CryoEM structure of the spliceosome immediately after branching

Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat–intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5′-splice site is cleaved but remains close to the catalytic Mg2+ site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5′-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2′OH. The 5′-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson–Crick interactions between the branch helix and 5′-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation.

• Publication year

  2016

• Venue

  Nature

• Publication date

  2016-07-26

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1038/nature19316PMID 27459055PMCID 5156311
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• The 3.8 Å structure of the U4/U6.U5 tri-snRNP: Insights into spliceosome assembly and catalysis

  2016cited by this paper
• CryoEM structures of two spliceosomal complexes: starter and dessert at the spliceosome feast

  2016cited by this paper
• Spliceosomal DEAH-Box ATPases Remodel Pre-mRNA to Activate Alternative Splice Sites.

  2016cited by this paper
• CryoEM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution

  2016cited by this paper
• 113Cd NMR Experiments Reveal an Unusual Metal Cluster in the Solution Structure of the Yeast Splicing Protein Bud31p†

  2015cited by this paper
• The exon junction complex as a node of post-transcriptional networks

  2015cited by this paper
• Sampling the conformational space of the catalytic subunit of human γ-secretase

  2015cited by this paper
• Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions

  2015cited by this paper
• 113Cd NMR Experiments Reveal an Unusual Metal Cluster in the Solution Structure of the Yeast Splicing Protein Bud31p**

  2015cited by this paper
• Assembly and dynamics of the U4/U6 di-snRNP by single-molecule FRET

  2015cited by this paper
• The architecture of the spliceosomal U4/U6.U5 tri-snRNP

  2015cited by this paper
• CTFFIND4: Fast and accurate defocus estimation from electron micrographs

  2015cited by this paper
• Semi-automated selection of cryo-EM particles in RELION-1.3

  2015cited by this paper
• Structure of a yeast spliceosome at 3.6-angstrom resolution

  2015cited by this paper
• Crystal structure of a eukaryotic group II intron lariat

  2014cited by this paper
• Faculty Opinions recommendation of RNA polymerase II termination involves C-terminal-domain tyrosine dephosphorylation by CPF subunit Glc7.

  2014cited by this paper
• The I-TASSER Suite: protein structure and function prediction

  2014cited by this paper
• Endogenous U2·U5·U6 snRNA complexes in S. pombe are intron lariat spliceosomes

  2014cited by this paper
• Evidence for a group II intron-like catalytic triplex in the spliceosome

  2014cited by this paper
• SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information

  2014cited by this paper
• Conformation-independent structural comparison of macromolecules with ProSMART

  2014cited by this paper
• A homolog of lariat-debranching enzyme modulates turnover of branched RNA

  2014cited by this paper
• RNA polymerase II termination involves CTD tyrosine dephosphorylation by CPF subunit Glc7

  2014cited by this paper
• Structural Basis of Brr2-Prp8 Interactions and Implications for U5 snRNP Biogenesis and the Spliceosome Active Site

  2013cited by this paper
• Crystal structure of the human eIF4AIII–CWC22 complex shows how a DEAD-box protein is inhibited by a MIF4G domain

  2013cited by this paper
• Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system

  2013cited by this paper
• The Local Resolution of Cryo-EM Density Maps

  2013cited by this paper
• Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing

  2013cited by this paper
• Electron counting and beam-induced motion correction enable near atomic resolution single particle cryoEM

  2013cited by this paper
• High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy☆

  2013cited by this paper
• Visualizing group II intron catalysis through the stages of splicing.

  2012cited by this paper
• Prevention of overfitting in cryo-EM structure determination

  2012cited by this paper
• CWC22 connects pre-mRNA splicing and exon junction complex assembly.

  2012cited by this paper
• Cwc2 and its human homologue RBM22 promote an active conformation of the spliceosome catalytic centre

  2012cited by this paper
• Structure of the mRNA splicing complex component Cwc2: insights into RNA recognition.

