bridges. U4/U6.U5 tri-snRNP proteins, which are located in the main body, undergo significant rearrangements during tri-snRNP integration into the B complex. These include formation of a partially closed Prp8 conformation that creates, together with Dim1, a 5′ splice site (ss) binding pocket, displacement of Sad1, and rearrangement of Brr2 such that it contacts its U4/U6 substrate and is poised for the subsequent Spliceosome activation step. The molecular organization of several B-specific proteins suggests that they are involved in negatively regulating Brr2, positioning the U6/5′ss helix, and stabilizing the B complex structure. Our results indicate significant differences between the early activation phase of human and yeast Spliceosomes.
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the rna helicase aquarius exhibits structural adaptations mediating its recruitment to Spliceosomes
Nature Structural & Molecular Biology, 2015Co-Authors: Inessa De, Sergey Bessonov, Karine Dos Santos, Cindy L Will, Reinhard Luhrmann, Romina V Hofele, Henning Urlaub, Vladimir PenaAbstract:Aquarius is an RNA helicase associated with Spliceosomes. Luhrmann, Pena and colleagues now provide structural insights into how Aquarius is recruited to the Spliceosome, revealing a new spliceosomal building block that aids in Aquarius positioning.
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dissection of the factor requirements for Spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system
Genes & Development, 2013Co-Authors: Jeanbaptiste Fourmann, Ralf Ficner, Patrizia Fabrizio, Henning Urlaub, Jana Schmitzová, Henning Christian, Kumloong Boon, Reinhard LuhrmannAbstract: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. Incubation of the isolated ILS with the RNA helicase Prp43 plus Ntr1/Ntr2 and ATP generates defined spliceosomal dissociation products: the intron-lariat, U6 snRNA, a 20–25S U2 snRNP containing SF3a/b, an 18S U5 snRNP, and the ‘‘nineteen complex’’ associated with both the released U2 snRNP and intron-lariat RNA. Our system reproduces the entire ordered disassembly phase of the Spliceosome with purified components, which defines the minimum set of agents required for this process. It enabled us to characterize the proteins of the ILS by mass spectrometry and identify the ATPase action of Prp43 as necessary and sufficient for dissociation of the ILS without the involvement of Brr2 ATPase.
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small nuclear ribonucleoprotein remodeling during catalytic activation of the Spliceosome
Science, 2002Co-Authors: Evgeny M Makarov, Cindy L Will, Henning Urlaub, Olga V Makarova, Marc Gentzel, Matthias Wilm, Reinhard LuhrmannAbstract:Major structural changes occur in the Spliceosome during its activation just before catalyzing the splicing of pre-messenger RNAs (pre-mRNAs). Whereas changes in small nuclear RNA (snRNA) conformation are well documented, little is known about remodeling of small nuclear ribonucleoprotein (snRNP) structures during Spliceosome activation. Here, human 45S activated Spliceosomes and a previously unknown 35S U5 snRNP were isolated by immunoaffinity selection and were characterized by mass spectrometry. Comparison of their protein components with those of other snRNP and spliceosomal complexes revealed a major change in protein composition during Spliceosome activation. Our data also suggest that the U5 snRNP is dramatically remodeled at this stage, with the Prp19 complex and other factors tightly associating, possibly in exchange for other U5 proteins, and suggest that after catalysis the remodeled U5 is eventually released from the postsplicing complex as a 35S snRNP particle.
Yigong Shi - One of the best experts on this subject based on the ideXlab platform.
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How Is Precursor Messenger RNA Spliced by the Spliceosome
Annual Review of Biochemistry, 2019Co-Authors: Ruixue Wan, Rui Bai, Xiechao Zhan, Yigong ShiAbstract:Splicing of the precursor messenger RNA, involving intron removal and exon ligation, is mediated by the Spliceosome. Together with biochemical and genetic investigations of the past four decades, structural studies of the intact Spliceosome at atomic resolution since 2015 have led to mechanistic delineation of RNA splicing with remarkable insights. The Spliceosome is proven to be a protein-orchestrated metalloribozyme. Conserved elements of small nuclear RNA (snRNA) constitute the splicing active site with two catalytic metal ions and recognize three conserved intron elements through duplex formation, which are delivered into the splicing active site for branching and exon ligation. The protein components of the Spliceosome stabilize the conformation of the snRNA, drive Spliceosome remodeling, orchestrate the movement of the RNA elements, and facilitate the splicing reaction. The overall organization of the Spliceosome and the configuration of the splicing active site are strictly conserved between human and yeast.
