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- PDB-9u47: Cryo-EM structure of spMETTL16 in complex with U6 snRNA_delta17 -

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Basic information

Entry
Database: PDB / ID: 9u47
TitleCryo-EM structure of spMETTL16 in complex with U6 snRNA_delta17
Components
  • U6 small nuclear RNA
  • U6 small nuclear RNA (adenine-(43)-N(6))-methyltransferase
KeywordsTRANSFERASE/RNA / m6A methyltransferase / U6 snRNA / protein-RNA complex / splicing / TRANSFERASE / TRANSFERASE-RNA complex
Function / homology
Function and homology information


snRNA (adenine-N6)-methylation / U6 snRNA m6A methyltransferase / U6 snRNA (adenine-(43)-N(6))-methyltransferase activity / rRNA base methylation / mRNA splicing, via spliceosome / nucleus / cytosol
Similarity search - Function
Methyltransferase METTL16/PsiM / RNA methyltransferase / METTL16/RlmF family / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
: / RNA / RNA (> 10) / U6 small nuclear RNA (adenine-(43)-N(6))-methyltransferase
Similarity search - Component
Biological speciesSchizosaccharomyces pombe (fission yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsJu, J. / Tomita, K.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)23H00368 Japan
CitationJournal: Nat Commun / Year: 2025
Title: Structures and mechanisms of U6 snRNA mA modification by METTL16.
Authors: Jue Ju / Kozo Tomita /
Abstract: The N-methyladenosine (mA) modification in U6 snRNA, catalyzed by METTL16 using S-adenosylmethionine (SAM) as the methyl donor, is required for efficient and accurate pre-mRNA splicing. However, the ...The N-methyladenosine (mA) modification in U6 snRNA, catalyzed by METTL16 using S-adenosylmethionine (SAM) as the methyl donor, is required for efficient and accurate pre-mRNA splicing. However, the mechanism by which METTL16 modifies U6 snRNA with mA remains elusive. Here, we present cryo-EM structures of METTL16 in complex with U6 snRNA, providing insights into the METTL16-mediated modification of U6 snRNA with mA. The structures reveal that U6 snRNA is recruited to METTL16 through specific interactions between the C-terminal kinase-associated 1 (KA-1) domain of METTL16 and the internal stem-loop (ISL) of U6 snRNA. Upon SAM binding to the catalytic pocket within the N-terminal methyltransferase domain (MTD), U6 snRNA undergoes a structural rearrangement that positions the target adenine-containing motif at the catalytic site. This conformational change is followed by an additional structural adjustment of U6 snRNA into a productive conformation, bringing the target adenosine closer to SAM within the catalytic pocket and thereby ensuring efficient mA modification. The KA-1 domain functions as a scaffold for initial substrate recognition and facilitates the subsequent dynamic methylation process within the MTD, highlighting the cooperative roles of METTL16 domains for U6 snRNA modification.
History
DepositionMar 19, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 6, 2025Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Aug 6, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Sep 24, 2025Group: Data collection / Database references / Category: citation / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: U6 small nuclear RNA
A: U6 small nuclear RNA (adenine-(43)-N(6))-methyltransferase


Theoretical massNumber of molelcules
Total (without water)74,9192
Polymers74,9192
Non-polymers00
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: RNA chain U6 small nuclear RNA


Mass: 26476.871 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Schizosaccharomyces pombe (fission yeast) / References: GenBank: 929984519
#2: Protein U6 small nuclear RNA (adenine-(43)-N(6))-methyltransferase


Mass: 48442.223 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Residues MET A -22 to SER A 0 are expression tags. MET A 1 to PHE A 23 are part of the protein sequence, based on publication (DOI: 10.1038/s41467-021-23457-6).
Source: (gene. exp.) Schizosaccharomyces pombe (fission yeast)
Strain: ED668 / Gene: SPAC27D7.08c / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Rosetta2 / References: UniProt: O42662, U6 snRNA m6A methyltransferase
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: Schizosaccharomyces pombe N6-methyladenosine methyltransferase METTL16 in complex with U6 snRNA_delta17
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Rosetta2
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 1 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6010
Image scansWidth: 4092 / Height: 5760

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6.0particle selection
2EPUimage acquisition
4cryoSPARC4.6.0CTF correction
7UCSF ChimeraX1.8model fitting
9PHENIX1.18.2_3874model refinement
10cryoSPARC4.6.0initial Euler assignment
11cryoSPARC4.6.0final Euler assignment
13cryoSPARC4.6.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4868850
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173139 / Symmetry type: POINT
Atomic model building
ID 3D fitting-IDSource nameType
11AlphaFoldin silico model
21
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 94.59 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00643531
ELECTRON MICROSCOPYf_angle_d0.89344964
ELECTRON MICROSCOPYf_chiral_restr0.0541580
ELECTRON MICROSCOPYf_plane_restr0.0063471
ELECTRON MICROSCOPYf_dihedral_angle_d13.37921464

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