The Experts below are selected from a list of 9753 Experts worldwide ranked by ideXlab platform
Reinhard Luhrmann - One of the best experts on this subject based on the ideXlab platform.
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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, H. 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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functional organization of the sm core in the crystal structure of human u1 SnRNP
The EMBO Journal, 2010Co-Authors: Reinhard Luhrmann, Berthold Kastner, Gert Weber, Simon Trowitzsch, Markus C WahlAbstract:U1 small nuclear ribonucleoprotein (SnRNP) recognizes the 5′-splice site early during spliceosome assembly. It represents a prototype spliceosomal subunit containing a paradigmatic Sm core RNP. The crystal structure of human U1 SnRNP obtained from natively purified material by in situ limited proteolysis at 4.4 A resolution reveals how the seven Sm proteins, each recognize one nucleotide of the Sm site RNA using their Sm1 and Sm2 motifs. Proteins D1 and D2 guide the snRNA into and out of the Sm ring, and proteins F and E mediate a direct interaction between the Sm site termini. Terminal extensions of proteins D1, D2 and B/B′, and extended internal loops in D2 and B/B′ support a four-way RNA junction and a 3′-terminal stem-loop on opposite sides of the Sm core RNP, respectively. On a higher organizational level, the core RNP presents multiple attachment sites for the U1-specific 70K protein. The intricate, multi-layered interplay of proteins and RNA rationalizes the hierarchical assembly of U SnRNPs in vitro and in vivo.
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Coilin-dependent SnRNP assembly is essential for zebrafish embryogenesis
Nature Structural & Molecular Biology, 2010Co-Authors: Magdalena Strzelecka, Reinhard Luhrmann, Gert Weber, Simon Trowitzsch, Andrew C Oates, Karla M. NeugebauerAbstract:The function of many non-membrane bound aggregates (Cajal bodies, P-bodies, stress granules, etc.) is poorly understood. Now studies in zebrafish embryos show that coilin promotes spliceosomal small nuclear ribonucleoproteins (SnRNPs) assembly, most likely by concentrating SnRNP components in Cajal bodies and to overcome a rate-limiting step(s) in assembly. Spliceosomal small nuclear ribonucleoproteins (SnRNPs), comprised of small nuclear RNAs (snRNAs) in complex with SnRNP-specific proteins, are essential for pre-mRNA splicing. Coilin is not a SnRNP protein but concentrates SnRNPs and their assembly intermediates in Cajal bodies (CBs). Here we show that depletion of coilin in zebrafish embryos leads to CB dispersal, deficits in SnRNP biogenesis and expression of spliced mRNA, as well as reduced cell proliferation followed by developmental arrest. Notably, injection of purified mature human SnRNPs restored mRNA expression and viability. snRNAs were necessary but not sufficient for rescue, showing that only assembled SnRNPs can bypass the requirement for coilin. Thus, coilin's essential function in embryos is to promote macromolecular assembly of SnRNPs, likely by concentrating SnRNP components in CBs to overcome rate-limiting assembly steps.
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Exon definition complexes contain the tri-SnRNP and can be directly converted into B-like precatalytic splicing complexes
Molecular Cell, 2010Co-Authors: M. Schneider, J. Tazi, C. L. Will, M. Anokhina, H. Urlaub, Reinhard LuhrmannAbstract:The first step in splicing of pre-mRNAs with long introns is exon definition, where U1 and U2 SnRNPs bind at opposite ends of an exon. After exon definition, these SnRNPs must form a complex across the upstream intron to allow splicing catalysis. Exon definition and conversion of cross-exon to cross-intron spliceosomal complexes are poorly understood. Here we demonstrate that, in addition to U1 and U2 SnRNPs, cross-exon complexes contain U4, U5, and U6 (which form the tri-SnRNP). Tri-SnRNP docking involves the formation of U2/U6 helix II. This interaction is stabilized by a 5' splice site (SS)-containing oligonucleotide, which can bind the tri-SnRNP and convert the cross-exon complex into a cross-intron, B-like complex. Our data suggest that the switch from cross-exon to cross-intron complexes can occur directly when an exon-bound tri-SnRNP interacts with an upstream 5'SS, without prior formation of a cross-intron A complex, revealing an alternative spliceosome assembly pathway.