  2012cited by this paper
• Crystal structure of Prp8 reveals active site cavity of the spliceosome

  2012cited by this paper
• Human spliceosomal protein CWC22 plays a role in coupling splicing to exon junction complex deposition and nonsense-mediated decay

  2012cited by this paper
• Link of NTR-Mediated Spliceosome Disassembly with DEAH-Box ATPases Prp2, Prp16, and Prp22

  2012cited by this paper
• RELION: Implementation of a Bayesian approach to cryo-EM structure determination

  2012cited by this paper
• Human CWC22 escorts the helicase eIF4AIII to spliceosomes and promotes exon junction complex assembly

  2012cited by this paper
• Structure, function and regulation of spliceosomal RNA helicases.

  2012cited by this paper
• Spliceosome structure and function.

  2011cited by this paper
• DEAH-box ATPase Prp16 has dual roles in remodeling of the spliceosome in catalytic steps.

  2011cited by this paper
• Group II introns: mobile ribozymes that invade DNA.

  2011cited by this paper
• Cross-linking, ligation, and sequencing of hybrids reveals RNA–RNA interactions in yeast

  2011cited by this paper
• Conformational dynamics of single pre-mRNA molecules during in vitro splicing

  2010cited by this paper
• Prp43p contains a processive helicase structural architecture with a specific regulatory domain

  2010cited by this paper
• Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome

  2009cited by this paper
• Cwc25 Is a Novel Splicing Factor Required after Prp2 and Yju2 To Facilitate the First Catalytic Reaction

  2009cited by this paper
• Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components

  2009cited by this paper
• The evolutionarily conserved core design of the catalytic activation step of the yeast spliceosome.

  2009cited by this paper
• Physical and genetic interactions of yeast Cwc21p, an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome.

  2009cited by this paper
• "Nought may endure but mutability": spliceosome dynamics and the regulation of splicing.

  2008cited by this paper
• Isolation of an active step I spliceosome and composition of its RNP core

  2008cited by this paper
• A conformational rearrangement in the spliceosome sets the stage for Prp22-dependent mRNA release.

  2008cited by this paper
• Visualizing density maps with UCSF Chimera.

  2007cited by this paper
• Rearrangement of competing U2 RNA helices within the spliceosome promotes multiple steps in splicing.

  2007cited by this paper
• U2 toggles iteratively between the stem IIa and stem IIc conformations to promote pre-mRNA splicing.

  2007cited by this paper
• Structure of the Exon Junction Core Complex with a Trapped DEAD-Box ATPase Bound to RNA

  2006cited by this paper
• The crystal structure of the exon junction complex reveals how it maintains a stable grip on mRNA.

  2006cited by this paper
• Repositioning of the reaction intermediate within the catalytic center of the spliceosome.

  2006cited by this paper
• The Isy1p component of the NineTeen complex interacts with the ATPase Prp16p to regulate the fidelity of pre-mRNA splicing.

  2005cited by this paper
• Spliceosome disassembly catalyzed by Prp43 and its associated components Ntr1 and Ntr2.

  2005cited by this paper
• MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes

  2004cited by this paper
• Interacts with Exon Sequences at 5 ’ and 3 ’ Splice Sites

  2003cited by this paper
• GeneSilico protein structure prediction meta-server

  2003cited by this paper
• The Prp19p-Associated Complex in Spliceosome Activation

  2003cited by this paper
• Comprehensive proteomic analysis of the human spliceosome

  2002cited by this paper
• Characterization of interactions among the Cef1p-Prp19p-associated splicing complex.

  2002cited by this paper
• Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae.

  2001cited by this paper
• RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2.

  1998cited by this paper
• Crystal structure of the spliceosomal U2B″–U2A′ protein complex bound to a fragment of U2 small nuclear RNA

  1998cited by this paper
• The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro.

  1998cited by this paper
• Refinement of macromolecular structures by the maximum-likelihood method.

  1997cited by this paper
• Site-specific crosslinks of yeast U6 snRNA to the pre-mRNA near the 5' splice site.

  1996cited by this paper
• Prp16p, Slu7p, and Prp8p interact with the 3' splice site in two distinct stages during the second catalytic step of pre-mRNA splicing.

