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- PDB-7oqc: The U1 part of Saccharomyces cerevisiae spliceosomal pre-A comple... -

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Basic information

Entry
Database: PDB / ID: 7oqc
TitleThe U1 part of Saccharomyces cerevisiae spliceosomal pre-A complex (delta BS-A ACT1)
Components
  • (Small nuclear ribonucleoprotein ...SnRNP) x 6
  • (U1 small nuclear ribonucleoprotein ...) x 5
  • 56 kDa U1 small nuclear ribonucleoprotein component
  • ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A)
  • Pre-mRNA-processing factor 39
  • Protein LUC7
  • Protein NAM8
  • Small nuclear ribonucleoprotein-associated protein B
  • U1 snRNAU1 spliceosomal RNA
KeywordsSPLICING / S. cerevisiae / pre-A complex / Prp5 / U1 snRNP / U2 snRNP / prespliceosome
Function / homology
Function and homology information


positive regulation of RNA binding / mRNA splice site recognition / splicing factor binding / U4/U6 snRNP / 7-methylguanosine cap hypermethylation / pICln-Sm protein complex / spliceosomal tri-snRNP complex / small nuclear ribonucleoprotein complex / SMN-Sm protein complex / positive regulation of mRNA splicing, via spliceosome ...positive regulation of RNA binding / mRNA splice site recognition / splicing factor binding / U4/U6 snRNP / 7-methylguanosine cap hypermethylation / pICln-Sm protein complex / spliceosomal tri-snRNP complex / small nuclear ribonucleoprotein complex / SMN-Sm protein complex / positive regulation of mRNA splicing, via spliceosome / U2-type prespliceosome assembly / commitment complex / U4 snRNP / U2 snRNP / poly(U) RNA binding / U1 snRNP / U2-type prespliceosome / pre-mRNA 5'-splice site binding / precatalytic spliceosome / spliceosomal complex assembly / mRNA 5'-splice site recognition / U5 snRNP / spliceosomal snRNP assembly / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / spliceosomal complex / mRNA splicing, via spliceosome / mRNA binding / RNA binding / zinc ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Luc7-related / LUC7 N_terminus / Snu56-like U1 small nuclear ribonucleoprotein component / Snu56-like U1 small nuclear ribonucleoprotein component / U1 small nuclear ribonucleoprotein C / U1 small nuclear ribonucleoprotein of 70kDa N-terminal / U1 small nuclear ribonucleoprotein of 70kDa MW N terminal / U1-C, C2H2-type zinc finger / U1 zinc finger / PWI domain ...Luc7-related / LUC7 N_terminus / Snu56-like U1 small nuclear ribonucleoprotein component / Snu56-like U1 small nuclear ribonucleoprotein component / U1 small nuclear ribonucleoprotein C / U1 small nuclear ribonucleoprotein of 70kDa N-terminal / U1 small nuclear ribonucleoprotein of 70kDa MW N terminal / U1-C, C2H2-type zinc finger / U1 zinc finger / PWI domain / PWI, domain in splicing factors / Matrin/U1-C, C2H2-type zinc finger / Zinc finger matrin-type profile. / Small nuclear ribonucleoprotein Sm D3 / Small nuclear ribonucleoprotein Sm D2 / Small nuclear ribonucleoprotein E / Small nuclear ribonucleoprotein G / Small nuclear ribonucleoprotein F / Sm-like protein Lsm7/SmG / Like-Sm (LSM) domain containing protein, LSm4/SmD1/SmD3 / Matrin/U1-C-like, C2H2-type zinc finger / U1-like zinc finger / Sm-like protein Lsm6/SmF / LSM domain / LSM domain, eukaryotic/archaea-type / snRNP Sm proteins / HAT (Half-A-TPR) repeat / HAT (Half-A-TPR) repeats / : / Sm domain profile. / LSM domain superfamily / Zinc finger C2H2 superfamily / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Tetratricopeptide-like helical domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / U1 small nuclear ribonucleoprotein A / Pre-mRNA-processing factor 39 / Small nuclear ribonucleoprotein-associated protein B / Small nuclear ribonucleoprotein G / Small nuclear ribonucleoprotein Sm D3 / U1 small nuclear ribonucleoprotein component SNU71 / Small nuclear ribonucleoprotein F ...RNA / RNA (> 10) / RNA (> 100) / U1 small nuclear ribonucleoprotein A / Pre-mRNA-processing factor 39 / Small nuclear ribonucleoprotein-associated protein B / Small nuclear ribonucleoprotein G / Small nuclear ribonucleoprotein Sm D3 / U1 small nuclear ribonucleoprotein component SNU71 / Small nuclear ribonucleoprotein F / Protein NAM8 / U1 small nuclear ribonucleoprotein 70 kDa homolog / Small nuclear ribonucleoprotein Sm D1 / U1 small nuclear ribonucleoprotein component PRP42 / 56 kDa U1 small nuclear ribonucleoprotein component / U1 small nuclear ribonucleoprotein C / Small nuclear ribonucleoprotein Sm D2 / Protein LUC7 / Small nuclear ribonucleoprotein E
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsZhang, Z. / Rigo, N. / Dybkov, O. / Fourmann, J. / Will, C.L. / Kumar, V. / Urlaub, H. / Stark, H. / Luehrmann, R.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB 860 Germany
CitationJournal: Nature / Year: 2021
Title: Structural insights into how Prp5 proofreads the pre-mRNA branch site.
Authors: Zhenwei Zhang / Norbert Rigo / Olexandr Dybkov / Jean-Baptiste Fourmann / Cindy L Will / Vinay Kumar / Henning Urlaub / Holger Stark / Reinhard Lührmann /
Abstract: During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex-a poorly ...During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex-a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. ). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2-BS helix), which is proofread by Prp5 at this stage through an unclear mechanism. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2-BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155 to the bulged BS-A of the U2-BS helix triggers closure of Hsh155, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing.
History
DepositionJun 3, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 29, 2021Group: Data collection / Database references
Category: citation / em_admin ...citation / em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod