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Organization of Core Spliceosomal Components U5 snRNA Loop I and U4/U6 Di-SnRNP within U4/U6.U5 Tri-SnRNP as Revealed by Electron Cryomicroscopy
Molecular cell, 2006Co-Authors: Bjoern Sander, Reinhard Luhrmann, Monika M. Golas, Evgeny M. Makarov, Hero Brahms, Berthold Kastner, Holger StarkAbstract:In eukaryotes, pre-mRNA exons are interrupted by large noncoding introns. Alternative selection of exons and nucleotide-exact removal of introns are performed by the spliceosome, a highly dynamic macromolecular machine. U4/U6.U5 tri-SnRNP is the largest and most conserved building block of the spliceosome. By 3D electron cryomicroscopy and labeling, the exon-aligning U5 snRNA loop I is localized at the center of the tetrahedrally shaped tri-SnRNP reconstructed to ∼2.1 nm resolution in vitrified ice. Independent 3D reconstructions of its subunits, U4/U6 and U5 SnRNPs, show how U4/U6 and U5 combine to form tri-SnRNP and, together with labeling experiments, indicate a close proximity of the spliceosomal core components U5 snRNA loop I and U4/U6 at the center of tri-SnRNP. We suggest that this central tri-SnRNP region may be the site to which the prespliceosomal U2 snRNA has to approach closely during formation of the catalytic core of the spliceosome.
Gideon Dreyfuss - One of the best experts on this subject based on the ideXlab platform.
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u1 SnRNP protects pre mrnas from premature cleavage and polyadenylation
Nature, 2010Co-Authors: Daisuke Kaida, Michael G Berg, Ihab Younis, Mumtaz Kasim, Larry N Singh, Lili Wan, Gideon DreyfussAbstract:In eukaryotes, U1 small nuclear ribonucleoprotein (SnRNP) forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6 SnRNPs; however, its abundance in human far exceeds that of the other SnRNPs. Here we used antisense morpholino oligonucleotide to U1 snRNA to achieve functional U1 SnRNP knockdown in HeLa cells, and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. In addition to inhibiting splicing, U1 SnRNP knockdown caused premature cleavage and polyadenylation in numerous pre-mRNAs at cryptic polyadenylation signals, frequently in introns near (<5 kilobases) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA antisense morpholino oligonucleotide or the U2-SnRNP-inactivating drug spliceostatin A unless U1 antisense morpholino oligonucleotide was also included. We further show that U1 snRNA-pre-mRNA base pairing was required to suppress premature cleavage and polyadenylation from nearby cryptic polyadenylation signals located in introns. These findings reveal a critical splicing-independent function for U1 SnRNP in protecting the transcriptome, which we propose explains its overabundance.
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U1 SnRNP protects pre-mRNAs from premature cleavage and polyadenylation
Nature, 2010Co-Authors: Daisuke Kaida, Michael G Berg, Ihab Younis, Mumtaz Kasim, Larry N Singh, Lili Wan, Gideon DreyfussAbstract:In eukaryotes, U1 small nuclear ribonucleoprotein (SnRNP) forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6 SnRNPs; however, its abundance in human far exceeds that of the other SnRNPs. Here we used antisense morpholino oligonucleotide to U1 snRNA to achieve functional U1 SnRNP knockdown in HeLa cells, and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. In addition to inhibiting splicing, U1 SnRNP knockdown caused premature cleavage and polyadenylation in numerous pre-mRNAs at cryptic polyadenylation signals, frequently in introns near (
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gemin5 delivers snrna precursors to the smn complex for SnRNP biogenesis
Molecular Cell, 2010Co-Authors: Jeongsik Yong, Mumtaz Kasim, Lili Wan, Jennifer L Bachorik, Gideon DreyfussAbstract:The SMN complex assembles Sm cores on snRNAs, a key step in the biogenesis of SnRNPs, the spliceosome's major components. Here, using SMN complex inhibitors identified by high-throughput screening and a ribo-proteomic strategy on formaldehyde crosslinked RNPs, we dissected this pathway in cells. We show that protein synthesis inhibition impairs the SMN complex, revealing discrete SMN and Gemin subunits and accumulating an snRNA precursor (pre-snRNA)-Gemin5 intermediate. By high-throughput sequencing of this transient intermediate's RNAs, we discovered the previously undetectable precursors of all the snRNAs and identified their Gemin5-binding sites. We demonstrate that pre-snRNA 3' sequences function to enhance SnRNP biogenesis. The SMN complex is also inhibited by oxidation, and we show that it stalls an inventory-complete SMN complex containing pre-snRNAs. We propose a stepwise pathway of SMN complex formation and SnRNP biogenesis, highlighting Gemin5's function in delivering pre-snRNAs as substrates for Sm core assembly and processing.
