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Reinhard Luhrmann - One of the best experts on this subject based on the ideXlab platform.
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smu1 and red are required for activation of spliceosomal b complexes assembled on short introns
Nature Communications, 2019Co-Authors: Sandra Keiper, Cindy L Will, Cyrille Girard, Panagiotis Papasaikas, Juan Valcarcel, Reinhard LuhrmannAbstract:Human pre-catalytic Spliceosomes contain several proteins that associate transiently just prior to spliceosome activation and are absent in yeast, suggesting that this critical step is more complex in higher eukaryotes. We demonstrate via RNAi coupled with RNA-Seq that two of these human-specific proteins, Smu1 and RED, function both as alternative splicing regulators and as general splicing factors and are required predominantly for efficient splicing of short introns. In vitro splicing assays reveal that Smu1 and RED promote spliceosome activation, and are essential for this step when the distance between the pre-mRNA's 5' splice site (SS) and branch site (BS) is sufficiently short. This Smu1-RED requirement can be bypassed when the 5' and 3' regions of short introns are physically separated. Our observations suggest that Smu1 and RED relieve physical constraints arising from a short 5'SS-BS distance, thereby enabling Spliceosomes to overcome structural challenges associated with the splicing of short introns.
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the target of the deah box ntp triphosphatase prp43 in saccharomyces cerevisiae Spliceosomes is the u2 snrnp intron interaction
eLife, 2016Co-Authors: Jeanbaptiste Fourmann, Marcel J Tauchert, Ralf Ficner, Patrizia Fabrizio, Olexandr Dybkov, Dmitry E Agafonov, Henning Urlaub, Reinhard LuhrmannAbstract: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. Unlike the NTR, Prp43_Ntr1GP disassembles earlier spliceosomal complexes (A, B, Bact), indicating that Ntr2/Ntr1-CTD prevents NTR from disrupting properly assembled Spliceosomes other than the ILS. The U2 snRNP-intron interaction is disrupted in all complexes by Prp43_Ntr1GP, and in the spliceosome contacts U2 proteins and the pre-mRNA, indicating that the U2 snRNP-intron interaction is Prp43’s major target.
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molecular architecture of the human u4 u6 u5 tri snrnp
Science, 2016Co-Authors: Dmitry E Agafonov, Romina V Hofele, Olexandr Dybkov, Berthold Kastner, Reinhard Luhrmann, Henning Urlaub, Wenti Liu, Holger StarkAbstract:The U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) is a major spliceosome building block. We obtained a three-dimensional structure of the 1.8-megadalton human tri-snRNP at a resolution of 7 angstroms using single-particle cryo–electron microscopy (cryo-EM). We fit all known high-resolution structures of tri-snRNP components into the EM density map and validated them by protein cross-linking. Our model reveals how the spatial organization of Brr2 RNA helicase prevents premature U4/U6 RNA unwinding in isolated human tri-snRNPs and how the ubiquitin C-terminal hydrolase–like protein Sad1 likely tethers the helicase Brr2 to its preactivation position. Comparison of our model with cryo-EM three-dimensional structures of the Saccharomyces cerevisiae tri-snRNP and Schizosaccharomyces pombe spliceosome indicates that Brr2 undergoes a marked conformational change during spliceosome activation, and that the scaffolding protein Prp8 is also rearranged to accommodate the spliceosome’s catalytic RNA network.
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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
PLOS Genetics, 2015Co-Authors: Cornelius Schneider, Patrizia Fabrizio, Jana Schmitzová, Dmitry E Agafonov, Klaus Hartmuth, Reinhard LuhrmannAbstract: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 Bact 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 Bact 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.
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rna structure analysis of human Spliceosomes reveals a compact 3d arrangement of snrnas at the catalytic core
The EMBO Journal, 2013Co-Authors: Maria Anokhina, Sergey Bessonov, Klaus Hartmuth, Eric Westhof, Zhichao Miao, Reinhard LuhrmannAbstract:Although U snRNAs play essential roles in splicing, little is known about the 3D arrangement of U2, U6, and U5 snRNAs and the pre-mRNA in active Spliceosomes. To elucidate their relative spatial organization and dynamic rearrangement, we examined the RNA structure of affinity-purified, human Spliceosomes before and after catalytic step 1 by chemical RNA structure probing. We found a stable 3-way junction of the U2/U6 snRNA duplex in active Spliceosomes that persists minimally through step 1. Moreover, the formation of alternating, mutually exclusive, U2 snRNA conformations, as observed in yeast, was not detected in different assembly stages of human spliceosomal complexes (that is, B, Bact, or C complexes). Psoralen crosslinking revealed an interaction during/after step 1 between internal loop 1 of the U5 snRNA, and intron nucleotides immediately downstream of the branchpoint. Using the experimentally derived structural constraints, we generated a model of the RNA network of the step 1 spliceosome, based on the crystal structure of a group II intron through homology modelling. The model is topologically consistent with current genetic, biochemical, and structural data.
