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- PDB-8w2o: Yeast U1 snRNP with humanized U1C Zinc-Finger domain -

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Basic information

Entry
Database: PDB / ID: 8w2o
TitleYeast U1 snRNP with humanized U1C Zinc-Finger domain
Components
  • (Small nuclear ribonucleoprotein ...) x 6
  • (U1 small nuclear ribonucleoprotein ...) x 5
  • 56 kDa U1 small nuclear ribonucleoprotein component
  • ACT1 pre-mRNA
  • Pre-mRNA-processing factor 39
  • Protein LUC7
  • Protein NAM8
  • Small nuclear ribonucleoprotein-associated protein B
  • U1 snRNA
KeywordsSPLICING / U1snRNP / U1C humanization
Function / homology
Function and homology information


mRNA splice site recognition / splicing factor binding / U4/U6 snRNP / 7-methylguanosine cap hypermethylation / pICln-Sm protein complex / positive regulation of mRNA splicing, via spliceosome / snRNP binding / small nuclear ribonucleoprotein complex / SMN-Sm protein complex / spliceosomal tri-snRNP complex ...mRNA splice site recognition / splicing factor binding / U4/U6 snRNP / 7-methylguanosine cap hypermethylation / pICln-Sm protein complex / positive regulation of mRNA splicing, via spliceosome / snRNP binding / small nuclear ribonucleoprotein complex / SMN-Sm protein complex / spliceosomal tri-snRNP complex / commitment complex / U2-type prespliceosome assembly / 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 / Prp19 complex / 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 ...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 / Matrin/U1-C-like, C2H2-type zinc finger / U1-like zinc finger / Sm-like protein Lsm7/SmG / Like-Sm (LSM) domain containing protein, LSm4/SmD1/SmD3 / 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 S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsShi, S.S. / Kuang, Z.L. / Zhao, R.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM145289 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM126157 (R.Z.) United States
CitationJournal: RNA / Year: 2024
Title: Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast.
Authors: Subbaiah Chalivendra / Shasha Shi / Xueni Li / Zhiling Kuang / Joseph Giovinazzo / Lingdi Zhang / John Rossi / Jingxin Wang / Anthony J Saviola / Robb Welty / Shiheng Liu / Katherine F Vaeth ...Authors: Subbaiah Chalivendra / Shasha Shi / Xueni Li / Zhiling Kuang / Joseph Giovinazzo / Lingdi Zhang / John Rossi / Jingxin Wang / Anthony J Saviola / Robb Welty / Shiheng Liu / Katherine F Vaeth / Z Hong Zhou / Kirk C Hansen / J Matthew Taliaferro / Rui Zhao /
Abstract: The recognition of the 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand, the mechanism and regulation of 5' ss recognition, we selectively humanized ...The recognition of the 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand, the mechanism and regulation of 5' ss recognition, we selectively humanized components of the yeast U1 (yU1) snRNP to reveal the function of these components in 5' ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yU1 snRNA and the 5' ss RNA duplex. We replaced the zinc-finger (ZnF) domain of yeast U1C (yU1C) with its human counterpart, which resulted in a cold-sensitive growth phenotype and moderate splicing defects. We next added an auxin-inducible degron to yeast Luc7 (yLuc7) protein (to mimic the lack of Luc7Ls in human U1 snRNP). We found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of Prp40 and Snu71 (two other essential yU1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yU1 snRNP, and splicing plays a major role in the regulation of mitochondrial function in yeast.
History
DepositionFeb 20, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 15, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: U1 small nuclear ribonucleoprotein 70 kDa homolog
B: U1 small nuclear ribonucleoprotein C
C: U1 small nuclear ribonucleoprotein A
D: U1 small nuclear ribonucleoprotein component PRP42
E: Pre-mRNA-processing factor 39
F: Protein NAM8
G: 56 kDa U1 small nuclear ribonucleoprotein component
H: U1 small nuclear ribonucleoprotein component SNU71
I: Protein LUC7
K: Small nuclear ribonucleoprotein-associated protein B
L: Small nuclear ribonucleoprotein Sm D1
M: Small nuclear ribonucleoprotein Sm D2
N: Small nuclear ribonucleoprotein Sm D3
O: Small nuclear ribonucleoprotein E
P: Small nuclear ribonucleoprotein F
Q: Small nuclear ribonucleoprotein G
r: ACT1 pre-mRNA
R: U1 snRNA


Theoretical massNumber of molelcules
Total (without water)725,14718
Polymers725,14718
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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