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snrnas contain specific smn binding domains that are essential for SnRNP assembly
Molecular and Cellular Biology, 2004Co-Authors: Jeongsik Yong, Livio Pellizzoni, Tracey J Golembe, Daniel J Battle, Gideon DreyfussAbstract:To serve in its function as an assembly machine for spliceosomal small nuclear ribonucleoprotein particles (SnRNPs), the survival of motor neurons (SMN) protein complex binds directly to the Sm proteins and the U snRNAs. A specific domain unique to U1 snRNA, stem-loop 1 (SL1), is required for SMN complex binding and U1 SnRNP Sm core assembly. Here, we show that each of the major spliceosomal U snRNAs (U2, U4, and U5), as well as the minor splicing pathway U11 snRNA, contains a domain to which the SMN complex binds directly and with remarkable affinity (low nanomolar concentration). The SMN-binding domains of the U snRNAs do not have any significant nucleotide sequence similarity yet they compete for binding to the SMN complex in a manner that suggests the presence of at least two binding sites. Furthermore, the SMN complex-binding domain and the Sm site are both necessary and sufficient for Sm core assembly and their relative positions are critical for SnRNP assembly. These findings indicate that the SMN complex stringently scrutinizes RNAs for specific structural features that are not obvious from the sequence of the RNAs but are required for their identification as bona fide snRNAs. It is likely that this surveillance capacity of the SMN complex ensures assembly of Sm cores on the correct RNAs only and prevents illicit, potentially deleterious, assembly of Sm cores on random RNAs.
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The SMN Complex Is Associated with SnRNPs throughout Their Cytoplasmic Assembly Pathway
Molecular and cellular biology, 2002Co-Authors: Severine Massenet, Iain W. Mattaj, Livio Pellizzoni, Sergey Paushkin, Gideon DreyfussAbstract:The common neurodegenerative disease spinal muscular atrophy is caused by reduced levels of the survival of motor neurons (SMN) protein. SMN associates with several proteins (Gemin2 to Gemin6) to form a large complex which is found both in the cytoplasm and in the nucleus. The SMN complex functions in the assembly and metabolism of several RNPs, including spliceosomal SnRNPs. The SnRNP core assembly takes place in the cytoplasm from Sm proteins and newly exported snRNAs. Here, we identify three distinct cytoplasmic SMN complexes, each representing a defined intermediate in the SnRNP biogenesis pathway. We show that the SMN complex associates with newly exported snRNAs containing the nonphosphorylated form of the snRNA export factor PHAX. The second SMN complex identified contains assembled Sm cores and m3G-capped snRNAs. Finally, the SMN complex is associated with a preimport complex containing m3G-capped SnRNP cores bound to the SnRNP nuclear import mediator snurportin1. Thus, the SMN complex is associated with SnRNPs during the entire process of their biogenesis in the cytoplasm and may have multiple functions throughout this process.
Karla M. Neugebauer - One of the best experts on this subject based on the ideXlab platform.