  1995cited by this paper
• Involvement of U6 snRNA in 5' splice site selection.

  1993cited by this paper
• The U5 and U6 small nuclear RNAs as active site components of the spliceosome.

  1993cited by this paper
• Role of U6 snRNA in 5' splice site selection

  1993cited by this paper
• Mutations in U6 snRNA that alter splice site specificity: implications for the active site.

  1993cited by this paper
• A general two-metal-ion mechanism for catalytic RNA.

  1993cited by this paper
• A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis.

  1992cited by this paper
• A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome.

  1992cited by this paper
• Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes

  1991cited by this paper
• Yeast mRNA splicing in vitro.

  1985cited by this paper

• Monovalent ions aid catalysis of two-metal-ion dependent RNA editors

  2026cites this paper
• DEAH‐Box RNA Helicases in the Spliceosome: Advances in Structure and Function

  2026cites this paper
• The spliceosome assembles on excised linear introns to protect them from degradation

  2026cites this paper
• Decoding Spliceosome Dynamics through Computation and Experiment.

  2025cites this paper
• Structural evidence for metal ion catalysis in the ribosome

  2025influential citation
• Unraveling gene expression: a beginner’s guide from chromatin modifications to mRNA export in Saccharomyces cerevisiae

  2025cites this paper
• Control of 3′ splice site selection in S. cerevisiae by a highly conserved amino acid within the Prp8 α-finger domain

  2025cites this paper
• New mechanistic insights into Prp22-mediated exon ligation and mRNA release

  2025cites this paper
• Structural insights into spliceosome fidelity: DHX35–GPATCH1- mediated rejection of aberrant splicing substrates

  2025cites this paper
• Retinitis pigmentosa-linked mutations impair the snRNA unwinding activity of SNRNP200 and reduce pre-mRNA binding of PRPF8

  2025cites this paper
• Design of an orally bioavailable small molecule that modulates the microtubule-associated protein tau’s pre-mRNA splicing

  2025cites this paper
• Brr2p-mediated unwinding of U4/U6 is promoted by a mutually exclusive intra-molecular stem loop in U4 and involves destabilization of the 5’ stem-loop of U4

  2025cites this paper
• Comprehensive analysis of Saccharomyces cerevisiae intron structures in vivo

  2025cites this paper
• Control of 3′ Splice Site Selection in S. cerevisiae by a Highly Conserved Amino Acid within the Prp8 α-finger Domain

  2025cites this paper
• Intron lariat spliceosomes convert lariats to true circles: implications for intron transposition

  2024cites this paper
• Characterization of Cwc2, U6 snRNA, and Prp8 interactions destabilized by Prp16 ATPase at the transition between the first and second steps of splicing

  2024influential citation
• Mechanisms and regulation of spliceosome-mediated pre-mRNA splicing in Saccharomyces cerevisiae

  2024cites this paper
• Intron-lariat spliceosomes convert lariats to true circles: implications for intron transposition

  2024cites this paper
• Structures of aberrant spliceosome intermediates on their way to disassembly

  2024cites this paper
• Variation of C-terminal domain governs RNA polymerase II genomic locations and alternative splicing in eukaryotic transcription

  2024cites this paper
• Branch site recognition by the spliceosome

  2024influential citation
• Prp16 enables efficient splicing of introns with diverse exonic consensus elements in the short-intron rich Cryptococcus neoformans transcriptome

  2024cites this paper
• Variation of C-terminal domain governs RNA polymerase II genomic locations and alternative splicing in eukaryotic transcription

  2024cites this paper
• The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis

  2024cites this paper
• Structural basis of branching during RNA splicing

  2023cites this paper
• Cryo-EM of a heterogeneous biochemical fraction elucidates multiple protein complexes from a multicellular thermophilic eukaryote

  2023cites this paper
• An ATP-independent role for Prp16 in promoting aberrant splicing

  2023cites this paper
• RNA recognition by Npl3p reveals U2 snRNA-binding compatible with a chaperone role during splicing

  2023cites this paper
• Spliceosome assembly and regulation: insights from analysis of highly reduced spliceosomes

  2023cites this paper
• Structure and function of spliceosomal DEAH-box ATPases

  2023cites this paper
• Monovalent metal ion binding promotes the first transesterification reaction in the spliceosome

  2023cites this paper
• Harmonizing the growing fluorogenic RNA aptamer toolbox for RNA detection and imaging.

