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- EMDB-61237: Cryo-EM structure of human TUT1 complexed with U6 snRNA -

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Basic information

Entry
Database: EMDB / ID: EMD-61237
TitleCryo-EM structure of human TUT1 complexed with U6 snRNA
Map data
Sample
  • Complex: Complex of human TUT1 and U6 snRNA
    • Protein or peptide: Speckle targeted PIP5K1A-regulated poly(A) polymerase
    • RNA: U6 snRNA
  • Ligand: ZINC ION
KeywordsComplex / TUTase / TRANSFERASE
Function / homology
Function and homology information


U6 snRNA 3'-end processing / RNA uridylyltransferase / 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 ...U6 snRNA 3'-end processing / RNA uridylyltransferase / 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
Speckle targeted PIP5K1A-regulated poly(A) polymerase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsYamashita S / Tomita K
Funding support Japan, 1 items
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, 2024-
Header (metadata) releaseJan 1, 2025-
Map releaseJan 1, 2025-
UpdateFeb 5, 2025-
Current statusFeb 5, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61237.png

Downloads & links

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Map

FileDownload / File: emd_61237.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.25 Å/pix.
x 128 pix.
= 159.36 Å		1.25 Å/pix.
x 128 pix.
= 159.36 Å		1.25 Å/pix.
x 128 pix.
= 159.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.245 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum0.0 - 13.990384000000001
Average (Standard dev.)0.10665803 (±0.7029009)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 159.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Complex of human TUT1 and U6 snRNA

EntireName: Complex of human TUT1 and U6 snRNA
Components
  • Complex: Complex of human TUT1 and U6 snRNA
    • Protein or peptide: Speckle targeted PIP5K1A-regulated poly(A) polymerase
    • RNA: U6 snRNA
  • Ligand: ZINC ION

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Supramolecule #1: Complex of human TUT1 and U6 snRNA

SupramoleculeName: Complex of human TUT1 and U6 snRNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Speckle targeted PIP5K1A-regulated poly(A) polymerase

MacromoleculeName: Speckle targeted PIP5K1A-regulated poly(A) polymerase / type: protein_or_peptide / ID: 1 / Details: hTUT1 (1-874 and expression tag of pET22b) / Number of copies: 1 / Enantiomer: LEVO / EC number: polynucleotide adenylyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 95.026133 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAAVDSDVES LPRGGFRCCL CHVTTANRPS LDAHLGGRKH RHLVELRAAR KAQGLRSVFV SGFPRDVDSA QLSEYFLAFG PVASVVMDK DKGVFAIVEM GDVGAREAVL SQSQHSLGGH RLRVRPREQK EFQSPASKSP KGAAPDSHQL AKALAEAADV G AQMIKLVG ...String:
MAAVDSDVES LPRGGFRCCL CHVTTANRPS LDAHLGGRKH RHLVELRAAR KAQGLRSVFV SGFPRDVDSA QLSEYFLAFG PVASVVMDK DKGVFAIVEM GDVGAREAVL SQSQHSLGGH RLRVRPREQK EFQSPASKSP KGAAPDSHQL AKALAEAADV G AQMIKLVG LRELSEAERQ LRSLVVALMQ EVFTEFFPGC VVHPFGSSIN SFDVHGCDLD LFLDLGDLEE PQPVPKAPES PS LDSALAS PLDPQALACT PASPPDSQPP ASPQDSEALD FETPSSSLAP QTPDSALASE TLASPQSLPP ASPLLEDREE GDL GKASEL AETPKEEKAE GAAMLELVGS ILRGCVPGVY RVQTVPSARR PVVKFCHRPS GLHGDVSLSN RLALHNSRFL SLCS ELDGR VRPLVYTLRC WAQGRGLSGS GPLLSNYALT LLVIYFLQTR DPPVLPTVSQ LTQKAGEGEQ VEVDGWDCSF PRDAS RLEP SINVEPLSSL LAQFFSCVSC WDLRGSLLSL REGQALPVAG GLPSNLWEGL RLGPLNLQDP FDLSHNVAAN VTSRVA GRL QNCCRAAANY CRSLQYQRRS SRGRDWGLLP LLQPSSPSSL LSATPIPLPL APFTQLTAAL VQVFREALGC HIEQATK RT RSEGGGTGES SQGGTSKRLK VDGQKNCCEE GKEEQQGCAG DGGEDRVEEM VIEVGEMVQD WAMQSPGQPG DLPLTTGK H GAPGEEGQPS HAALAERGPK GHEAAQEWSQ GEAGKGASLP SSASWRCALW HRVWQGRRRA RRRLQQQTKE GAGGGAGTR AGWLATEAQV TQELKGLSGG EERPETEPLL SFVASVSPAD RMLTVTPLQD PQGLFPDLHH FLQVFLPQAI RHLKLEHHHH HH

UniProtKB: Speckle targeted PIP5K1A-regulated poly(A) polymerase

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Macromolecule #2: U6 snRNA

MacromoleculeName: U6 snRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 34.09827 KDa
SequenceString:
GUGCUCGCUU CGGCAGCACA UAUACUAAAA UUGGAACGAU ACAGAGAAGA UUAGCAUGGC CCCUGCGCAA GGAUGACACG CAAAUUCGU GAAGCGUUCC AUAUUUU

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #0 - Number grids imaged: 1 / #0 - Number real images: 5903 / #0 - Average electron dose: 50.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #1 - Number grids imaged: 1 / #1 - Number real images: 9967 / #1 - Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Image recording ID1
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF
Details: This map (1300050518) is a composite map prepared with 1300050515, 1300050516, and 1300050517.
Number images used: 263158
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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