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the nuclear cap binding complex interacts with the u4 u6 u5 tri SnRNP and promotes spliceosome assembly in mammalian cells
RNA, 2013Co-Authors: Marta Pabis, Noa Neufeld, Michaela C Steiner, Teodora Bojic, Yaron Shavtal, Karla M. NeugebauerAbstract:The nuclear cap-binding complex (CBC) binds to the 7-methyl guanosine cap present on every RNA polymerase II transcript. CBC has been implicated in many aspects of RNA biogenesis; in addition to roles in miRNA biogenesis, nonsense-mediated decay, 3′-end formation, and snRNA export from the nucleus, CBC promotes pre-mRNA splicing. An unresolved question is how CBC participates in splicing. To investigate CBC’s role in splicing, we used mass spectrometry to identify proteins that copurify with mammalian CBC. Numerous components of spliceosomal SnRNPs were specifically detected. Among these, three U4/U6·U5 SnRNP proteins (hBrr2, hPrp4, and hPrp31) copurified with CBC in an RNA-independent fashion, suggesting that a significant fraction of CBC forms a complex with the U4/U6·U5 SnRNP and that the activity of CBC might be associated with SnRNP recruitment to pre-mRNA. To test this possibility, CBC was depleted from HeLa cells by RNAi. Chromatin immunoprecipitation and live-cell imaging assays revealed decreased cotranscriptional accumulation of U4/U6·U5 SnRNPs on active transcription units, consistent with a requirement for CBC in cotranscriptional spliceosome assembly. Surprisingly, recruitment of U1 and U2 SnRNPs was also affected, indicating that RNA-mediated interactions between CBC and SnRNPs contribute to splicing. On the other hand, CBC depletion did not impair SnRNP biogenesis, ruling out the possibility that decreased SnRNP recruitment was due to changes in nuclear SnRNP concentration. Taken together, the data support a model whereby CBC promotes pre-mRNA splicing through a network of interactions with and among spliceosomal SnRNPs during cotranscriptional spliceosome assembly.
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The differential interaction of SnRNPs with pre-mRNA reveals splicing kinetics in living cells
The Journal of cell biology, 2010Co-Authors: Martina Huranova, Karla M. Neugebauer, Ivan Ivani, Aleš Benda, Ina Poser, Yehuda Brody, Martin Hof, Yaron Shav-tal, David StaněkAbstract:Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (SnRNPs) and additional proteins. Using live cell imaging of GFP-tagged SnRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of SnRNP dynamics in the cell nucleus enabled us to determine SnRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual SnRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 SnRNPs, associated with pre-mRNA for 15–30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 SnRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual SnRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.
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Coilin-dependent SnRNP assembly is essential for zebrafish embryogenesis
Nature Structural & Molecular Biology, 2010Co-Authors: Magdalena Strzelecka, Reinhard Luhrmann, Gert Weber, Simon Trowitzsch, Andrew C Oates, Karla M. NeugebauerAbstract:The function of many non-membrane bound aggregates (Cajal bodies, P-bodies, stress granules, etc.) is poorly understood. Now studies in zebrafish embryos show that coilin promotes spliceosomal small nuclear ribonucleoproteins (SnRNPs) assembly, most likely by concentrating SnRNP components in Cajal bodies and to overcome a rate-limiting step(s) in assembly. Spliceosomal small nuclear ribonucleoproteins (SnRNPs), comprised of small nuclear RNAs (snRNAs) in complex with SnRNP-specific proteins, are essential for pre-mRNA splicing. Coilin is not a SnRNP protein but concentrates SnRNPs and their assembly intermediates in Cajal bodies (CBs). Here we show that depletion of coilin in zebrafish embryos leads to CB dispersal, deficits in SnRNP biogenesis and expression of spliced mRNA, as well as reduced cell proliferation followed by developmental arrest. Notably, injection of purified mature human SnRNPs restored mRNA expression and viability. snRNAs were necessary but not sufficient for rescue, showing that only assembled SnRNPs can bypass the requirement for coilin. Thus, coilin's essential function in embryos is to promote macromolecular assembly of SnRNPs, likely by concentrating SnRNP components in CBs to overcome rate-limiting assembly steps.
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Spliceosomal SnRNPs Repeatedly Cycle through Cajal Bodies
Molecular biology of the cell, 2008Co-Authors: David Staněk, Jarmila Přidalová, Ivan Novotný, Martina Huranova, Michaela Blažíková, Xin Wen, Aparna K. Sapra, Karla M. NeugebauerAbstract:The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (SnRNPs). Here, we tested whether CBs also contain mature SnRNPs and whether CB integrity depends on the ongoing SnRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor SnRNP behavior in living cells over time; mature SnRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported SnRNPs. To test whether CB integrity depends on the SnRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 SnRNPs in CBs, suggesting that reassembly of the U4/U6·U5 tri-SnRNP was delayed. Accordingly, a relative decrease in U5 SnRNPs compared with U4/U6 SnRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-SnRNP occurring in CBs.