  2023cites this paper
• Crystal structure of Prp16 in complex with ADP

  2023cites this paper
• IARA: a complete and curated atlas of the biogenesis of spliceosome machinery during RNA splicing

  2023influential citation
• Structural basis of catalytic activation in human splicing

  2023cites this paper
• The PRP19 Ubiquitin Ligase, Standing at the Cross-Roads of mRNA Processing and Genome Stability

  2022cites this paper
• Phytophthora effector PSR1 hijacks the host pre-mRNA splicing machinery to modulate small RNA biogenesis and plant immunity.

  2022cites this paper
• m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site

  2022cites this paper
• Structural and functional investigation of the human snRNP assembly factor AAR2 in complex with the RNase H-like domain of PRPF8

  2022cites this paper
• Insights into the Mechanism of Pre-mRNA Splicing of Tiny Introns from the Genome of a Giant Ciliate Stentor coeruleus

  2022cites this paper
• Kiyoshi Nagai. 25 June 1949—27 September 2019

  2022cites this paper
• Contrast variation SAXS: Sample preparation protocols, experimental procedures, and data analysis.

  2022cites this paper
• Structure-based thermodynamics of ion selectivity (Mg2+versus Ca2+ and K+versus Na+) in the active site of the eukaryotic lariat group II intron from algae Pylaiella littoralis.

  2022cites this paper
• Structural studies of the spliceosome: Bridging the gaps

  2022cites this paper
• CryoEM reveals oligomeric isomers of a multienzyme complex and assembly mechanics

  2022cites this paper
• Group II Intron-Encoded Proteins (IEPs/Maturases) as Key Regulators of Nad1 Expression and Complex I Biogenesis in Land Plant Mitochondria

  2022cites this paper
• The structure of yeast Npl3 bound to RNA reveals a cooperative sequence-specific recognition and an RNA chaperone role in splicing

  2022cites this paper
• Zinc finger structure determination by NMR: Why zinc fingers can be a handful

  2022cites this paper
• m 6 A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site

  2022cites this paper
• On the diverse roles of Class II nucleobase protonation in shaping structures and functions of RNA base pairs – a QM-based insight

  2022cites this paper
• m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5’ splice site

  2022cites this paper
• m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site

  2022cites this paper
• Structural basis of branch site recognition by the human spliceosome

  2021cites this paper
• U2 snRNA structure is influenced by SF3A and SF3B proteins but not by SF3B inhibitors

  2021cites this paper
• Organellar Introns in Fungi, Algae, and Plants

  2021cites this paper
• Long-range allostery mediates cooperative adenine nucleotide binding by the Ski2-like RNA helicase Brr2

  2021cites this paper
• Biology of the mRNA Splicing Machinery and Its Dysregulation in Cancer Providing Therapeutic Opportunities

  2021influential citation
• Synergistic roles for human U1 snRNA stem-loops in pre-mRNA splicing

  2021cites this paper
• The structure of Prp2 bound to RNA and ADP-BeF3 − reveals structural features important for RNA unwinding by DEAH-box ATPases

  2021cites this paper
• Evidence for complex dynamics during U2 snRNP selection of the intron branchpoint

  2021cites this paper
• Transcriptome-Wide Identification of Coding and Noncoding RNA-Binding Proteins Defines the Comprehensive RNA Interactome of Leishmania mexicana

  2021cites this paper
• Regulation of RNA helicase activity: principles and examples

  2021cites this paper
• Long-range allosteric effects - How adenine nucleotides shape the conformational landscape of the Ski2-like RNA helicase, Brr2

  2021cites this paper
• Network Theory Reveals Principles of Spliceosome Structure and Dynamics

  2021cites this paper
• Macromolecular Protein Complexes III: Structure and Function

  2021cites this paper
• U5 snRNA Interactions With Exons Ensure Splicing Precision

  2021cites this paper
• An RNA-centric historical narrative around the Protein Data Bank

  2021cites this paper
• The SF3b Complex is an Integral Component of the Spliceosome and Targeted by Natural Product-Based Inhibitors.

