+Open data
-Basic information
Entry | Database: PDB / ID: 8c6j | ||||||
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Title | Human spliceosomal PM5 C* complex | ||||||
Components |
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Keywords | SPLICING / spliceosome / nucleus | ||||||
Function / homology | Function and homology information regulation of homologous chromosome segregation / endoplasmic reticulum membrane organization / second spliceosomal transesterification activity / exon-exon junction subcomplex mago-y14 / negative regulation of selenocysteine incorporation / regulation of nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / post-spliceosomal complex / negative regulation of nitric-oxide synthase activity / cellular response to selenite ion / spliceosomal complex disassembly ...regulation of homologous chromosome segregation / endoplasmic reticulum membrane organization / second spliceosomal transesterification activity / exon-exon junction subcomplex mago-y14 / negative regulation of selenocysteine incorporation / regulation of nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / post-spliceosomal complex / negative regulation of nitric-oxide synthase activity / cellular response to selenite ion / spliceosomal complex disassembly / exon-exon junction complex / selenocysteine insertion sequence binding / protein exit from endoplasmic reticulum / NOSIP mediated eNOS trafficking / pre-mRNA 3'-splice site binding / granulocyte differentiation / regulation of translation at postsynapse, modulating synaptic transmission / negative regulation of toll-like receptor signaling pathway / post-mRNA release spliceosomal complex / regulation of retinoic acid receptor signaling pathway / alternative mRNA splicing, via spliceosome / U2 snRNP binding / renal system process / endonucleolytic cleavage of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / negative regulation of interleukin-8 production / U7 snRNA binding / negative regulation of excitatory postsynaptic potential / histone pre-mRNA DCP binding / 3'-5' RNA helicase activity / negative regulation of catalytic activity / U7 snRNP / negative regulation of phosphorylation / generation of catalytic spliceosome for first transesterification step / Z-decay: degradation of maternal mRNAs by zygotically expressed factors / regulation of vitamin D receptor signaling pathway / histone pre-mRNA 3'end processing complex / negative regulation of lipopolysaccharide-mediated signaling pathway / cis assembly of pre-catalytic spliceosome / SLBP independent Processing of Histone Pre-mRNAs / regulation of mRNA processing / SLBP Dependent Processing of Replication-Dependent Histone Pre-mRNAs / Deadenylation of mRNA / embryonic cranial skeleton morphogenesis / molecular sequestering activity / protein methylation / negative regulation of interferon-beta production / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / U12-type spliceosomal complex / embryonic brain development / methylosome / nuclear retinoic acid receptor binding / 7-methylguanosine cap hypermethylation / positive regulation of androgen receptor activity / poly(A) binding / M-decay: degradation of maternal mRNAs by maternally stored factors / Prp19 complex / U1 snRNP binding / mRNA 3'-end processing / ATP-dependent activity, acting on RNA / pICln-Sm protein complex / snRNP binding / sno(s)RNA-containing ribonucleoprotein complex / RNA splicing, via transesterification reactions / U2-type catalytic step 1 spliceosome / small nuclear ribonucleoprotein complex / pre-mRNA binding / C2H2 zinc finger domain binding / regulation of mRNA splicing, via spliceosome / SMN-Sm protein complex / telomerase RNA binding / spliceosomal tri-snRNP complex / telomerase holoenzyme complex / P granule / regulation of nitric oxide biosynthetic process / U2-type spliceosomal complex / U2-type precatalytic spliceosome / positive regulation by host of viral transcription / mRNA cis splicing, via spliceosome / positive regulation of vitamin D receptor signaling pathway / commitment complex / U2-type prespliceosome assembly / nuclear vitamin D receptor binding / Transport of Mature mRNA derived from an Intron-Containing Transcript / U2-type catalytic step 2 spliceosome / Notch binding / positive regulation of alpha-beta T cell differentiation / Regulation of gene expression in late stage (branching morphogenesis) pancreatic bud precursor cells / U4 snRNP / positive regulation of mRNA splicing, via spliceosome / RUNX3 regulates NOTCH signaling / U2 snRNP / RNA Polymerase II Transcription Termination / NOTCH4 Intracellular Domain Regulates Transcription / exploration behavior / U1 snRNP / ubiquitin-ubiquitin ligase activity / positive regulation of neurogenesis / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / NOTCH3 Intracellular Domain Regulates Transcription / WD40-repeat domain binding Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å | ||||||
Authors | Dybkov, O. / Kastner, B. / Luehrmann, R. | ||||||
Funding support | Germany, 1items
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Citation | Journal: Sci Adv / Year: 2023 Title: Regulation of 3' splice site selection after step 1 of splicing by spliceosomal C* proteins. Authors: Olexandr Dybkov / Marco Preußner / Leyla El Ayoubi / Vivi-Yun Feng / Caroline Harnisch / Kilian Merz / Paula Leupold / Peter Yudichev / Dmitry E Agafonov / Cindy L Will / Cyrille Girard / ...Authors: Olexandr Dybkov / Marco Preußner / Leyla El Ayoubi / Vivi-Yun Feng / Caroline Harnisch / Kilian Merz / Paula Leupold / Peter Yudichev / Dmitry E Agafonov / Cindy L Will / Cyrille Girard / Christian Dienemann / Henning Urlaub / Berthold Kastner / Florian Heyd / Reinhard Lührmann / Abstract: Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3' splice site (3'ss) usage contribute to human disease. We demonstrate by ...Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3'ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3' region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3'ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3'ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3'ss choices. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8c6j.cif.gz | 2.9 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8c6j.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 8c6j.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8c6j_validation.pdf.gz | 2 MB | Display | wwPDB validaton report |
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Full document | 8c6j_full_validation.pdf.gz | 2 MB | Display | |
Data in XML | 8c6j_validation.xml.gz | 295.4 KB | Display | |
Data in CIF | 8c6j_validation.cif.gz | 557.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/c6/8c6j ftp://data.pdbj.org/pub/pdb/validation_reports/c6/8c6j | HTTPS FTP |
-Related structure data
Related structure data | 16452MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
+RNA chain , 5 types, 5 molecules 256EI
+Splicing factor ... , 3 types, 3 molecules 4CTF
+Protein , 24 types, 25 molecules 789ACCDCECFCNDGJKLOPRSUVZbkjz
+U5 small nuclear ribonucleoprotein ... , 2 types, 2 molecules BN
+Peptidyl-prolyl cis-trans isomerase-like ... , 2 types, 2 molecules C3i
+Pre-mRNA-splicing factor ... , 7 types, 7 molecules CIHMThsy
+U2 small nuclear ribonucleoprotein ... , 2 types, 2 molecules WY
+Small nuclear ribonucleoprotein ... , 6 types, 12 molecules amcldnepfqgr
+Pre-mRNA-processing factor ... , 2 types, 5 molecules otuvw
+Non-polymers , 6 types, 18 molecules
+Details
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: human spliceosomal C* complex assembled on PM5 pre-mRNA Type: COMPLEX / Entity ID: #1-#53 / Source: NATURAL |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Homo sapiens (human) |
Buffer solution | pH: 7.8 |
Specimen | Conc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: 4 microliter sample was applied to a grid. After 15 s, the grid was blotted for 3 s with a blot force of 5 and vitrified by plunging into liquid ethane using FEI Vitrobot Mark IV. |
Specimen support | Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 1.465 sec. / Electron dose: 44.24 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 14388 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1643674 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1150057 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | 3D fitting-ID: 1
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