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- PDB-7ele: Cryo-EM structure of Arabidopsis DCL1 in complex with pre-miRNA 166f -

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Entry
Database: PDB / ID: 7ele
TitleCryo-EM structure of Arabidopsis DCL1 in complex with pre-miRNA 166f
Components
  • Endoribonuclease Dicer homolog 1
  • pre-miRNA 166f
KeywordsHYDROLASE / MicroRNA / miRNA / Endonuclease / Helicase / Nuclease / RNA-binding
Function / homology
Function and homology information


ta-siRNA processing / ribonuclease III activity / Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters / helicase activity / rRNA processing / double-stranded RNA binding / DNA binding / ATP binding / metal ion binding / nucleus
Similarity search - Function
double strand RNA binding domain from DEAD END PROTEIN 1 / Ribonuclease III / 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 ...double strand RNA binding domain from DEAD END PROTEIN 1 / Ribonuclease III / 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 / Helicase/UvrB, N-terminal / Type III restriction enzyme, res subunit / Double-stranded RNA binding motif / Double-stranded RNA binding motif / Ribonuclease III, endonuclease domain superfamily / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / 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 homolog 1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsWei, X. / Ke, H. / Feng, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31970040 China
CitationJournal: Nat Plants / Year: 2021
Title: Structural basis of microRNA processing by Dicer-like 1.
Authors: Xiaobin Wei / Huanhuan Ke / Aijia Wen / Bo Gao / Jing Shi / Yu Feng /
Abstract: MicroRNAs (miRNAs) are short non-coding RNAs that inhibit the expression of target genes by directly binding to their mRNAs. In animals, pri-miRNAs are cleaved by Drosha to generate pre-miRNAs, which ...MicroRNAs (miRNAs) are short non-coding RNAs that inhibit the expression of target genes by directly binding to their mRNAs. In animals, pri-miRNAs are cleaved by Drosha to generate pre-miRNAs, which are subsequently cleaved by Dicer to generate mature miRNAs. Instead of being cleaved by two different enzymes, both cleavages in plants are performed by Dicer-like 1 (DCL1). With a similar domain architecture as human Dicer, it is mysterious how DCL1 recognizes pri-miRNAs and performs two cleavages sequentially. Here, we report the single-particle cryo-electron microscopy structures of Arabidopsis DCL1 complexed with a pri-miRNA and a pre-miRNA, respectively, in cleavage-competent states. These structures uncover the plasticity of the PAZ domain, which is critical for the recognition of both pri-miRNA and pre-miRNA. These structures suggest that the helicase module serves as an engine that transfers the substrate between two sequential cleavage events. This study lays a foundation for dissecting the regulation mechanism of miRNA biogenesis in plants and provides insights into the dicing state of human Dicer.
History
DepositionApr 9, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID
Revision 1.2Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Assembly

Deposited unit
A: Endoribonuclease Dicer homolog 1
G: pre-miRNA 166f


Theoretical massNumber of molelcules
Total (without water)242,3142
Polymers242,3142
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Endoribonuclease Dicer homolog 1 / Dicer-like protein 1 / AtDCL1 / Protein ABNORMAL SUSPENSOR 1 / Protein CARPEL FACTORY / Protein ...Dicer-like protein 1 / AtDCL1 / Protein ABNORMAL SUSPENSOR 1 / Protein CARPEL FACTORY / Protein SHORT INTEGUMENTS 1 / Protein SUSPENSOR 1


Mass: 213859.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: DCL1, ASU1, CAF SIN1, SUS1, At1g01040, T25K16.4 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q9SP32, Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters
#2: RNA chain pre-miRNA 166f


Mass: 28454.812 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Arabidopsis thaliana (thale cress) / References: GenBank: 3449316

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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: Binary complex of DCL1 in complex with pre-miRNA 166f / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD
Image recordingElectron dose: 64 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 300148 / Symmetry type: POINT

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