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The Cajal body: a meeting place for spliceosomal SnRNPs in the nuclear maze
Chromosoma, 2006Co-Authors: David Staněk, Karla M. NeugebauerAbstract:Spliceosomal small nuclear ribonucleoprotein particles (SnRNPs) are essential pre-mRNA splicing factors that consist of small nuclear RNAs (snRNAs) complexed with specific sets of proteins. A considerable body of evidence has established that SnRNP assembly is accomplished after snRNA synthesis in the nucleus through a series of steps involving cytoplasmic and nuclear phases. Recent work indicates that SnRNPs transiently localize to the Cajal body (CB), a nonmembrane-bound inclusion present in the nuclei of most cells, for the final steps in SnRNP maturation, including snRNA base modification, U4/U6 snRNA annealing, and snRNA-protein assembly. Here, we review these findings that suggest a crucial role for CBs in the spliceosome cycle in which production of new SnRNPs—and perhaps regenerated SnRNPs after splicing—is promoted by the concentration of substrates in this previously mysterious subnuclear organelle. These insights allow us to speculate on the role of nuclear bodies in regulating the dynamics of RNP assembly to maintain a functional pool of factors available for key steps in gene expression.
Albrecht Bindereif - One of the best experts on this subject based on the ideXlab platform.
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snRNA-specific role of SMN in trypanosome SnRNP biogenesis in vivo.
RNA biology, 2011Co-Authors: Nicolas Jaé, Christian Preußer, Timothy Krüger, Itai Dov Tkacz, Markus Engstler, Shulamit Michaeli, Albrecht BindereifAbstract:Pre-mRNA splicing in trypanosomes requires the SMN-mediated assembly of small nuclear ribonucleoproteins (SnRNPs). In contrast to higher eukaryotes, the cellular localization of SnRNP biogenesis and the involvement of nuclear-cytoplasmic trafficking in trypanosomes are controversial. By using RNAi knockdown of SMN in T.brucei to investigate its functional role in SnRNP assembly, we found dramatic changes in the steady-state levels of snRNAs and SnRNPs: The SL RNA accumulates, whereas U1, U4, and U5 snRNA levels decrease, and Sm core assembly in particular of the SL RNA is strongly reduced. In addition, SMN depletion blocks U4/U6 di-SnRNP formation; the variant Sm core of the U2 SnRNP, however, still forms efficiently after SMN knockdown. Concerning the longstanding question, whether nuclear-cytoplasmic trafficking is involved in trypanosomal SnRNP biogenesis, fluorescence in situ hybridization (FISH) and immunofluorescence assays revealed that the SL RNA genes and transcripts colocalize with SMN. Remarkab...
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Recycling of the U12-type spliceosome requires p110, a component of the U6atac SnRNP.
Molecular and cellular biology, 2004Co-Authors: Andrey Damianov, Silke Schreiner, Albrecht BindereifAbstract:U12-dependent introns are spliced by the so-called minor spliceosome, requiring the U11, U12, and U4atac/U6atac SnRNPs in addition to the U5 SnRNP. We have recently identified U6-p110 (SART3) as a novel human recycling factor that is related to the yeast splicing factor Prp24. U6-p110 transiently associates with the U6 and U4/U6 SnRNPs during the spliceosome cycle, regenerating functional U4/U6 SnRNPs from singular U4 and U6 SnRNPs. Here we investigated the involvement of U6-p110 in recycling of the U4atac/U6atac SnRNP. In contrast to the major U6 and U4/U6 SnRNPs, p110 is primarily associated with the U6atac SnRNP but is almost undetectable in the U4atac/U6atac SnRNP. Since p110 does not occur in U5 snRNA-containing complexes, it appears to be transiently associated with U6atac during the cycle of the minor spliceosome. The p110 binding site was mapped to U6 nucleotides 38 to 57 and U6atac nucleotides 10 to 30, which are highly conserved between these two functionally related snRNAs. With a U12-dependent in vitro splicing system, we demonstrate that p110 is required for recycling of the U4atac/U6atac SnRNP.
