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- PDB-7zpi: Mammalian Dicer in the "dicing state" with pre-miR-15a substrate -

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Basic information

Entry
Database: PDB / ID: 7zpi
TitleMammalian Dicer in the "dicing state" with pre-miR-15a substrate
Components
  • 59-nt precursor of miR-15a
  • Endoribonuclease Dicer
KeywordsRNA BINDING PROTEIN / endoribonuclease / dsRNA / complex / gene silencing / post-transcriptional / catalytic complex / cytoplasm
Function / homology
Function and homology information


regulation of muscle cell apoptotic process / MicroRNA (miRNA) biogenesis / Small interfering RNA (siRNA) biogenesis / ganglion development / hair follicle cell proliferation / zygote asymmetric cell division / regulation of oligodendrocyte differentiation / cardiac neural crest cell development involved in outflow tract morphogenesis / olfactory bulb interneuron differentiation / regulation of RNA metabolic process ...regulation of muscle cell apoptotic process / MicroRNA (miRNA) biogenesis / Small interfering RNA (siRNA) biogenesis / ganglion development / hair follicle cell proliferation / zygote asymmetric cell division / regulation of oligodendrocyte differentiation / cardiac neural crest cell development involved in outflow tract morphogenesis / olfactory bulb interneuron differentiation / regulation of RNA metabolic process / trophectodermal cell proliferation / regulation of odontogenesis of dentin-containing tooth / regulation of enamel mineralization / regulation of miRNA metabolic process / spermatogonial cell division / peripheral nervous system myelin formation / regulation of epithelial cell differentiation / regulation of regulatory T cell differentiation / global gene silencing by mRNA cleavage / spinal cord motor neuron differentiation / negative regulation of Schwann cell proliferation / epidermis morphogenesis / reproductive structure development / positive regulation of myelination / regulation of Notch signaling pathway / ribonuclease III / myoblast differentiation involved in skeletal muscle regeneration / nerve development / positive regulation of Schwann cell differentiation / inner ear receptor cell development / meiotic spindle organization / RISC-loading complex / regulatory ncRNA-mediated post-transcriptional gene silencing / RISC complex assembly / intestinal epithelial cell development / miRNA processing / ribonuclease III activity / pre-miRNA processing / siRNA processing / pericentric heterochromatin formation / regulation of stem cell differentiation / regulation of viral genome replication / RISC complex / mRNA stabilization / digestive tract development / embryonic limb morphogenesis / embryonic hindlimb morphogenesis / cardiac muscle cell development / miRNA binding / positive regulation of miRNA metabolic process / cartilage development / regulation of neuron differentiation / regulation of myelination / negative regulation of glial cell proliferation / hair follicle morphogenesis / branching morphogenesis of an epithelial tube / stem cell population maintenance / regulation of neurogenesis / hair follicle development / postsynaptic density, intracellular component / RNA processing / spindle assembly / spleen development / neuron projection morphogenesis / post-embryonic development / helicase activity / lung development / multicellular organism growth / cerebral cortex development / rRNA processing / regulation of gene expression / regulation of inflammatory response / gene expression / angiogenesis / defense response to virus / cell population proliferation / regulation of cell cycle / positive regulation of gene expression / perinuclear region of cytoplasm / glutamatergic synapse / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / DNA binding / ATP binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Dicer, double-stranded RNA-binding domain / : / Dicer, partner-binding domain / Dicer, dsRNA-binding domain / Ribonuclease III / : / Dicer, platform domain / Dicer dimerisation domain / Dicer dimerisation domain superfamily / Dicer dimerisation domain ...Dicer, double-stranded RNA-binding domain / : / Dicer, partner-binding domain / Dicer, dsRNA-binding domain / Ribonuclease III / : / 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 / Helicase/UvrB, N-terminal / Type III restriction enzyme, res subunit / 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
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.91 Å
AuthorsZanova, M. / Zapletal, D. / Kubicek, K. / Stefl, R. / Pinkas, M. / Novacek, J.
Funding support Czech Republic, 2items
OrganizationGrant numberCountry
Czech Science FoundationGA22-19896S Czech Republic
Ministry of Education, Youth and Sports of the Czech RepublicLM2018127 Czech Republic
CitationJournal: Mol Cell / Year: 2022
Title: Structural and functional basis of mammalian microRNA biogenesis by Dicer.
Authors: David Zapletal / Eliska Taborska / Josef Pasulka / Radek Malik / Karel Kubicek / Martina Zanova / Christian Much / Marek Sebesta / Valeria Buccheri / Filip Horvat / Irena Jenickova / ...Authors: David Zapletal / Eliska Taborska / Josef Pasulka / Radek Malik / Karel Kubicek / Martina Zanova / Christian Much / Marek Sebesta / Valeria Buccheri / Filip Horvat / Irena Jenickova / Michaela Prochazkova / Jan Prochazka / Matyas Pinkas / Jiri Novacek / Diego F Joseph / Radislav Sedlacek / Carrie Bernecky / Dónal O'Carroll / Richard Stefl / Petr Svoboda /
Abstract: MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is ...MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer's DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer•-miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.
History
DepositionApr 27, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Endoribonuclease Dicer
B: 59-nt precursor of miR-15a