Cindy L Will - One of the best experts on this subject based on the ideXlab platform.
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smu1 and red are required for activation of spliceosomal b complexes assembled on short introns
Nature Communications, 2019Co-Authors: Sandra Keiper, Cindy L Will, Cyrille Girard, Panagiotis Papasaikas, Juan Valcarcel, Reinhard LuhrmannAbstract:Human pre-catalytic Spliceosomes contain several proteins that associate transiently just prior to spliceosome activation and are absent in yeast, suggesting that this critical step is more complex in higher eukaryotes. We demonstrate via RNAi coupled with RNA-Seq that two of these human-specific proteins, Smu1 and RED, function both as alternative splicing regulators and as general splicing factors and are required predominantly for efficient splicing of short introns. In vitro splicing assays reveal that Smu1 and RED promote spliceosome activation, and are essential for this step when the distance between the pre-mRNA's 5' splice site (SS) and branch site (BS) is sufficiently short. This Smu1-RED requirement can be bypassed when the 5' and 3' regions of short introns are physically separated. Our observations suggest that Smu1 and RED relieve physical constraints arising from a short 5'SS-BS distance, thereby enabling Spliceosomes to overcome structural challenges associated with the splicing of short introns.
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cryo em structure of a pre catalytic human spliceosome primed for activation
Cell, 2017Co-Authors: Karl Bertram, Cindy L Will, 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, Romina V Hofele, 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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Spliceosome structure and function
Cold Spring Harbor Perspectives in Biology, 2011Co-Authors: Cindy L WillAbstract:Pre-mRNA splicing is catalyzed by the spliceosome, a multimegadalton ribonucleoprotein (RNP) complex comprised of five snRNPs and numerous proteins. Intricate RNA-RNA and RNP networks, which serve to align the reactive groups of the pre-mRNA for catalysis, are formed and repeatedly rearranged during spliceosome assembly and catalysis. Both the conformation and composition of the spliceosome are highly dynamic, affording the splicing machinery its accuracy and flexibility, and these remarkable dynamics are largely conserved between yeast and metazoans. Because of its dynamic and complex nature, obtaining structural information about the spliceosome represents a major challenge. Electron microscopy has revealed the general morphology of several spliceosomal complexes and their snRNP subunits, and also the spatial arrangement of some of their components. X-ray and NMR studies have provided high resolution structure information about spliceosomal proteins alone or complexed with one or more binding partners. The extensive interplay of RNA and proteins in aligning the pre-mRNA's reactive groups, and the presence of both RNA and protein at the core of the splicing machinery, suggest that the spliceosome is an RNP enzyme. However, elucidation of the precise nature of the spliceosome's active site, awaits the generation of a high-resolution structure of its RNP core.
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conservation of the protein composition and electron microscopy structure of drosophila melanogaster and human spliceosomal complexes
Molecular and Cellular Biology, 2009Co-Authors: Nadine Herold, Cindy L Will, Berthold Kastner, Henning Urlaub, Elmar Wolf, Reinhard LuhrmannAbstract:Comprehensive proteomics analyses of spliceosomal complexes are currently limited to those in humans, and thus, it is unclear to what extent the spliceosome's highly complex composition and compositional dynamics are conserved among metazoans. Here we affinity purified Drosophila melanogaster spliceosomal B and C complexes formed in Kc cell nuclear extract. Mass spectrometry revealed that their composition is highly similar to that of human B and C complexes. Nonetheless, a number of Drosophila-specific proteins were identified, suggesting that there may be novel factors contributing specifically to splicing in flies. Protein recruitment and release events during the B-to-C transition were also very similar in both organisms. Electron microscopy of Drosophila B complexes revealed a high degree of structural similarity with human B complexes, indicating that higher-order interactions are also largely conserved. A comparison of Drosophila Spliceosomes formed on a short versus long intron revealed only small differences in protein composition but, nonetheless, clear structural differences under the electron microscope. Finally, the characterization of affinity-purified Drosophila mRNPs indicated that exon junction complex proteins are recruited in a splicing-dependent manner during C complex formation. These studies provide insights into the evolutionarily conserved composition and structure of the metazoan spliceosome, as well as its compositional dynamics during catalytic activation.
Scott William Roy - One of the best experts on this subject based on the ideXlab platform.
