The Experts below are selected from a list of 294 Experts worldwide ranked by ideXlab platform
Yigong Shi - One of the best experts on this subject based on the ideXlab platform.
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structures of the catalytically activated yeast spliceosome reveal the mechanism of Branching
Cell, 2019Co-Authors: Ruixue Wan, Jianlin Lei, Chuangye Yan, Rui Bai, Yigong ShiAbstract:Summary Pre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B∗) is pivotal for understanding the Branching Reaction. In this study, we assembled the B∗ complexes on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B∗ complexes at overall resolutions of 2.9–3.8 A. The duplex between U2 small nuclear RNA (snRNA) and the branch point sequence (BPS) is discretely away from the 5′-splice site (5′SS) in the three B∗ complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5′SS, with the BPS nucleophile positioned 4 A away from the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in Branching. These structures on different pre-mRNAs reveal substrate-specific conformations of the spliceosome in a major functional state.
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structures of the catalytically activated yeast spliceosome reveal the mechanism of Branching
bioRxiv, 2018Co-Authors: Ruixue Wan, Jianlin Lei, Chuangye Yan, Rui Bai, Yigong ShiAbstract:Summary Pre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B*) is pivotal for mechanistic understanding of catalysis of the Branching Reaction by the spliceosome. In this study, we assembled the B* complex on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B complexes at overall resolutions of 2.9-3.8 A. The duplex between U2 snRNA and the branch point sequence (BPS) is located 13-20 A away from the 5’-splice site (5’SS) in the B* complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5’SS, ready for Branching. In the absence of Cwc25, the nucleophile from BPS is positioned about 4 A away from, and remains to be activated by, the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in Branching. These four structures constitute compelling evidence for substrate-specific conformations of the spliceosome in a major functional state.
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structure of a human catalytic step i spliceosome
Science, 2018Co-Authors: Xiechao Zhan, Chuangye Yan, Xiaofeng Zhang, Jianlin Lei, Yigong ShiAbstract:Splicing by the spliceosome involves Branching and exon ligation. The Branching Reaction leads to the formation of the catalytic step I spliceosome (C complex). Here we report the cryo-electron microscopy structure of the human C complex at an average resolution of 4.1 angstroms. Compared with the Saccharomyces cerevisiae C complex, the human complex contains 11 additional proteins. The step I splicing factors CCDC49 and CCDC94 (Cwc25 and Yju2 in S. cerevisiae, respectively) closely interact with the DEAH-family adenosine triphosphatase/helicase Prp16 and bridge the gap between Prp16 and the active-site RNA elements. These features, together with structural comparison of the human C and C* complexes, provide mechanistic insights into ribonucleoprotein remodeling and allow the proposition of a working mechanism for the C-to-C* transition.
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structure of the human activated spliceosome in three conformational states
Cell Research, 2018Co-Authors: Xiaofeng Zhang, Chuangye Yan, Xiechao Zhan, Jianlin Lei, Yigong ShiAbstract:During each cycle of pre-mRNA splicing, the pre-catalytic spliceosome (B complex) is converted into the activated spliceosome (Bact complex), which has a well-formed active site but cannot proceed to the Branching Reaction. Here, we present the cryo-EM structure of the human Bact complex in three distinct conformational states. The EM map allows atomic modeling of nearly all protein components of the U2 small nuclear ribonucleoprotein (snRNP), including three of the SF3a complex and seven of the SF3b complex. The structure of the human Bact complex contains 52 proteins, U2, U5, and U6 small nuclear RNA (snRNA), and a pre-mRNA. Three distinct conformations have been captured, representing the early, mature, and late states of the human Bact complex. These complexes differ in the orientation of the Switch loop of Prp8, the splicing factors RNF113A and NY-CO-10, and most components of the NineTeen complex (NTC) and the NTC-related complex. Analysis of these three complexes and comparison with the B and C complexes reveal an ordered flux of components in the B-to-Bact and the Bact-to-B* transitions, which ultimately prime the active site for the Branching Reaction.
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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.
Klaas J Hellingwerf - One of the best experts on this subject based on the ideXlab platform.