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Molecular choreography of pre-mRNA splicing by the Spliceosome.
Current Opinion in Structural Biology, 2019Co-Authors: Ruixue Wan, Rui Bai, Yigong ShiAbstract:The Spliceosome executes eukaryotic precursor messenger RNA (pre-mRNA) splicing to remove noncoding introns through two sequential transesterification reactions, branching and exon ligation. The fidelity of this process is based on the recognition of the conserved sequences in the intron and dynamic compositional and structural rearrangement of this multi-megadalton machinery. Since atomic visualization of the splicing active site in an endogenous Schizosaccharomyces pombe Spliceosome in 2015, high-resolution cryoelectron microscopy (cryo-EM) structures of other Spliceosome intermediates began to uncover the molecular mechanism. Recent advances in the structural biology of the Spliceosome make it clearer the mechanisms of its assembly, activation, disassembly and exon ligation. Together, these discrete structural images give rise to a molecular choreography of the Spliceosome.
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mechanistic insights into precursor messenger rna splicing by the Spliceosome
Nature Reviews Molecular Cell Biology, 2017Co-Authors: Yigong ShiAbstract:Precursor messenger RNA (pre-mRNA) splicing is an essential step in the flow of information from DNA to protein in all eukaryotes. Research over the past four decades has molecularly delineated the splicing pathway, including characterization of the detailed splicing reaction, definition of the Spliceosome and identification of its components, and biochemical analysis of the various splicing complexes and their regulation. Structural information is central to mechanistic understanding of pre-mRNA splicing by the Spliceosome. X-ray crystallography of the spliceosomal components and subcomplexes is complemented by electron microscopy of the intact Spliceosome. In this Review, I discuss recent atomic-resolution structures of the intact Spliceosome at different stages of the splicing cycle. These structures have provided considerable mechanistic insight into pre-mRNA splicing and have corroborated and explained a large body of genetic and biochemical data. Together, the structural data have proved that the Spliceosome is a protein-directed metalloribozyme.
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The Spliceosome: A Protein-Directed Metalloribozyme.
Journal of Molecular Biology, 2017Co-Authors: Yigong ShiAbstract:Abstract Pre-mRNA splicing is executed by the ribonucleoprotein machinery Spliceosome. Nearly 40 years after the discovery of pre-mRNA splicing, the atomic structure of the Spliceosome has finally come to light. Four distinct conformational states of the yeast Spliceosome have been captured at atomic or near-atomic resolutions. Two catalytic metal ions at the active site are specifically coordinated by the U6 small nuclear RNA (snRNA) and catalyze both the branching reaction and the exon ligation. Of the three snRNAs in the fully assembled Spliceosome, U5 and U6, along with 30 contiguous nucleotides of U2 at its 5′-end, remain structurally rigid throughout the splicing reaction. The rigidity of these RNA elements is safeguarded by Prp8 and 16 core protein components, which maintain the same overall conformation in all structurally characterized Spliceosomes during the splicing reaction. Only the sequences downstream of nucleotide 30 of U2 snRNA are mobile; their movement, directed by the protein components, delivers the intron branch site into the close proximity of the 5′-splice site for the branching reaction. A set of additional structural rearrangement is required for exon ligation, and the lariat junction is moved out of the active site for recruitment of the 3′-splice site and 3′-exon. The Spliceosome is proven to be a protein-directed metalloribozyme.
Soo-chen Cheng - One of the best experts on this subject based on the ideXlab platform.
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Structural requirement of Ntc77 for Spliceosome activation and first catalytic step
Nucleic Acids Research, 2014Co-Authors: Hsin-chou Chen, Kae-jiun Chang, Yu-hsin Huang, Soo-chen ChengAbstract:The Prp19-associated complex is required for Spliceosome activation by stabilizing the binding of U5 and U6 on the Spliceosome after the release of U4. The complex comprises at least eight proteins, among which Ntc90 and Ntc77 contain multiple tetratricopeptide repeat (TPR) elements. We have previously shown that Ntc90 is not involved in Spliceosome activation, but is required for the recruitment of essential first-step factor Yju2 to the Spliceosome. We demonstrate here that Ntc77 has dual functions in both Spliceosome activation and the first catalytic step in recruiting Yju2. We have identified an amino-terminal region of Ntc77, which encompasses the N-terminal domain and the first three TPR motifs, dispensable for Spliceosome activation but required for stable interaction of Yju2 with the Spliceosome. Deletion of this region had no severe effect on the integrity of the NTC, binding of NTC to the Spliceosome or Spliceosome activation, but impaired splicing and exhibited a dominant-negative growth phenotype. Our data reveal functional roles of Ntc77 in both Spliceosome activation and the first catalytic step, and distinct structural domains of Ntc77 required for these two steps.