#1: Protein U1 small nuclear ribonucleoprotein 70 kDa homolog / U1-70K / U1 small nuclear ribonucleoprotein SNP1 / U1 snRNP protein SNP1


Mass: 34506.148 Da / Num. of mol.: 1 / Mutation: 0
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SNP1, YIL061C / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: Q00916
#2: Protein U1 small nuclear ribonucleoprotein C / U1 snRNP C / U1-C / U1C


Mass: 27084.949 Da / Num. of mol.: 1
Mutation: T2P,R3K,Y4F,E7D,H10D,S11T,T17S,L18P,S23T,L25C,V26S,K28R,N29K,L31K,R32E,I33N,T34V,A35K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: YHC1, YLR298C, L8003.21 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: Q05900
#3: Protein U1 small nuclear ribonucleoprotein A / U1A / Mutant U1 die protein 1


Mass: 40413.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: MUD1, YBR119W, YBR0915 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P32605
#4: Protein U1 small nuclear ribonucleoprotein component PRP42 / U1 snRNP protein PRP42 / 65 kDa snRNP protein / Pre-mRNA-processing factor 42


Mass: 65222.020 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: PRP42, MUD16, SNU65, YDR235W, YD8419.02 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: Q03776
#8: Protein U1 small nuclear ribonucleoprotein component SNU71


Mass: 5974.760 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SNU71, YGR013W / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P53207

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Protein , 5 types, 5 molecules EFGIK

#5: Protein Pre-mRNA-processing factor 39


Mass: 74834.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: PRP39, YML046W, YM9827.06 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P39682
#6: Protein Protein NAM8


Mass: 57010.840 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: NAM8, MRE2, YHR086W / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: Q00539
#7: Protein 56 kDa U1 small nuclear ribonucleoprotein component


Mass: 56575.277 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SNU56, MUD10, YDR240C / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: Q03782
#9: Protein Protein LUC7


Mass: 30245.885 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: LUC7, EPE1, EXM2, YDL087C / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: Q07508
#10: Protein Small nuclear ribonucleoprotein-associated protein B / snRNP-B / Sm protein B / SmB


Mass: 22426.990 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SMB1, YER029C / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P40018

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

#11: Protein Small nuclear ribonucleoprotein Sm D1 / Sm-D1 / snRNP core protein D1


Mass: 16296.798 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SMD1, YGR074W / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: Q02260
#12: Protein Small nuclear ribonucleoprotein Sm D2 / Sm-D2 / snRNP core protein D2


Mass: 12876.066 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SMD2, YLR275W, L9328.5 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: Q06217
#13: Protein Small nuclear ribonucleoprotein Sm D3 / Sm-D3 / snRNP core protein D3


Mass: 11240.139 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SMD3, YLR147C, L9634.6 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P43321
#14: Protein Small nuclear ribonucleoprotein E / snRNP-E / Sm protein E / SmE


Mass: 10385.098 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SME1, YOR159C / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: Q12330
#15: Protein Small nuclear ribonucleoprotein F / snRNP-F / Sm protein F / SmF


Mass: 9669.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SMX3, YPR182W, P9705.4 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P54999
#16: Protein Small nuclear ribonucleoprotein G / snRNP-G / Sm protein G / SmG


Mass: 8490.809 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SMX2, SNP2, YFL017W-A, YFL017Bw / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P40204

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RNA chain , 2 types, 2 molecules rR

#17: RNA chain ACT1 pre-mRNA


Mass: 59778.191 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Production host: Saccharomyces cerevisiae S288C (yeast)
#18: RNA chain U1 snRNA


Mass: 182114.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Production host: Saccharomyces cerevisiae S288C (yeast)

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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: U1snRNP / Type: COMPLEX / Entity ID: all / Source: NATURAL
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Buffer solutionpH: 7.9 / Details: 20 mM Hepes, pH7.9, 120 mM KCl, 2 mM EGTA
Buffer component
IDConc.NameFormulaBuffer-ID
1120 mMsodium chlorideNaCl1
220 mMHepesC8H18N2O4S1
32 mMEGTAC14H24N2O101
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample is monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: The grids were obtained with the chamber at 100% humidity, 2.5 s blotting time, -6 blotting force and 15 s wait time and flash-frozen into liquid ethane with a Vitrobot Mark IV (Thermo Fisher Scientific)

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 138997 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00830066
ELECTRON MICROSCOPYf_angle_d0.94942403
ELECTRON MICROSCOPYf_dihedral_angle_d21.1657223
ELECTRON MICROSCOPYf_chiral_restr0.0465164
ELECTRON MICROSCOPYf_plane_restr0.0064067

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