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influence of the crystalline state on photoinduced dynamics of photoactive yellow protein studied by ultraviolet visible transient absorption spectroscopy
Biophysical Journal, 2006Co-Authors: Sergey Yeremenko, Ivo H. M. Van Stokkum, Keith Moffat, Klaas J HellingwerfAbstract:Time-resolved ultraviolet-visible spectroscopy was used to characterize the photocycle transitions in single crystals of wild-type and the E-46Q mutant of photoactive yellow protein (PYP) with microsecond time resolution. The results were compared with the results of similar measurements on aqueous solutions of these two variants of PYP, with and without the components present in the mother liquor of crystals. The experimental data were analyzed with global and target analysis. Distinct differences in the Reaction path of a PYP molecule are observed between these conditions when it progresses through its photocycle. In the crystalline state i), much faster relaxation of the late blue-shifted photocycle intermediate back to the ground state is observed; ii), this intermediate in crystalline PYP absorbs at 380 nm, rather than at 350-360 nm in solution; and iii), for various intermediates of this photocycle the forward Reaction through the photocycle directly competes with a Branching Reaction that leads directly to the ground state. Significantly, with these altered characteristics, the spectroscopic data on PYP are fully consistent with the structural data obtained for this photoreceptor protein with time-resolved x-ray diffraction analysis, particularly for wild-type PYP.
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Kinetics of and intermediates in a photocycle Branching Reaction of the photoactive yellow protein from Ectothiorhodospira halophila.
FEBS letters, 1999Co-Authors: Johnny Hendriks, Ivo H. M. Van Stokkum, Wim Crielaard, Klaas J HellingwerfAbstract:We have studied the kinetics of the blue light-induced Branching Reaction in the photocycle of photoactive yellow protein (PYP) from Ectothiorhodospira halophila, by nanosecond time-resolved UV/Vis spectroscopy. As compared to the parallel dark recovery Reaction of the presumed blue-shifted signaling state pB, the light-induced Branching Reaction showed a 1000-fold higher rate. In addition, a new intermediate was detected in this Branching pathway, which, compared to pB, showed a larger extinction coefficient and a blue-shifted absorption maximum. This substantiates the conclusion that isomerization of the chromophore is the rate-controlling step in the thermal photocycle Reactions of PYP and implies that absorption of a blue photon leads to cis-->trans isomerization of the 4-hydroxy-cinnamyl chromophore of PYP in its pB state.
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a light dependent Branching Reaction in the photocycle of the yellow protein from ectothiorhodospira halophila
Biochimica et Biophysica Acta, 1993Co-Authors: A Miller, H Leigeber, Wouter D Hoff, Klaas J HellingwerfAbstract:Abstract The photoactive yellow protein (PYP) from Ectothiorhodospira halophila was studied with transient absorption spectroscopy in order to investigate the presence of a light-induced Branching Reaction in its photocycle. Such a Branching Reaction is indeed initiated by blue light, from a long-lived blue-shifted intermediate back to the ground state. Using a two-state model for the photocycle of the yellow protein, we have determined the quantum yield of the Branching Reaction with two different methods. The value determined for this quantum yield (0.5 ± 0.2) is of the same order of magnitude as the quantum yield of the excitation of the ground state. This latter observation may suggest that this photochemical Branching Reaction has a biological function.
Charles C. Query - One of the best experts on this subject based on the ideXlab platform.
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Catalytic spliceosome captured
Nature, 2016Co-Authors: Brian Kosmyna, Charles C. QueryAbstract:The excision of introns from RNA is not a concerted process, but is rather an ordered one involving two transesterification Reactions by the spliceosome. In the first step, the 5′-splice site is cleaved and the intron end is joined to make a lariat structure. Kiyoshi Nagai and colleagues have captured the Saccharomyces cerevisiae spliceosome stalled immediately after this first transesterification (Branching) Reaction by cryo-electron microscopy single-particle reconstruction at an overall resolution of 3.8 Å. The configuration of the RNA within the complex suggests that remodelling occurs before the second step, exon ligation. Spliceosome complexes remove non-coding sequences from RNA transcripts in two steps. A structure of a spliceosome after the first step reveals active-site interactions and evolutionary constraints on these non-coding regions. See Article p.197
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Structural biology: Catalytic spliceosome captured
Nature, 2016Co-Authors: Brian Kosmyna, Charles C. QueryAbstract:Spliceosome complexes remove non-coding sequences from RNA transcripts in two steps. A structure of a spliceosome after the first step reveals active-site interactions and evolutionary constraints on these non-coding regions. See Article p.197 The excision of introns from RNA is not a concerted process, but is rather an ordered one involving two transesterification Reactions by the spliceosome. In the first step, the 5′-splice site is cleaved and the intron end is joined to make a lariat structure. Kiyoshi Nagai and colleagues have captured the Saccharomyces cerevisiae spliceosome stalled immediately after this first transesterification (Branching) Reaction by cryo-electron microscopy single-particle reconstruction at an overall resolution of 3.8 A. The configuration of the RNA within the complex suggests that remodelling occurs before the second step, exon ligation.
