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- PDB-9j8p: Cryo-EM structure of human TUT1 complexed with U6 snRNA -

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Basic information

Entry
Database: PDB / ID: 9j8p
TitleCryo-EM structure of human TUT1 complexed with U6 snRNA
Components
  • Speckle targeted PIP5K1A-regulated poly(A) polymerase
  • U6 snRNA
KeywordsTRANSFERASE / Complex / TUTase
Function / homology
Function and homology information


U6 snRNA 3'-end processing / RNA uridylyltransferase / regulation of RNA metabolic process / co-transcriptional mRNA 3'-end processing, cleavage and polyadenylation pathway / RNA 3'-end processing / RNA uridylyltransferase activity / snRNA processing / mRNA cleavage and polyadenylation specificity factor complex / polynucleotide adenylyltransferase / poly(A) RNA polymerase activity ...U6 snRNA 3'-end processing / RNA uridylyltransferase / regulation of RNA metabolic process / co-transcriptional mRNA 3'-end processing, cleavage and polyadenylation pathway / RNA 3'-end processing / RNA uridylyltransferase activity / snRNA processing / mRNA cleavage and polyadenylation specificity factor complex / polynucleotide adenylyltransferase / poly(A) RNA polymerase activity / mRNA 3'-end processing / enzyme-substrate adaptor activity / U6 snRNA binding / mRNA 3'-UTR binding / nuclear speck / nucleolus / enzyme binding / RNA binding / zinc ion binding / nucleoplasm / ATP binding / cytosol
Similarity search - Function
Star-PAP, RNA recognition motif / PAP/25A-associated / Cid1 family poly A polymerase / : / Poly(A) RNA polymerase, mitochondrial-like, central palm domain / Zinc-finger of C2H2 type / Nucleotidyltransferase superfamily / Zinc finger C2H2 superfamily / Zinc finger C2H2-type / RNA recognition motif ...Star-PAP, RNA recognition motif / PAP/25A-associated / Cid1 family poly A polymerase / : / Poly(A) RNA polymerase, mitochondrial-like, central palm domain / Zinc-finger of C2H2 type / Nucleotidyltransferase superfamily / Zinc finger C2H2 superfamily / Zinc finger C2H2-type / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / Speckle targeted PIP5K1A-regulated poly(A) polymerase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsYamashita, S. / Tomita, K.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Cryo-EM structure of human TUT1:U6 snRNA complex.
Authors: Seisuke Yamashita / Kozo Tomita /
Abstract: U6 snRNA (small nuclear ribonucleic acid) is a ribozyme that catalyzes pre-messenger RNA (pre-mRNA) splicing and undergoes epitranscriptomic modifications. After transcription, the 3'-end of U6 snRNA ...U6 snRNA (small nuclear ribonucleic acid) is a ribozyme that catalyzes pre-messenger RNA (pre-mRNA) splicing and undergoes epitranscriptomic modifications. After transcription, the 3'-end of U6 snRNA is oligo-uridylylated by the multi-domain terminal uridylyltransferase (TUTase), TUT1. The 3'- oligo-uridylylated tail of U6 snRNA is crucial for U4/U6 di-snRNP (small nuclear ribonucleoprotein) formation and pre-mRNA splicing. Here, we present the cryo-electron microscopy structure of the human TUT1:U6 snRNA complex. The AUA-rich motif between the 5'-short stem-loop and the telestem of U6 snRNA is clamped by the N-terminal zinc finger (ZF)-RNA recognition motif and the catalytic Palm of TUT1, and the telestem is gripped by the N-terminal ZF and the Fingers, positioning the 3'-end of the telestem in the catalytic pocket. The internal stem-loop in the 3'-stem-loop of U6 snRNA is anchored by the C-terminal kinase-associated 1 domain, preventing U6 snRNA from dislodging on the TUT1 surface during oligo-uridylylation. TUT1 recognizes the sequence and structural features of U6 snRNA, and holds the entire U6 snRNA body using multiple domains to ensure oligo-uridylylation. This highlights the specificity of TUT1 as a U6 snRNA-targeting TUTase.
History
DepositionAug 21, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 1, 2025Provider: repository / Type: Initial release
Revision 1.1Feb 5, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Speckle targeted PIP5K1A-regulated poly(A) polymerase
B: U6 snRNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)129,1903
Polymers129,1242
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Speckle targeted PIP5K1A-regulated poly(A) polymerase / Star-PAP / RNA-binding motif protein 21 / RNA-binding protein 21 / U6 snRNA-specific terminal ...Star-PAP / RNA-binding motif protein 21 / RNA-binding protein 21 / U6 snRNA-specific terminal uridylyltransferase 1 / U6-TUTase


Mass: 95026.133 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: hTUT1 (1-874 and expression tag of pET22b) / Source: (gene. exp.) Homo sapiens (human) / Gene: TUT1, RBM21 / Production host: Escherichia coli (E. coli)
References: UniProt: Q9H6E5, polynucleotide adenylyltransferase, RNA uridylyltransferase
#2: RNA chain U6 snRNA


Mass: 34098.270 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: Complex of human TUT1 and U6 snRNA / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen support
IDSpecimen-IDGrid materialGrid mesh size (divisions/in.)Grid type
11COPPER300Quantifoil R1.2/1.3
21GOLD300UltrAuFoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 %

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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: 1800 nm / Nominal defocus min: 800 nm
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector modelNum. of grids imagedNum. of real images
1150GATAN K3 BIOQUANTUM (6k x 4k)15903
2150GATAN K3 BIOQUANTUM (6k x 4k)19967

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 263158
Details: This map (1300050518) is a composite map prepared with 1300050515, 1300050516, and 1300050517.
Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 147.94 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0067431
ELECTRON MICROSCOPYf_angle_d0.898910582
ELECTRON MICROSCOPYf_chiral_restr0.04941268
ELECTRON MICROSCOPYf_plane_restr0.0062984
ELECTRON MICROSCOPYf_dihedral_angle_d20.70653088

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