Theoretical massNumber of molelcules
Total (without water)245,9432
Polymers245,9432
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: immunoprecipitation, Pull-down experiment
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2740 Å2
ΔGint-23 kcal/mol
Surface area42460 Å2
MethodPISA

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Components

#1: Protein Endoribonuclease Dicer / Double-strand-specific ribonuclease mDCR-1


Mass: 226925.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Dicer1, Dicer, Mdcr / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q8R418, ribonuclease III
#2: RNA chain 59-nt precursor of miR-15a


Mass: 19017.279 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: from Mus musculus / Source: (synth.) Mus musculus (house mouse) / References: GenBank: 262205589

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Dicing state of mouse somatic dicer with pre-mir-15aCOMPLEXTARBP2 present in the sample but its density was not observed in final 3D EM mapall0MULTIPLE SOURCES
2Dicing state of mouse somatic dicerCOMPLEX#11RECOMBINANT
3Pre-mir-15aCOMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Mus musculus (house mouse)10090
33Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Trichoplusia ni (cabbage looper)7111
33synthetic construct (others)32630
Buffer solutionpH: 8
Details: The buffer was always prepared fresh in RNAse-free manner.
Buffer component
IDConc.NameBuffer-ID
150 mMTris1
2100 mMSodium Chloride1
31 mMDithiotreitol1
42 mMCalcium Chloride1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Described in STAR methods

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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 / Nominal defocus max: 3500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60.198 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 48253

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Processing

EM software
IDNameVersionCategoryDetails
1Topazparticle selection
4cryoSPARCv3.3.1CTF correction
7UCSF Chimera1.15model fittingFit to Map
9ISOLDE1.1.0model refinement
12cryoSPARCv3.3.1classification
13cryoSPARCv3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2219694
3D reconstructionResolution: 5.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 64038 / Algorithm: FOURIER SPACE / Num. of class averages: 27 / Symmetry type: POINT
Atomic model buildingB value: 520 / Protocol: FLEXIBLE FIT / Space: REAL
Target criteria: Ramachandran Plot, Rotamer Analysis, Density Fit Analysis, Correlation coefficient
Details: We used our previous deposition 7YYN as an initial model source. Initial local fitting was done using Chimera. ISOLDE was used for flexible fitting with torsion restraints defined for ...Details: We used our previous deposition 7YYN as an initial model source. Initial local fitting was done using Chimera. ISOLDE was used for flexible fitting with torsion restraints defined for polypeptide chain and distance restraints for polyribonucleotides.
Atomic model building

3D fitting-ID: 1 / Accession code: 7YYN / Initial refinement model-ID: 1 / PDB-ID: 7YYN

7yyn

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B

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