Jianlin Lei - One of the best experts on this subject based on the ideXlab platform.
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structures of the catalytically activated yeast spliceosome reveal the mechanism of Branching
Cell, 2019Co-Authors: Ruixue Wan, Jianlin Lei, Chuangye Yan, Rui Bai, Yigong ShiAbstract:Summary Pre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B∗) is pivotal for understanding the Branching Reaction. In this study, we assembled the B∗ complexes on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B∗ complexes at overall resolutions of 2.9–3.8 A. The duplex between U2 small nuclear RNA (snRNA) and the branch point sequence (BPS) is discretely away from the 5′-splice site (5′SS) in the three B∗ complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5′SS, with the BPS nucleophile positioned 4 A away from the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in Branching. These structures on different pre-mRNAs reveal substrate-specific conformations of the spliceosome in a major functional state.
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structures of the catalytically activated yeast spliceosome reveal the mechanism of Branching
bioRxiv, 2018Co-Authors: Ruixue Wan, Jianlin Lei, Chuangye Yan, Rui Bai, Yigong ShiAbstract:Summary Pre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B*) is pivotal for mechanistic understanding of catalysis of the Branching Reaction by the spliceosome. In this study, we assembled the B* complex on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B complexes at overall resolutions of 2.9-3.8 A. The duplex between U2 snRNA and the branch point sequence (BPS) is located 13-20 A away from the 5’-splice site (5’SS) in the B* complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5’SS, ready for Branching. In the absence of Cwc25, the nucleophile from BPS is positioned about 4 A away from, and remains to be activated by, the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in Branching. These four structures constitute compelling evidence for substrate-specific conformations of the spliceosome in a major functional state.
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structure of a human catalytic step i spliceosome
Science, 2018Co-Authors: Xiechao Zhan, Chuangye Yan, Xiaofeng Zhang, Jianlin Lei, Yigong ShiAbstract:Splicing by the spliceosome involves Branching and exon ligation. The Branching Reaction leads to the formation of the catalytic step I spliceosome (C complex). Here we report the cryo-electron microscopy structure of the human C complex at an average resolution of 4.1 angstroms. Compared with the Saccharomyces cerevisiae C complex, the human complex contains 11 additional proteins. The step I splicing factors CCDC49 and CCDC94 (Cwc25 and Yju2 in S. cerevisiae, respectively) closely interact with the DEAH-family adenosine triphosphatase/helicase Prp16 and bridge the gap between Prp16 and the active-site RNA elements. These features, together with structural comparison of the human C and C* complexes, provide mechanistic insights into ribonucleoprotein remodeling and allow the proposition of a working mechanism for the C-to-C* transition.
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structure of the human activated spliceosome in three conformational states
Cell Research, 2018Co-Authors: Xiaofeng Zhang, Chuangye Yan, Xiechao Zhan, Jianlin Lei, Yigong ShiAbstract:During each cycle of pre-mRNA splicing, the pre-catalytic spliceosome (B complex) is converted into the activated spliceosome (Bact complex), which has a well-formed active site but cannot proceed to the Branching Reaction. Here, we present the cryo-EM structure of the human Bact complex in three distinct conformational states. The EM map allows atomic modeling of nearly all protein components of the U2 small nuclear ribonucleoprotein (snRNP), including three of the SF3a complex and seven of the SF3b complex. The structure of the human Bact complex contains 52 proteins, U2, U5, and U6 small nuclear RNA (snRNA), and a pre-mRNA. Three distinct conformations have been captured, representing the early, mature, and late states of the human Bact complex. These complexes differ in the orientation of the Switch loop of Prp8, the splicing factors RNF113A and NY-CO-10, and most components of the NineTeen complex (NTC) and the NTC-related complex. Analysis of these three complexes and comparison with the B and C complexes reveal an ordered flux of components in the B-to-Bact and the Bact-to-B* transitions, which ultimately prime the active site for the Branching Reaction.