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expansion and transformation of the minor spliceosomal system in the slime mold physarum polycephalum
Current Biology, 2021Co-Authors: Graham E Larue, Marek Eliáš, Scott William RoyAbstract:Summary Spliceosomal introns interrupt nuclear genes and are removed from RNA transcripts ("spliced") by machinery called Spliceosomes. Although the vast majority of spliceosomal introns are removed by the so-called major (or "U2") spliceosome, diverse eukaryotes also contain a rare second form, the minor ("U12") spliceosome, and associated ("U12-type") introns.1–3 In all characterized species, U12-type introns are distinguished by several features, including being rare in the genome (∼0.5% of all introns),4–6 containing extended evolutionarily conserved splicing motifs,4,5,7,8 being generally ancient,9,10 and being inefficiently spliced.11–13 Here, we report a remarkable exception in the slime mold Physarum polycephalum. The P. polycephalum genome contains >20,000 U12-type introns—25 times more than any other species—enriched in a diversity of non-canonical splice boundaries as well as transformed splicing signals that appear to have co-evolved with the spliceosome due to massive gain of efficiently spliced U12-type introns. These results reveal an unappreciated dynamism of minor spliceosomal introns and spliceosomal introns in general.
Matthias Wilm - One of the best experts on this subject based on the ideXlab platform.
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a subset of human 35s u5 proteins including prp19 function prior to catalytic step 1 of splicing
The EMBO Journal, 2004Co-Authors: Olga V Makarova, Cindy L Will, Evgeny M Makarov, Marc Gentzel, Matthias Wilm, Henning Urlaub, Reinhard LuhrmannAbstract:During catalytic activation of the spliceosome, snRNP remodeling events occur, leading to the formation of a 35S U5 snRNP that contains a large group of proteins, including Prp19 and CDC5, not found in 20S U5 snRNPs. To investigate the function of 35S U5 proteins, we immunoaffinity purified human Spliceosomes that had not yet undergone catalytic activation (designated BΔU1), which contained U2, U4, U5, and U6, but lacked U1 snRNA. Comparison of the protein compositions of BΔU1 and activated B* Spliceosomes revealed that, whereas U4/U6 snRNP proteins are stably associated with BΔU1 Spliceosomes, 35S U5-associated proteins (which are present in B*) are largely absent, suggesting that they are dispensable for complex B formation. Indeed, immunodepletion/complementation experiments demonstrated that a subset of 35S U5 proteins including Prp19, which form a stable heteromeric complex, are required prior to catalytic step 1 of splicing, but not for stable integration of U4/U6.U5 tri-snRNPs. Thus, comparison of the proteomes of spliceosomal complexes at defined stages can provide information as to which proteins function as a group at a particular step of splicing.
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small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome
Science, 2002Co-Authors: Evgeny M Makarov, Cindy L Will, Olga V Makarova, Marc Gentzel, Matthias WilmAbstract: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.
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a novel u2 and u11 u12 snrnp protein that associates with the pre mrna branch site
The EMBO Journal, 2001Co-Authors: Cindy L Will, Nikos F. Katopodis, Andrew M Macmillan, Gitte Neubauer, Claudia Schneider, Matthias Wilm, C. C. QueryAbstract:Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa protein (p14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this protein by microsequencing a 14 kDa protein isolated from U2-type Spliceosomes. This protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded protein precipitated the 14 kDa protein cross-linked to the branch adenosine, confirming the identity of the p14 cDNA. A combination of immunoblotting, protein microsequencing and immunoprecipitation revealed that p14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2- and U12-type pre-mRNAs, respectively. p14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immuno precipitations indicated that p14 is present in U12-type Spliceosomes, consistent with the idea that branch point selection is similar in the major and minor Spliceosomes.
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partial purification of the yeast u2 snrnp reveals a novel yeast pre mrna splicing factor required for pre spliceosome assembly
The EMBO Journal, 1999Co-Authors: Friederike Caspary, Matthias Wilm, Anna Shevchenko, Bertrand SeraphinAbstract:We have partially purified the U2 snRNP of Saccharomyces cerevisiae. Identification of some proteins consistently found in the purified fractions by nanoelectrospray mass spectrometry indicated the presence of a novel splicing factor named Rse1p. The RSE1 gene is essential and codes for a 148.2 kDa protein. We demonstrated that Rse1p associates specifically with U2 snRNA at low salt concentrations. In addition, we showed that Rse1p is a component of the pre-spliceosome. Depletion of Rse1p and analysis of a conditional mutant indicated that Rse1p was required for efficient splicing in vivo. In vitro Rse1p is required for the formation of pre-Spliceosomes. Database searches revealed that Rse1p is conserved in humans and that it belongs to a large protein family that includes polyadenylation factors and DNA repair proteins. The characteristics of Rse1p suggest that its human homologue could be a subunit of the SF3 splicing factor.
Melissa J. Moore - One of the best experts on this subject based on the ideXlab platform.