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The Spliceosome catalyzes debranching in competition with reverse of the first chemical reaction
RNA, 2013Co-Authors: Chi-kang Tseng, Soo-chen ChengAbstract:Splicing of nuclear pre-mRNA occurs via two steps of the transesterification reaction, forming a lariat intermediate and product. The reactions are catalyzed by the Spliceosome, a large ribonucleoprotein complex composed of five small nuclear RNAs and numerous protein factors. The Spliceosome shares a similar catalytic core structure with that of fungal group II introns, which can self-splice using the same chemical mechanism. Like group II introns, both catalytic steps of pre-mRNA splicing can efficiently reverse on the affinity-purified Spliceosome. The Spliceosome also catalyzes a hydrolytic spliced-exon reopening reaction as observed in group II introns, indicating a strong link in their evolutionary relationship. We show here that, by arresting splicing after the first catalytic step, the purified Spliceosome can catalyze debranching of lariat-intron-exon 2. The debranching reaction, although not observed in group II introns, has similar monovalent cation preferences as those for splicing catalysis of group II introns. The debranching reaction is in competition with the reverse Step 1 reaction influenced by the ionic environment and the structure of components binding near the catalytic center, suggesting that the catalytic center of the Spliceosome can switch between different conformations to direct different chemical reactions.
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Link of NTR-Mediated Spliceosome Disassembly with DEAH-Box ATPases Prp2, Prp16, and Prp22
Molecular and Cellular Biology, 2012Co-Authors: Hsin-chou Chen, Chi-kang Tseng, Rong-tzong Tsai, Che-sheng Chung, Soo-chen ChengAbstract: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. We found that NTR could catalyze the disassembly of affinity-purified Spliceosomes arrested specifically after the ATP-dependent action of DEAH-box ATPase Prp2, Prp16, or Prp22 but not at steps before the action of these ATPases or upon their binding to the Spliceosome. These results link Spliceosome disassembly to the functioning of splicing ATPases. Analysis of the binding of Ntr2 to each splicing complex has revealed that the presence of Prp16 and Slu7, which also interact with Brr2, has a negative impact on Ntr2 binding. Our study provides insights into the mechanism by which NTR can be recruited to the Spliceosome to mediate the disassembly of Spliceosome intermediates when the Spliceosome pathway is retarded, while disassembly is prevented in normal reactions.
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Spliceosome disassembly catalyzed by prp43 and its associated components ntr1 and ntr2
Genes & Development, 2005Co-Authors: Rong-tzong Tsai, Chi-kang Tseng, Soo-chen Cheng, Yu-hsin Huang, Fu Lung Yeh, Yu Chieh LinAbstract:Two novel yeast splicing factors required for Spliceosome disassembly have been identified. Ntr1 and Ntr2 (NineTeen complex-Related proteins) were identified for their weak association with components of the Prp19-associated complex. Unlike other Prp19-associated components, these two proteins were primarily associated with the intron-containing Spliceosome during the splicing reaction. Extracts depleted of Ntr1 or Ntr2 exhibited full splicing activity, but accumulated large amounts of lariat-intron in the Spliceosome after splicing, indicating that the normal function of the Prp19-associated complex in Spliceosome activation was not affected, but Spliceosome disassembly was hindered. Immunoprecipitation analysis revealed that Ntr1 and Ntr2 formed a stable complex with DExD/H-box RNA helicase Prp43 in the splicing extract. Ntr1 interacted with Prp43 through the N-terminal G-patch domain, with Ntr2 through a middle region, and with itself through the carboxyl half of the protein. The affinity-purified Ntr1–Ntr2–Prp43 complex could catalyze disassembly of the Spliceosome in an ATP-dependent manner, separating U2, U5, U6, NTC (NineTeen Complex), and lariat-intron. This is the first demonstration of physical disassembly of the Spliceosome, catalyzed by a complex containing a DExD/H-box RNA helicase and two accessory factors, which might function in targeting the helicase to the correct substrate.