Chuangye Yan - One of the best experts on this subject based on the ideXlab platform.
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structures of the catalytically activated yeast spliceosome reveal the mechanism of Branching
Cell, 2019Co-Authors: Ruixue Wan, Jianlin Lei, Chuangye Yan, Rui Bai, Yigong ShiAbstract:Summary Pre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B∗) is pivotal for understanding the Branching Reaction. In this study, we assembled the B∗ complexes on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B∗ complexes at overall resolutions of 2.9–3.8 A. The duplex between U2 small nuclear RNA (snRNA) and the branch point sequence (BPS) is discretely away from the 5′-splice site (5′SS) in the three B∗ complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5′SS, with the BPS nucleophile positioned 4 A away from the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in Branching. These structures on different pre-mRNAs reveal substrate-specific conformations of the spliceosome in a major functional state.
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structures of the catalytically activated yeast spliceosome reveal the mechanism of Branching
bioRxiv, 2018Co-Authors: Ruixue Wan, Jianlin Lei, Chuangye Yan, Rui Bai, Yigong ShiAbstract:Summary Pre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B*) is pivotal for mechanistic understanding of catalysis of the Branching Reaction by the spliceosome. In this study, we assembled the B* complex on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B complexes at overall resolutions of 2.9-3.8 A. The duplex between U2 snRNA and the branch point sequence (BPS) is located 13-20 A away from the 5’-splice site (5’SS) in the B* complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5’SS, ready for Branching. In the absence of Cwc25, the nucleophile from BPS is positioned about 4 A away from, and remains to be activated by, the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in Branching. These four structures constitute compelling evidence for substrate-specific conformations of the spliceosome in a major functional state.
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structure of a human catalytic step i spliceosome
Science, 2018Co-Authors: Xiechao Zhan, Chuangye Yan, Xiaofeng Zhang, Jianlin Lei, Yigong ShiAbstract:Splicing by the spliceosome involves Branching and exon ligation. The Branching Reaction leads to the formation of the catalytic step I spliceosome (C complex). Here we report the cryo-electron microscopy structure of the human C complex at an average resolution of 4.1 angstroms. Compared with the Saccharomyces cerevisiae C complex, the human complex contains 11 additional proteins. The step I splicing factors CCDC49 and CCDC94 (Cwc25 and Yju2 in S. cerevisiae, respectively) closely interact with the DEAH-family adenosine triphosphatase/helicase Prp16 and bridge the gap between Prp16 and the active-site RNA elements. These features, together with structural comparison of the human C and C* complexes, provide mechanistic insights into ribonucleoprotein remodeling and allow the proposition of a working mechanism for the C-to-C* transition.
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structure of the human activated spliceosome in three conformational states
Cell Research, 2018Co-Authors: Xiaofeng Zhang, Chuangye Yan, Xiechao Zhan, Jianlin Lei, Yigong ShiAbstract:During each cycle of pre-mRNA splicing, the pre-catalytic spliceosome (B complex) is converted into the activated spliceosome (Bact complex), which has a well-formed active site but cannot proceed to the Branching Reaction. Here, we present the cryo-EM structure of the human Bact complex in three distinct conformational states. The EM map allows atomic modeling of nearly all protein components of the U2 small nuclear ribonucleoprotein (snRNP), including three of the SF3a complex and seven of the SF3b complex. The structure of the human Bact complex contains 52 proteins, U2, U5, and U6 small nuclear RNA (snRNA), and a pre-mRNA. Three distinct conformations have been captured, representing the early, mature, and late states of the human Bact complex. These complexes differ in the orientation of the Switch loop of Prp8, the splicing factors RNF113A and NY-CO-10, and most components of the NineTeen complex (NTC) and the NTC-related complex. Analysis of these three complexes and comparison with the B and C complexes reveal an ordered flux of components in the B-to-Bact and the Bact-to-B* transitions, which ultimately prime the active site for the Branching Reaction.