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p110, a novel human U6 SnRNP protein and U4/U6 SnRNP recycling factor
The EMBO journal, 2002Co-Authors: Mathias Bell, Silke Schreiner, Andrey Damianov, Ram Reddy, Albrecht BindereifAbstract:During each spliceosome cycle, the U6 snRNA undergoes extensive structural rearrangements, alternating between singular, U4–U6 and U6–U2 base-paired forms. In Saccharomyces cerevisiae, Prp24 functions as an SnRNP recycling factor, reannealing U4 and U6 snRNAs. By database searching, we have identified a Prp24-related human protein previously described as p110nrb or SART3. p110 contains in its C-terminal region two RNA recognition motifs (RRMs). The N-terminal two-thirds of p110, for which there is no counterpart in the S.cerevisiae Prp24, carries seven tetratricopeptide repeat (TPR) domains. p110 homologs sharing the same domain structure also exist in several other eukaryotes. p110 is associated with the mammalian U6 and U4/U6 SnRNPs, but not with U4/U5/U6 tri-SnRNPs nor with spliceosomes. Recom binant p110 binds in vitro specifically to human U6 snRNA, requiring an internal U6 region. Using an in vitro recycling assay, we demonstrate that p110 functions in the reassembly of the U4/U6 SnRNP. In summary, p110 represents the human ortholog of Prp24, and associates only transiently with U6 and U4/U6 SnRNPs during the recycling phase of the spliceosome cycle.
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p110 a novel human u6 SnRNP protein and u4 u6 SnRNP recycling factor
The EMBO Journal, 2002Co-Authors: Mathias Bell, Silke Schreiner, Andrey Damianov, Ram Reddy, Albrecht BindereifAbstract:During each spliceosome cycle, the U6 snRNA undergoes extensive structural rearrangements, alternating between singular, U4–U6 and U6–U2 base-paired forms. In Saccharomyces cerevisiae, Prp24 functions as an SnRNP recycling factor, reannealing U4 and U6 snRNAs. By database searching, we have identified a Prp24-related human protein previously described as p110nrb or SART3. p110 contains in its C-terminal region two RNA recognition motifs (RRMs). The N-terminal two-thirds of p110, for which there is no counterpart in the S.cerevisiae Prp24, carries seven tetratricopeptide repeat (TPR) domains. p110 homologs sharing the same domain structure also exist in several other eukaryotes. p110 is associated with the mammalian U6 and U4/U6 SnRNPs, but not with U4/U5/U6 tri-SnRNPs nor with spliceosomes. Recom binant p110 binds in vitro specifically to human U6 snRNA, requiring an internal U6 region. Using an in vitro recycling assay, we demonstrate that p110 functions in the reassembly of the U4/U6 SnRNP. In summary, p110 represents the human ortholog of Prp24, and associates only transiently with U6 and U4/U6 SnRNPs during the recycling phase of the spliceosome cycle.
Michael Rosbash - One of the best experts on this subject based on the ideXlab platform.
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cotranscriptional spliceosome assembly dynamics and the role of u1 snrna 5 ss base pairing in yeast
Molecular Cell, 2005Co-Authors: Scott A Lacadie, Michael RosbashAbstract:To investigate the mechanism of spliceosome assembly in vivo, we performed chromatin immunoprecipitation (ChIP) analysis of U1, U2, and U5 small nuclear ribonucleoprotein particles (SnRNPs) to intron-containing yeast (S. cerevisiae) genes. The SnRNPs display patterns that indicate a cotranscriptional assembly model: U1 first, then U2, and the U4/U6*U5 tri-SnRNP followed by U1 destabilization. cis-splicing mutations also support a role of U2 and/or the tri-SnRNP in U1 destabilization. Moreover, they indicate that splicing efficiency has a major impact on cotranscriptional SnRNP recruitment and suggest that cotranscriptional recruitment of U2 or the tri-SnRNP is required to commit the pre-mRNA to splicing. Branchpoint (BP) mutations had a major effect on the U1 pattern, whereas 5' splice site (5'ss) mutations had a stronger effect on the U2 pattern. A 5'ss-U1 snRNA complementation experiment suggests that pairing between U1 and the 5'ss occurs after U1 recruitment and contributes to a specific U1:substrate conformation required for efficient U2 and tri-SnRNP recruitment.
