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- PDB-7s7c: Human Nuclear Exosome Targeting (NEXT) complex bound to RNA (subs... -

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Basic information

Entry
Database: PDB / ID: 7s7c
TitleHuman Nuclear Exosome Targeting (NEXT) complex bound to RNA (substrate 2)
Components
  • Exosome RNA helicase MTR4
  • RNA (30-MER)
  • RNA-binding protein 7
  • Zinc finger CCHC domain-containing protein 8
KeywordsRNA BINDING PROTEIN/RNA / Helicase / ATPase / RNA / Exosome / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


: / snRNA catabolic process / TRAMP complex / snRNA binding / RNA catabolic process / maturation of 5.8S rRNA / regulation of alternative mRNA splicing, via spliceosome / Major pathway of rRNA processing in the nucleolus and cytosol / pre-mRNA intronic binding / catalytic step 2 spliceosome ...: / snRNA catabolic process / TRAMP complex / snRNA binding / RNA catabolic process / maturation of 5.8S rRNA / regulation of alternative mRNA splicing, via spliceosome / Major pathway of rRNA processing in the nucleolus and cytosol / pre-mRNA intronic binding / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / 14-3-3 protein binding / meiotic cell cycle / mRNA splicing, via spliceosome / rRNA processing / RNA helicase activity / single-stranded RNA binding / nuclear body / RNA helicase / nuclear speck / DNA damage response / nucleolus / ATP hydrolysis activity / RNA binding / zinc ion binding / nucleoplasm / ATP binding / nucleus
Similarity search - Function
RBM7, RNA recognition motif / rRNA-processing arch domain / Mtr4-like, beta-barrel domain / : / Exosome RNA helicase MTR4-like, stalk / ATP-dependent RNA helicase Ski2, C-terminal / ATP-dependent RNA helicase Ski2-like / DSHCT (NUC185) domain / DSHCT / PSP, proline-rich ...RBM7, RNA recognition motif / rRNA-processing arch domain / Mtr4-like, beta-barrel domain / : / Exosome RNA helicase MTR4-like, stalk / ATP-dependent RNA helicase Ski2, C-terminal / ATP-dependent RNA helicase Ski2-like / DSHCT (NUC185) domain / DSHCT / PSP, proline-rich / PSP / proline-rich domain in spliceosome associated proteins / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / Helicase conserved C-terminal domain / RNA-binding domain superfamily / zinc finger / Zinc knuckle / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Nucleotide-binding alpha-beta plait domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RNA / RNA (> 10) / Exosome RNA helicase MTR4 / Zinc finger CCHC domain-containing protein 8 / RNA-binding protein 7
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.62 Å
AuthorsPuno, M.R. / Lima, C.D.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118080 United States
CitationJournal: Cell / Year: 2022
Title: Structural basis for RNA surveillance by the human nuclear exosome targeting (NEXT) complex.
Authors: M Rhyan Puno / Christopher D Lima /
Abstract: RNA quality control relies on co-factors and adaptors to identify and prepare substrates for degradation by ribonucleases such as the 3' to 5' ribonucleolytic RNA exosome. Here, we determined ...RNA quality control relies on co-factors and adaptors to identify and prepare substrates for degradation by ribonucleases such as the 3' to 5' ribonucleolytic RNA exosome. Here, we determined cryogenic electron microscopy structures of human nuclear exosome targeting (NEXT) complexes bound to RNA that reveal mechanistic insights to substrate recognition and early steps that precede RNA handover to the exosome. The structures illuminate ZCCHC8 as a scaffold, mediating homodimerization while embracing the MTR4 helicase and flexibly anchoring RBM7 to the helicase core. All three subunits collaborate to bind the RNA, with RBM7 and ZCCHC8 surveying sequences upstream of the 3' end to facilitate RNA capture by MTR4. ZCCHC8 obscures MTR4 surfaces important for RNA binding and extrusion as well as MPP6-dependent recruitment and docking onto the RNA exosome core, interactions that contribute to RNA surveillance by coordinating RNA capture, translocation, and extrusion from the helicase to the exosome for decay.
History
DepositionSep 15, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 15, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 22, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Exosome RNA helicase MTR4
B: Zinc finger CCHC domain-containing protein 8
C: RNA-binding protein 7
D: RNA (30-MER)
L: RNA (30-MER)
E: Exosome RNA helicase MTR4
F: Zinc finger CCHC domain-containing protein 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)402,5338
Polymers402,4677
Non-polymers651
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Exosome RNA helicase MTR4 / MTR4 / MTREX / ATP-dependent RNA helicase DOB1 / ATP-dependent RNA helicase SKIV2L2 / Superkiller ...MTR4 / MTREX / ATP-dependent RNA helicase DOB1 / ATP-dependent RNA helicase SKIV2L2 / Superkiller viralicidic activity 2-like 2 / TRAMP-like complex helicase


Mass: 118224.961 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MTREX, DOB1, KIAA0052, MTR4, SKIV2L2 / Production host: Escherichia coli (E. coli) / References: UniProt: P42285, RNA helicase
#2: Protein Zinc finger CCHC domain-containing protein 8 / TRAMP-like complex RNA-binding factor ZCCHC8


Mass: 69259.805 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ZCCHC8 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6NZY4
#3: Protein RNA-binding protein 7 / RNA-binding motif protein 7


Mass: 9462.986 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RBM7 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9Y580
#4: RNA chain RNA (30-MER)


Mass: 9017.486 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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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: human Nuclear Exosome Targeting (NEXT) complex bound to RNA (substrate 2)
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: 30 s wait time, blot for 2.5 s before plunging

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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 FIELDBright-field microscopy
Image recording
IDImaging-IDElectron dose (e/Å2)Detector modeFilm or detector modelDetails
1177.5SUPER-RESOLUTIONGATAN K2 SUMMIT (4k x 4k)Dataset 1
2166GATAN K3 (6k x 4k)Datasets 2 and 3

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Processing

EM software
IDNameCategory
2Leginonimage acquisition
3SerialEMimage acquisition
5GctfCTF correction
14RELION3D reconstruction
Image processingDetails: 3 datasets collected using Gatan K2 Summit and Gatan K3 image detectors were used for the reconstructions.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 252638
Details: A total of 252,638 gave rise to an overall map at 3.62 Angstrom resolution (FSC 0.143 cut off). A composite reconstruction (deposited main map) was generated by combining the overall map ...Details: A total of 252,638 gave rise to an overall map at 3.62 Angstrom resolution (FSC 0.143 cut off). A composite reconstruction (deposited main map) was generated by combining the overall map with focused refinement maps of MTR4 core (3.5 Angstrom FSC=0.143), MTR4 core-ZCCHC8 PSP-RBM7-RNA (3.42 Angstrom, FSC=0.143), RBM7-RNA (3.94 Angstrom, FSC=0.143), MTR4-ZCCHC8 HD/KID (3.62 Angstrom, FSC=0.143), and MTR4 KOW-ZCCHC8 HD/KID (3.34 Angstrom, FSC=0.143) regions.
Symmetry type: POINT

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