  2021cites this paper
• TITLE : Saccharomyces cerevisiae Ecm 2 Modulates the Catalytic Steps of pre-mRNA 1 Splicing 2

  2020cites this paper
• DEAH-Box RNA Helicases in Pre-mRNA Splicing.

  2020cites this paper
• Structural Insights into the Roles of Metazoan-Specific Splicing Factors in the Human Step 1 Spliceosome.

  2020cites this paper
• Key Points to Consider When Studying RNA Remodeling by Proteins.

  2020cites this paper
• Structural basis for conformational equilibrium of the catalytic spliceosome

  2020influential citation
• The integral spliceosomal component CWC15 is required for development in Arabidopsis

  2020cites this paper
• Saccharomyces cerevisiae Ecm2 modulates the catalytic steps of pre-mRNA splicing

  2020cites this paper
• psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation

  2020cites this paper
• Decrypting the information exchange pathways across the spliceosome machinery.

  2020cites this paper
• RNA-Seq approach for accurate characterization of splicing efficiency of yeast introns.

  2020cites this paper
• RNA Conformation Capture by Proximity Ligation.

  2020cites this paper
• Transcriptional analysis supports the expression of human snRNA variants and reveals U2 snRNA homeostasis by an abundant U2 variant

  2020cites this paper
• Structural basis for DEAH-helicase activation by G-patch proteins

  2020cites this paper
• A Day in the Life of the Exon Junction Complex

  2020cites this paper
• Auxiliary domains of the HrpB bacterial DExH-box helicase shape its RNA preferences

  2020cites this paper
• Retroelement origins of pre‐mRNA splicing

  2020cites this paper
• mRNA Editing, Processing and Quality Control in Caenorhabditis elegans

  2020cites this paper
• Cryo-EM snapshots of the human spliceosome reveal structural adaptions for splicing regulation.

  2020cites this paper
• Crystal structure of DEAH-box ATPase Prp22 with bound ssRNA

  2019cites this paper
• Cryo-EM structure of the yeast B*-a1 complex at an average resolution of 3.6 angstrom

  2019cites this paper
• Cryo-EM Structures of a Group II Intron Reverse Splicing into DNA.

  2019influential citation
• Prp8 in a Reduced Spliceosome Lacks a Conserved Toggle that Correlates with Splicing Complexity across Diverse Taxa.

  2019cites this paper
• Global donor and acceptor splicing site kinetics in human cells

  2019cites this paper
• Topology of the U12–U6atac snRNA Complex of the Minor Spliceosome and Binding by NTC-Related Protein RBM22

  2019cites this paper
• Identification of a noncanonical RNA binding domain in the U2 snRNP protein SF3A1

  2019cites this paper
• Understanding human DNA variants affecting pre-mRNA splicing in the NGS era.

  2019cites this paper
• Structural basis for RNA translocation by DEAH-box ATPases

  2019cites this paper
• Early splicing functions of fission yeast Prp16 and its unexpected requirement for gene Silencing is governed by intronic features

  2019influential citation
• Rearrangements within the U6 snRNA Core during the Transition between the Two Catalytic Steps of Splicing.

  2019cites this paper
• Writing a wrong: Coupled RNA polymerase II transcription and RNA quality control

  2019cites this paper
• Structural Basis of Nuclear pre-mRNA Splicing: Lessons from Yeast.

  2019influential citation
• The inactive C-terminal cassette of the dual-cassette RNA helicase BRR2 both stimulates and inhibits the activity of the N-terminal helicase unit

  2019cites this paper