+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-16452 | |||||||||
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Title | Human spliceosomal PM5 C* complex | |||||||||
Map data | map of the human spliceosomal PM5 C* complex | |||||||||
Sample |
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Keywords | spliceosome / nucleus / splicing | |||||||||
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 / post-spliceosomal complex / negative regulation of selenocysteine incorporation / regulation of nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / spliceosomal complex disassembly / cellular response to selenite ion / negative regulation of nitric-oxide synthase activity ...regulation of homologous chromosome segregation / endoplasmic reticulum membrane organization / second spliceosomal transesterification activity / exon-exon junction subcomplex mago-y14 / post-spliceosomal complex / negative regulation of selenocysteine incorporation / regulation of nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / spliceosomal complex disassembly / cellular response to selenite ion / negative regulation of nitric-oxide synthase activity / exon-exon junction complex / NOSIP mediated eNOS trafficking / pre-mRNA 3'-splice site binding / granulocyte differentiation / selenocysteine insertion sequence binding / post-mRNA release spliceosomal complex / regulation of retinoic acid receptor signaling pathway / negative regulation of toll-like receptor signaling pathway / regulation of translation at postsynapse, modulating synaptic transmission / 3'-5' RNA helicase activity / snRNP binding / U2 snRNP binding / U7 snRNA binding / histone pre-mRNA DCP binding / renal system process / generation of catalytic spliceosome for first transesterification step / negative regulation of excitatory postsynaptic potential / U7 snRNP / alternative mRNA splicing, via spliceosome / endonucleolytic cleavage of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of vitamin D receptor signaling pathway / Z-decay: degradation of maternal mRNAs by zygotically expressed factors / negative regulation of catalytic activity / histone pre-mRNA 3'end processing complex / negative regulation of interleukin-8 production / cis assembly of pre-catalytic spliceosome / regulation of mRNA processing / SLBP independent Processing of Histone Pre-mRNAs / SLBP Dependent Processing of Replication-Dependent Histone Pre-mRNAs / negative regulation of lipopolysaccharide-mediated signaling pathway / Deadenylation of mRNA / embryonic brain development / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / protein methylation / STING mediated induction of host immune responses / U12-type spliceosomal complex / negative regulation of phosphorylation / methylosome / nuclear retinoic acid receptor binding / negative regulation of interferon-beta production / Prp19 complex / 7-methylguanosine cap hypermethylation / positive regulation of androgen receptor activity / poly(A) binding / U1 snRNP binding / M-decay: degradation of maternal mRNAs by maternally stored factors / pICln-Sm protein complex / mRNA 3'-end processing / pre-mRNA binding / ATP-dependent activity, acting on RNA / U2-type catalytic step 1 spliceosome / embryonic cranial skeleton morphogenesis / RNA splicing, via transesterification reactions / small nuclear ribonucleoprotein complex / P granule / sno(s)RNA-containing ribonucleoprotein complex / IRF3-mediated induction of type I IFN / SMN-Sm protein complex / regulation of mRNA splicing, via spliceosome / C2H2 zinc finger domain binding / spliceosomal tri-snRNP complex / positive regulation of mRNA splicing, via spliceosome / telomerase holoenzyme complex / mRNA cis splicing, via spliceosome / U2-type spliceosomal complex / telomerase RNA binding / positive regulation by host of viral transcription / U2-type precatalytic spliceosome / commitment complex / positive regulation of vitamin D receptor signaling pathway / Transport of Mature mRNA derived from an Intron-Containing Transcript / U2-type prespliceosome assembly / U2-type catalytic step 2 spliceosome / nuclear vitamin D receptor binding / U4 snRNP / Notch binding / Regulation of gene expression in late stage (branching morphogenesis) pancreatic bud precursor cells / RUNX3 regulates NOTCH signaling / U2 snRNP / positive regulation of alpha-beta T cell differentiation / RNA Polymerase II Transcription Termination / NOTCH4 Intracellular Domain Regulates Transcription / U1 snRNP / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / ubiquitin-ubiquitin ligase activity / WD40-repeat domain binding / NOTCH3 Intracellular Domain Regulates Transcription / positive regulation of neurogenesis / exploration behavior / U2-type prespliceosome Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) / human (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.8 Å | |||||||||
Authors | Dybkov O / Kastner B / Luehrmann R | |||||||||
Funding support | Germany, 1 items
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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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_16452.map.gz | 373.6 MB | EMDB map data format | |