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the u1 SnRNP protein u1c recognizes the 5 splice site in the absence of base pairing
Nature, 2002Co-Authors: Michael RosbashAbstract:Splicing of precursor messenger RNA takes place in the spliceosome, a large RNA/protein macromolecular machine. Spliceosome assembly occurs in an ordered pathway in vitro and is conserved between yeast and mammalian systems. The earliest step is commitment complex formation in yeast or E complex formation in mammals, which engages the pre-mRNA in the splicing pathway and involves interactions between U1 small nuclear ribonucleoprotein (SnRNP) and the pre-mRNA 5' splice site. Complex formation depends on highly conserved base pairing between the 5' splice site and the 5' end of U1 snRNA, both in vivo and in vitro. U1 SnRNP proteins also contribute to U1 SnRNP activity. Here we show that U1 SnRNP lacking the 5' end of its snRNA retains 5'-splice-site sequence specificity. We also show that recombinant yeast U1C protein, a U1 SnRNP protein, selects a 5'-splice-site-like sequence in which the first four nucleotides, GUAU, are identical to the first four nucleotides of the yeast 5'-splice-site consensus sequence. We propose that a U1C 5'-splice-site interaction precedes pre-mRNA/U1 snRNA base pairing and is the earliest step in the splicing pathway.
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a comprehensive biochemical and genetic analysis of the yeast u1 SnRNP reveals five novel proteins
RNA, 1998Co-Authors: Alexander Gottschalk, Hildur V. Colot, Michael Rosbash, Jie Tang, Oscar Puig, Josefa Salgado, Gitte Neubauer, Matthias Mann, Bertrand Seraphin, Reinhard LuhrmannAbstract:The U1 SnRNP is essential for recognition of the pre-mRNA 5'-splice site and the subsequent assembly of the spliceosome. Yeast U1 SnRNP is considerably more complex than its metazoan counterpart, which suggests possible differences between yeast and metazoa in early splicing events. We have comprehensively analyzed the composition of yeast U1 SnRNPs using a combination of biochemical, mass spectrometric, and genetic methods. We demonstrate the specific association of four novel U1 SnRNP proteins, Snu71p, Snu65p, Nam8p, and Snu56p, that have no known metazoan homologues. A fifth protein, Npl3p, is an abundant cellular component that reproducibly co-purifies with the U1 SnRNP, but its association is salt-sensitive. Therefore, we are unable to establish conclusively whether it binds specifically to the U1 SnRNP. Interestingly, Nam8p and Npl3p were previously assigned functions in (pre-m)RNA-metabolism; however, so far, no association with U1 SnRNP has been demonstrated or proposed. We also show that the yeast SmB protein is a U1 SnRNP component. Yeast U1 SnRNP therefore contains 16 different proteins, including seven SnRNP core proteins, three homologues of the metazoan U1 SnRNP-specific proteins, and six yeast-specific U1 SnRNP proteins. We have simultaneously continued the characterization of additional mutants isolated in a synthetic lethal (MUD) screen for genes that functionally cooperate with U1 snRNA. Consistent with the biochemical results, mud10, mud15, and mud16 are alleles of SNU56, NAM8, and SNU65, respectively. mud10 and mud15 affect the in vivo splicing efficiency of noncanonical introns. Moreover, mud10p strongly affects the in vitro formation of splicing complexes, and extracts from the mud15 strain contain a U1 SnRNP that migrates aberrantly on native gels. Finally, we show that Nam8p/Mud15p contributes to the stability of U1 SnRNP.
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Requirements for U2 SnRNP addition to yeast pre-mRNA
Nucleic acids research, 1992Co-Authors: Xiaoling C. Liao, Hildur V. Colot, Yue Wang, Michael RosbashAbstract:The in vitro spliceosome assembly pathway is conserved between yeast and mammals as U1 and U2 SnRNPs associate with the pre-mRNA prior to U5 and U4/U6 SnRNPs. In yeast, U1 SnRNP-pre-mRNA complexes are the first splicing complexes visualized on native gels, and association with U1 SnRNP apparently commits pre-mRNA to the spliceosome assembly pathway. The current study addresses U2 SnRNP addition to commitment complexes. We show that commitment complex formation is relatively slow and does not require ATP, whereas U2 SnRNP adds to the U1 SnRNP complexes in a reaction that is relatively fast and requires ATP or hydrolyzable ATP analogs. In vitro spliceosome assembly was assayed in extracts derived from strains containing several U1 sRNA mutations. The results were consistent with a critical role for U1 SnRNP in early complex formation. A mutation that disrupts the base-pairing between the 5' end of U1 snRNA and the 5' splice site allows some U2 SnRNP addition to bypass the ATP requirement, suggesting that ATP may be used to destabilize certain U1 SnRNP:pre-mRNA interactions to allow subsequent U2 SnRNP addition.
