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- PDB-7xw2: Cryo-EM structure of human DICER-pre-miRNA in a dicing state -

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Basic information

Entry
Database: PDB / ID: 7xw2
TitleCryo-EM structure of human DICER-pre-miRNA in a dicing state
Components
  • Endoribonuclease Dicer
  • RNA (73-MER)
KeywordsGENE REGULATION/RNA / Dicer / RNaseIII / RNA-binding / micro-RNA processing / GENE REGULATION / GENE REGULATION-RNA complex
Function / homology
Function and homology information


peripheral nervous system myelin formation / global gene silencing by mRNA cleavage / pre-miRNA binding / negative regulation of Schwann cell proliferation / tRNA-derived small RNA (tsRNA or tRNA-related fragment, tRF) biogenesis / Small interfering RNA (siRNA) biogenesis / apoptotic DNA fragmentation / tRNA decay / ribonuclease III / deoxyribonuclease I activity ...peripheral nervous system myelin formation / global gene silencing by mRNA cleavage / pre-miRNA binding / negative regulation of Schwann cell proliferation / tRNA-derived small RNA (tsRNA or tRNA-related fragment, tRF) biogenesis / Small interfering RNA (siRNA) biogenesis / apoptotic DNA fragmentation / tRNA decay / ribonuclease III / deoxyribonuclease I activity / positive regulation of Schwann cell differentiation / nerve development / RISC-loading complex / positive regulation of myelination / miRNA metabolic process / RISC complex assembly / ribonuclease III activity / miRNA processing / pre-miRNA processing / siRNA processing / siRNA binding / M-decay: degradation of maternal mRNAs by maternally stored factors / Regulation of MITF-M-dependent genes involved in apoptosis / RISC complex / MicroRNA (miRNA) biogenesis / negative regulation of tumor necrosis factor production / negative regulation of tumor necrosis factor-mediated signaling pathway / neuron projection morphogenesis / RNA endonuclease activity / helicase activity / double-stranded RNA binding / protein domain specific binding / negative regulation of gene expression / perinuclear region of cytoplasm / negative regulation of transcription by RNA polymerase II / RNA binding / extracellular exosome / ATP binding / metal ion binding / nucleus / cytoplasm / cytosol
Similarity search - Function
Dicer, double-stranded RNA-binding domain / : / Dicer, partner-binding domain / Dicer, dsRNA-binding domain / : / Dicer, platform domain / Dicer dimerisation domain / Dicer dimerisation domain superfamily / Dicer dimerisation domain / Dicer double-stranded RNA-binding fold domain profile. ...Dicer, double-stranded RNA-binding domain / : / Dicer, partner-binding domain / Dicer, dsRNA-binding domain / : / Dicer, platform domain / Dicer dimerisation domain / Dicer dimerisation domain superfamily / Dicer dimerisation domain / Dicer double-stranded RNA-binding fold domain profile. / Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain / PAZ domain superfamily / PAZ / PAZ domain / PAZ domain profile. / PAZ domain / Double-stranded RNA binding motif / Ribonuclease III, endonuclease domain superfamily / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RNA / RNA (> 10) / Endoribonuclease Dicer
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsLee, H. / Roh, S.-H.
Funding support Korea, Republic Of, 1items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea) Korea, Republic Of
CitationJournal: Nature / Year: 2023
Title: Structure of the human DICER-pre-miRNA complex in a dicing state.
Authors: Young-Yoon Lee / Hansol Lee / Haedong Kim / V Narry Kim / Soung-Hun Roh /
Abstract: Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs). Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as ...Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs). Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs-unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified 'GYM motif'. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5' end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5' terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5' pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.
History
DepositionMay 26, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 8, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 22, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Endoribonuclease Dicer
C: RNA (73-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)242,3714
Polymers242,2912
Non-polymers802
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Endoribonuclease Dicer / Helicase with RNase motif / Helicase MOI


Mass: 218947.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DICER1, DICER, HERNA, KIAA0928 / Production host: Homo sapiens (human) / References: UniProt: Q9UPY3, ribonuclease III
#2: RNA chain RNA (73-MER)


Mass: 23343.783 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Protein-RNA complex of human DICER with pre-miRNACOMPLEX#1-#20MULTIPLE SOURCES
2human DICERCOMPLEX#11RECOMBINANT
3pre-miRNACOMPLEX#21SYNTHETIC
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid 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: 288 K / Details: blot force 5 and blot for 2 seconds

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 900 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 44.163 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1386301 / Symmetry type: POINT

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