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

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Basic information

Entry
Database: PDB / ID: 7yyn
TitleMammalian Dicer in the dicing state with pre-miR-15a substrate
Components
  • 59-nt precursor of miR-15a
  • Isoform 2 of 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 / cardiac neural crest cell development involved in outflow tract morphogenesis / regulation of oligodendrocyte differentiation / olfactory bulb interneuron differentiation / regulation of odontogenesis of dentin-containing tooth ...regulation of muscle cell apoptotic process / MicroRNA (miRNA) biogenesis / Small interfering RNA (siRNA) biogenesis / ganglion development / hair follicle cell proliferation / zygote asymmetric cell division / cardiac neural crest cell development involved in outflow tract morphogenesis / regulation of oligodendrocyte differentiation / olfactory bulb interneuron differentiation / regulation of odontogenesis of dentin-containing tooth / trophectodermal cell proliferation / regulation of enamel mineralization / regulation of miRNA metabolic process / peripheral nervous system myelin formation / spermatogonial cell division / regulation of RNA metabolic process / regulation of epithelial cell differentiation / global gene silencing by mRNA cleavage / spinal cord motor neuron differentiation / negative regulation of Schwann cell proliferation / epidermis morphogenesis / reproductive structure development / ribonuclease III / regulation of Notch signaling pathway / myoblast differentiation involved in skeletal muscle regeneration / nerve development / regulation of regulatory T cell differentiation / positive regulation of Schwann cell differentiation / RISC-loading complex / positive regulation of myelination / meiotic spindle organization / intestinal epithelial cell development / RISC complex assembly / regulatory ncRNA-mediated post-transcriptional gene silencing / miRNA processing / ribonuclease III activity / pre-miRNA processing / pericentric heterochromatin formation / siRNA processing / regulation of stem cell differentiation / regulation of viral genome replication / RISC complex / inner ear receptor cell development / mRNA stabilization / cartilage development / embryonic limb morphogenesis / embryonic hindlimb morphogenesis / digestive tract development / positive regulation of miRNA metabolic process / cardiac muscle cell development / miRNA binding / regulation of neuron differentiation / regulation of myelination / negative regulation of glial cell proliferation / hair follicle morphogenesis / stem cell population maintenance / branching morphogenesis of an epithelial tube / hair follicle development / regulation of neurogenesis / RNA processing / postsynaptic density, intracellular component / spindle assembly / spleen development / neuron projection morphogenesis / lung development / post-embryonic development / helicase activity / cerebral cortex development / multicellular organism growth / rRNA processing / regulation of gene expression / regulation of inflammatory response / angiogenesis / defense response to virus / gene expression / 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: 6.21 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
DepositionFeb 18, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 17, 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

Downloads & links

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Assembly

Deposited unit
B: 59-nt precursor of miR-15a
A: Isoform 2 of Endoribonuclease Dicer


Theoretical massNumber of molelcules
Total (without water)219,0582
Polymers219,0582
Non-polymers00
Water00
1


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

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Components

#1: RNA chain 59-nt precursor of miR-15a


Mass: 19017.279 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mus musculus (house mouse) / References: GenBank: 66773608
#2: Protein Isoform 2 of Endoribonuclease Dicer / Double-strand-specific ribonuclease mDCR-1


Mass: 200040.938 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: Baculovirus expression vector pFastBac1-HM / References: UniProt: Q8R418, ribonuclease III

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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
IDNameTypeEntity IDParent-IDSource
1Dicing state of mouse oocyte dicer with pre-miR-15aCOMPLEXall0MULTIPLE SOURCES
259-nt precursor of miR-15aCOMPLEX#11RECOMBINANT
3Isoform 2 of Endoribonuclease DicerCOMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
13Baculovirus expression vector pFastBac1-HM274590
22Mus musculus (house mouse)10090
Buffer solutionpH: 8 / Details: The buffer was always fresh in RNAse-free manner
Buffer component
IDConc.NameBuffer-ID
150 mMTris1
2100 mMSodium Chloride1
31 mMDithiotreitol1
42 mMMagnesium 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: 1000 nm
Image recordingElectron dose: 55 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 9956

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Processing

EM software
IDNameVersionCategoryDetails
1crYOLO1.7.5particle selection
4cryoSPARCv3.3.1CTF correction
7UCSF ChimeraX1.15model fittingFit to Map
11cryoSPARCv3.3.1classification
12cryoSPARCv3.3.13D reconstruction
13ISOLDE1.1.0model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 230362
3D reconstructionResolution: 6.21 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 84697 / Algorithm: FOURIER SPACE / Num. of class averages: 36 / Symmetry type: POINT
Atomic model buildingB value: 451.4 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
Details: Initial model for mouse oocyte Dicer was calculated using AlphaFold 2.0. Initial rigid body fitting was done using Chimera. Further refinement was done using ISOLDE with distance restrains

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