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Header (meta data) | emd-16452-v30.xml emd-16452.xml | 92.1 KB 92.1 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_16452_fsc.xml | 20.1 KB | Display | FSC data file |
Images | emd_16452.png | 117.9 KB | ||
Others | emd_16452_additional_1.map.gz emd_16452_half_map_1.map.gz emd_16452_half_map_2.map.gz | 30.2 MB 690.5 MB 690.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-16452 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-16452 | HTTPS FTP |
-Related structure data
Related structure data | 8c6jMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_16452.map.gz / Format: CCP4 / Size: 744.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | map of the human spliceosomal PM5 C* complex | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.05 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: locally filtered map of the human spliceosomal PM5 C* complex
File | emd_16452_additional_1.map | ||||||||||||
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Annotation | locally filtered map of the human spliceosomal PM5 C* complex | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map A
File | emd_16452_half_map_1.map | ||||||||||||
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Annotation | half map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map B
File | emd_16452_half_map_2.map | ||||||||||||
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Annotation | half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : human spliceosomal C* complex assembled on PM5 pre-mRNA
+Supramolecule #1: human spliceosomal C* complex assembled on PM5 pre-mRNA
+Macromolecule #1: U2 snRNA
+Macromolecule #3: U5 snRNA
+Macromolecule #4: U6 snRNA
+Macromolecule #19: PM5 5' Exon
+Macromolecule #23: PM5 Intron
+Macromolecule #2: Splicing factor ESS-2 homolog
+Macromolecule #5: Eukaryotic initiation factor 4A-III
+Macromolecule #6: RNA-binding protein 8A
+Macromolecule #7: Protein mago nashi homolog
+Macromolecule #8: Pre-mRNA-processing-splicing factor 8
+Macromolecule #9: U5 small nuclear ribonucleoprotein 200 kDa helicase
+Macromolecule #10: 116 kDa U5 small nuclear ribonucleoprotein component
+Macromolecule #11: Peptidyl-prolyl cis-trans isomerase-like 3
+Macromolecule #12: Probable ATP-dependent RNA helicase DDX41
+Macromolecule #13: Peptidyl-prolyl cis-trans isomerase E
+Macromolecule #14: Protein FAM50A
+Macromolecule #15: Pre-mRNA-splicing factor ISY1 homolog
+Macromolecule #16: Nitric oxide synthase-interacting protein
+Macromolecule #17: Splicing factor C9orf78
+Macromolecule #18: PRKR-interacting protein 1
+Macromolecule #20: Splicing factor Cactin
+Macromolecule #21: Protein FAM32A
+Macromolecule #22: Pre-mRNA-splicing factor CWC22 homolog
+Macromolecule #24: Pleiotropic regulator 1
+Macromolecule #25: SNW domain-containing protein 1
+Macromolecule #26: Protein BUD31 homolog
+Macromolecule #27: Pre-mRNA-splicing factor RBM22
+Macromolecule #28: U5 small nuclear ribonucleoprotein 40 kDa protein
+Macromolecule #29: Cell division cycle 5-like protein
+Macromolecule #30: Spliceosome-associated protein CWC15 homolog
+Macromolecule #31: Serine/arginine repetitive matrix protein 2
+Macromolecule #32: Crooked neck-like protein 1
+Macromolecule #33: Pre-mRNA-splicing factor SYF1
+Macromolecule #34: Intron-binding protein aquarius
+Macromolecule #35: ATP-dependent RNA helicase DHX8
+Macromolecule #36: U2 small nuclear ribonucleoprotein A'
+Macromolecule #37: U2 small nuclear ribonucleoprotein B''
+Macromolecule #38: NF-kappa-B-activating protein
+Macromolecule #39: Small nuclear ribonucleoprotein Sm D2
+Macromolecule #40: Small nuclear ribonucleoprotein-associated proteins B and B'
+Macromolecule #41: Small nuclear ribonucleoprotein Sm D1
+Macromolecule #42: Small nuclear ribonucleoprotein Sm D3
+Macromolecule #43: Small nuclear ribonucleoprotein E
+Macromolecule #44: Small nuclear ribonucleoprotein F
+Macromolecule #45: Small nuclear ribonucleoprotein G
+Macromolecule #46: Pre-mRNA-splicing factor SLU7
+Macromolecule #47: Peptidyl-prolyl cis-trans isomerase-like 1
+Macromolecule #48: STING ER exit protein
+Macromolecule #49: Pre-mRNA-processing factor 17
+Macromolecule #50: Pre-mRNA-splicing factor SPF27
+Macromolecule #51: Pre-mRNA-processing factor 19
+Macromolecule #52: Pre-mRNA-splicing factor SYF2
+Macromolecule #53: Splicing regulator SDE2
+Macromolecule #54: POTASSIUM ION
+Macromolecule #55: MAGNESIUM ION
+Macromolecule #56: ADENOSINE-5'-TRIPHOSPHATE
+Macromolecule #57: INOSITOL HEXAKISPHOSPHATE
+Macromolecule #58: GUANOSINE-5'-TRIPHOSPHATE
+Macromolecule #59: ZINC ION
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.8 mg/mL |
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Buffer | pH: 7.8 |
Grid | Model: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 100 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV |
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. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 14388 / Average exposure time: 1.465 sec. / Average electron dose: 44.24 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
-Atomic model buiding 1
Initial model |
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Refinement | Space: REAL / Protocol: FLEXIBLE FIT | ||||||||||||||||||||||||
Output model | PDB-8c6j: |