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Structure visualization

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Assembly

Deposited unit
F: Protein NAM8
I: ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A)
E: U1 small nuclear ribonucleoprotein component PRP42
J: U1 small nuclear ribonucleoprotein component SNU71
1: U1 snRNA
G: 56 kDa U1 small nuclear ribonucleoprotein component
A: U1 small nuclear ribonucleoprotein A
C: U1 small nuclear ribonucleoprotein C
b: Small nuclear ribonucleoprotein-associated protein B
d: Small nuclear ribonucleoprotein Sm D3
e: Small nuclear ribonucleoprotein E
f: Small nuclear ribonucleoprotein F
g: Small nuclear ribonucleoprotein G
h: Small nuclear ribonucleoprotein Sm D1
i: Small nuclear ribonucleoprotein Sm D2
H: Protein LUC7
D: Pre-mRNA-processing factor 39
B: U1 small nuclear ribonucleoprotein 70 kDa homolog


Theoretical massNumber of molelcules
Total (without water)836,52618
Polymers836,52618
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 5 molecules FGbHD

#1: Protein Protein NAM8


Mass: 57010.840 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q00539
#6: Protein 56 kDa U1 small nuclear ribonucleoprotein component


Mass: 56575.277 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03782
#9: Protein Small nuclear ribonucleoprotein-associated protein B / snRNP-B / Sm protein B / SmB / U5 SmB


Mass: 22426.990 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40018
#16: Protein Protein LUC7


Mass: 30245.885 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q07508
#17: Protein Pre-mRNA-processing factor 39


Mass: 74834.742 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P39682

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RNA chain , 2 types, 2 molecules I1

#2: RNA chain ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A),ACT1 pre-mRNA (delta BS-A)


Mass: 111601.883 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast)
#5: RNA chain U1 snRNA / U1 spliceosomal RNA


Mass: 182114.516 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast)

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U1 small nuclear ribonucleoprotein ... , 5 types, 5 molecules EJACB

#3: Protein U1 small nuclear ribonucleoprotein component PRP42


Mass: 65222.020 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03776
#4: Protein U1 small nuclear ribonucleoprotein component SNU71


Mass: 71484.555 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53207
#7: Protein U1 small nuclear ribonucleoprotein A


Mass: 34438.105 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32605
#8: Protein U1 small nuclear ribonucleoprotein C


Mass: 27106.016 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q05900
#18: Protein U1 small nuclear ribonucleoprotein 70 kDa homolog


Mass: 34506.148 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q00916

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Small nuclear ribonucleoprotein ... , 6 types, 6 molecules defghi

#10: Protein Small nuclear ribonucleoprotein Sm D3 / Sm-D3 / snRNP core protein D3 / U5 SmD3


Mass: 11240.139 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P43321
#11: Protein Small nuclear ribonucleoprotein E / snRNP-E / Sm protein E / SmE / U5 SmE


Mass: 10385.098 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12330
#12: Protein Small nuclear ribonucleoprotein F / snRNP-F / Sm protein F / SmF


Mass: 9669.945 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P54999
#13: Protein Small nuclear ribonucleoprotein G / snRNP-G / Sm protein G / SmG


Mass: 8490.809 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40204
#14: Protein Small nuclear ribonucleoprotein Sm D1 / Sm-D1 / snRNP core protein D1 / U4 SmD1


Mass: 16296.798 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q02260
#15: Protein Small nuclear ribonucleoprotein Sm D2 / Sm-D2 / snRNP core protein D2 / SmD2


Mass: 12876.066 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q06217

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: S. cerevisiae spliceosomal pre-A complex / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R3.5/1
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / C2 aperture diameter: 100 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.02 sec. / Electron dose: 44 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
10RELION3initial Euler assignment
11RELION3final Euler assignment
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 217460 / Algorithm: FOURIER SPACE / Symmetry type: POINT

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