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- PDB-9no0: hPNPase RNA loading state -

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Basic information

Entry
Database: PDB / ID: 9no0
TitlehPNPase RNA loading state
Components
  • Polyribonucleotide nucleotidyltransferase 1, mitochondrial
  • RNA analog
KeywordsTRANSFERASE/RNA / RNase / Protein-RNA Complex / RNA degradation / mitochondria / phosphorolytic enzyme / TRANSFERASE-RNA complex
Function / homology
Function and homology information


RNA import into mitochondrion / mitochondrial mRNA polyadenylation / mitochondrial degradosome / mitochondrial mRNA catabolic process / positive regulation of mitochondrial RNA catabolic process / mitochondrial RNA 3'-end processing / Mitochondrial RNA degradation / positive regulation of miRNA catabolic process / mitochondrial RNA 5'-end processing / poly(G) binding ...RNA import into mitochondrion / mitochondrial mRNA polyadenylation / mitochondrial degradosome / mitochondrial mRNA catabolic process / positive regulation of mitochondrial RNA catabolic process / mitochondrial RNA 3'-end processing / Mitochondrial RNA degradation / positive regulation of miRNA catabolic process / mitochondrial RNA 5'-end processing / poly(G) binding / polyribonucleotide nucleotidyltransferase / polyribonucleotide nucleotidyltransferase activity / mitochondrial RNA catabolic process / nuclear polyadenylation-dependent mRNA catabolic process / positive regulation of mRNA catabolic process / regulation of cellular senescence / rRNA import into mitochondrion / regulation of cellular respiration / response to growth hormone / RNA catabolic process / miRNA binding / poly(U) RNA binding / protein homotrimerization / mRNA catabolic process / response to cAMP / cellular response to interferon-beta / liver regeneration / mitochondrion organization / protein homooligomerization / mitochondrial intermembrane space / mRNA processing / cellular response to oxidative stress / 3'-5'-RNA exonuclease activity / ribosome / mitochondrial matrix / endoplasmic reticulum membrane / mitochondrion / RNA binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Polyribonucleotide nucleotidyltransferase, RNA-binding domain superfamily / Polyribonucleotide nucleotidyltransferase / Polyribonucleotide nucleotidyltransferase, RNA-binding domain / Polyribonucleotide nucleotidyltransferase, RNA binding domain / Exoribonuclease, phosphorolytic domain 2 / 3' exoribonuclease family, domain 2 / Exoribonuclease, phosphorolytic domain 1 / PNPase/RNase PH domain superfamily / Exoribonuclease, PH domain 2 superfamily / 3' exoribonuclease family, domain 1 ...Polyribonucleotide nucleotidyltransferase, RNA-binding domain superfamily / Polyribonucleotide nucleotidyltransferase / Polyribonucleotide nucleotidyltransferase, RNA-binding domain / Polyribonucleotide nucleotidyltransferase, RNA binding domain / Exoribonuclease, phosphorolytic domain 2 / 3' exoribonuclease family, domain 2 / Exoribonuclease, phosphorolytic domain 1 / PNPase/RNase PH domain superfamily / Exoribonuclease, PH domain 2 superfamily / 3' exoribonuclease family, domain 1 / KH domain / K Homology domain, type 1 / Type-1 KH domain profile. / K Homology domain, type 1 superfamily / S1 domain profile. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / K Homology domain / K homology RNA-binding domain / Ribosomal protein S5 domain 2-type fold / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
PHOSPHATE ION / RNA / Polyribonucleotide nucleotidyltransferase 1, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.08 Å
AuthorsUnseld, O. / Das, H. / Hallberg, B.M.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Loop-mediated regulation and base flipping drive RNA cleavage by human mitochondrial PNPase.
Authors: Ole Unseld / Hrishikesh Das / B Martin Hällberg /
Abstract: Human polynucleotide phosphorylase (hPNPase), a trimeric exoribonuclease, is crucial for maintaining mitochondrial RNA metabolism, including the regulated degradation of RNA. Mutations in hPNPase ...Human polynucleotide phosphorylase (hPNPase), a trimeric exoribonuclease, is crucial for maintaining mitochondrial RNA metabolism, including the regulated degradation of RNA. Mutations in hPNPase have been linked to mitochondrial pathologies, underscoring its importance in mitochondrial RNA homeostasis. Despite this significance, the molecular basis of its catalytic mechanism and the structural consequences of active-site mutations remain poorly understood. We employed high-resolution electron cryo-microscopy to capture three distinct functional states of hPNPase during RNA degradation. In the loading state, flexible loops facilitate the recruitment of the substrate RNA and guide it toward the active site. During the pre-catalytic state, terminal nucleotides reorient within the active site, positioning the RNA backbone for cleavage, which is stabilized by Mg2+. Finally, the catalytic state reveals a nucleophilic attack of phosphate on the RNA backbone, mediated by key active-site residues. These results offer a clear biochemical framework for hPNPase-mediated RNA turnover, clarifying its catalytic mechanism and highlighting how active-site integrity is crucial for efficient RNA degradation.
History
DepositionMar 7, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 10, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polyribonucleotide nucleotidyltransferase 1, mitochondrial
B: Polyribonucleotide nucleotidyltransferase 1, mitochondrial
C: Polyribonucleotide nucleotidyltransferase 1, mitochondrial
D: RNA analog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)255,4197
Polymers255,1344
Non-polymers2853
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 Polyribonucleotide nucleotidyltransferase 1, mitochondrial / 3'-5' RNA exonuclease OLD35 / PNPase old-35 / Polynucleotide phosphorylase 1 / PNPase 1 / ...3'-5' RNA exonuclease OLD35 / PNPase old-35 / Polynucleotide phosphorylase 1 / PNPase 1 / Polynucleotide phosphorylase-like protein


Mass: 84417.086 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PNPT1, PNPASE / Plasmid: pDSG-103 / Cell line (production host): MEXi-293E / Production host: Homo sapiens (human)
References: UniProt: Q8TCS8, polyribonucleotide nucleotidyltransferase
#2: RNA chain RNA analog


Mass: 1883.213 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: PO4 / 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: Trimeric human PNPase bound to RNA in loading state / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.258 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: MEXi-293E / Plasmid: pDSG-103
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES1
250 mMsodium chlorideNaCl1
32 mMmagnesium chlorideMgCl21
42 mMsodium dihydrogen phosphateNaH2PO41
51 mMDTT1
SpecimenConc.: 0.38 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 60s back side and 120s front side glow discharching
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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: 165000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 20 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 58.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 20390
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6.2particle selection
2EPUimage acquisition
4cryoSPARC4.6.2CTF correction
7Coot0.9.8.95model fitting
9cryoSPARC4.6.2initial Euler assignment
10cryoSPARC4.6.2final Euler assignment
11RELION5.0-betaclassification
12cryoSPARC4.6.23D reconstruction
13PHENIX1.21.2_5419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 362937 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 77.31 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingAccession code: AF-Q8TCS8-F1 / Chain residue range: 1-783 / Source name: AlphaFold / Type: in silico model
RefinementHighest resolution: 2.08 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00714744
ELECTRON MICROSCOPYf_angle_d0.58519996
ELECTRON MICROSCOPYf_dihedral_angle_d8.3192089
ELECTRON MICROSCOPYf_chiral_restr0.0482344
ELECTRON MICROSCOPYf_plane_restr0.0052570

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