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synergistic assembly of human pre Spliceosomes across introns and exons
eLife, 2018Co-Authors: Joerg E Braun, Larry J. Friedman, Jeff Gelles, Melissa J. MooreAbstract:Most human genes contain multiple introns, necessitating mechanisms to effectively define exons and ensure their proper connection by Spliceosomes. Human spliceosome assembly involves both cross-intron and cross-exon interactions, but how these work together is unclear. We examined in human nuclear extracts dynamic interactions of single pre-mRNA molecules with individual fluorescently tagged spliceosomal subcomplexes to investigate how cross-intron and cross-exon processes jointly promote pre-spliceosome assembly. U1 subcomplex bound to the 5' splice site of an intron acts jointly with U1 bound to the 5' splice site of the next intron to dramatically increase the rate and efficiency by which U2 subcomplex is recruited to the branch site/3' splice site of the upstream intron. The flanking 5' splice sites have greater than additive effects implying distinct mechanisms facilitating U2 recruitment. This synergy of 5' splice sites across introns and exons is likely important in promoting correct and efficient splicing of multi-intron pre-mRNAs.
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Transcriptome-wide Interrogation of the Functional Intronome by Spliceosome Profiling.
Cell, 2018Co-Authors: Weijun Chen, Jill Moore, Hakan Ozadam, Hennady P. Shulha, Nicholas Rhind, Zhiping Weng, Melissa J. MooreAbstract:Summary Full understanding of eukaryotic transcriptomes and how they respond to different conditions requires deep knowledge of all sites of intron excision. Although RNA sequencing (RNA-seq) provides much of this information, the low abundance of many spliced transcripts (often due to their rapid cytoplasmic decay) limits the ability of RNA-seq alone to reveal the full repertoire of spliced species. Here, we present "spliceosome profiling," a strategy based on deep sequencing of RNAs co-purifying with late-stage Spliceosomes. Spliceosome profiling allows for unambiguous mapping of intron ends to single-nucleotide resolution and branchpoint identification at unprecedented depths. Our data reveal hundreds of new introns in S. pombe and numerous others that were previously misannotated. By providing a means to directly interrogate sites of spliceosome assembly and catalysis genome-wide, spliceosome profiling promises to transform our understanding of RNA processing in the nucleus, much as ribosome profiling has transformed our understanding mRNA translation in the cytoplasm.
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transcriptome wide analysis of the functional intronome using spliceosome profiling
bioRxiv, 2017Co-Authors: Weijun Chen, Jill Moore, Hakan Ozadam, Hennady P. Shulha, Nicholas Rhind, Zhiping Weng, Melissa J. MooreAbstract:Full understanding of eukaryotic transcriptomes and how they respond to different conditions requires deep knowledge of all sites of intron excision. Although RNA-Seq provides much of this information, the low abundance of many spliced transcripts (often due to their rapid cytoplasmic decay) limits the ability of RNA-Seq alone to reveal the full repertoire of spliced species. Here we present "spliceosome profiling", a strategy based on deep sequencing of RNAs copurifying with late stage Spliceosomes. Spliceosome profiling allows for unambiguous mapping of intron ends to single nucleotide resolution and branchpoint identification at unprecedented depths. Our data reveal hundreds of new introns in S. pombe and numerous others that were previously misannotated. By providing a means to directly interrogate sites of spliceosome assembly and catalysis genome-wide, spliceosome profiling may well transform our understanding of RNA processing in the nucleus much as ribosome profiling is transforming our understanding mRNA translation in the cytoplasm.
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Ordered and Dynamic Assembly of Single Spliceosomes
Science, 2011Co-Authors: Aaron A. Hoskins, Larry J. Friedman, Sarah S. Gallagher, Daniel J. Crawford, Eric G. Anderson, Richard Wombacher, Nicholas Ramirez, Virginia W. Cornish, Jeff Gelles, Melissa J. MooreAbstract:The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single Spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional Spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.
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Dynamic and Ordered Assembly of Single Spliceosomes
Biophysical Journal, 2011Co-Authors: Aaron A. Hoskins, Larry J. Friedman, Sarah S. Gallagher, Daniel J. Crawford, Eric G. Anderson, Richard Wombacher, Nicholas Ramirez, Virginia W. Cornish, Jeff Gelles, Melissa J. MooreAbstract:Spliceosome assembly is a highly dynamic and complex process involving the addition of 5 snRNAs and >100 proteins to a pre-mRNA transcript. This process has largely been analyzed by discontinuous assays that provide limited kinetic information. By combining yeast genetics, chemical biology, and a multi-wavelength single molecule microscopy technique (Colocalization Single Molecule Spectroscopy, CoSMoS), we monitored the formation of single Spliceosomes. This approach revealed that individual spliceosome subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional Spliceosomes. No single subcomplex binding event disproportionately limits the speed of the assembly reaction. While not all pre-mRNAs acquire a functional spliceosome, commitment of pre-mRNAs to splicing increases as assembly progresses. Therefore, spliceosome assembly is not a one-way, unbranched process as some models depict. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in complex environments.