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- PDB-7zpj: Mammalian Dicer in the "pre-dicing state" with pre-miR-15a substr... -

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Basic information

Entry
Database: PDB / ID: 7zpj
TitleMammalian Dicer in the "pre-dicing state" with pre-miR-15a substrate and TARBP2 subunit
Components
  • 59-nt precursor of miR-15a
  • Endoribonuclease Dicer
  • RISC-loading complex subunit TARBP2 isoform 1
KeywordsRNA BINDING PROTEIN / endoribonuclease / dsRNA / complex / gene silencing / post-transcriptional / catalytic complex / cytoplasm / RISC-loading complex / TARBP2 RNA BINDING PROTEIN
Function / homology
Function and homology information


regulation of muscle cell apoptotic process / MicroRNA (miRNA) biogenesis / Small interfering RNA (siRNA) biogenesis / ganglion development / regulation of siRNA processing / regulation of miRNA processing / hair follicle cell proliferation / zygote asymmetric cell division / cardiac neural crest cell development involved in outflow tract morphogenesis / regulation of oligodendrocyte differentiation ...regulation of muscle cell apoptotic process / MicroRNA (miRNA) biogenesis / Small interfering RNA (siRNA) biogenesis / ganglion development / regulation of siRNA processing / regulation of miRNA processing / 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 / negative regulation of cytoplasmic pattern recognition receptor signaling pathway / regulation of odontogenesis of dentin-containing tooth / regulation of viral transcription / trophectodermal cell proliferation / regulation of enamel mineralization / regulation of miRNA metabolic process / peripheral nervous system myelin formation / negative regulation of defense response to virus by host / spermatogonial cell division / regulation of RNA metabolic process / regulation of epithelial cell differentiation / PKR-mediated signaling / global gene silencing by mRNA cleavage / pre-miRNA binding / 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 / regulation of regulatory T cell differentiation / positive regulation of Schwann cell differentiation / nerve development / 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 / skeletal muscle tissue regeneration / pre-miRNA processing / pericentric heterochromatin formation / siRNA processing / siRNA binding / regulation of stem cell differentiation / regulation of viral genome replication / RISC complex / inner ear receptor cell development / mRNA stabilization / cartilage development / embryonic hindlimb morphogenesis / embryonic limb morphogenesis / digestive tract development / positive regulation of miRNA metabolic process / cardiac muscle cell development / miRNA binding / regulation of neuron differentiation / neural precursor cell proliferation / regulation of myelination / negative regulation of glial cell proliferation / hair follicle morphogenesis / positive regulation of muscle cell differentiation / stem cell population maintenance / branching morphogenesis of an epithelial tube / spermatid development / hair follicle development / regulation of neurogenesis / single fertilization / postsynaptic density, intracellular component / RNA processing / positive regulation of viral genome replication / spindle assembly / spleen development / neuron projection morphogenesis / lung development / protein sequestering activity / positive regulation of translation / post-embryonic development / helicase activity / cerebral cortex development / multicellular organism growth / rRNA processing / double-stranded RNA binding / regulation of gene expression / regulation of inflammatory response / angiogenesis / defense response to virus / gene expression / cell population proliferation / regulation of cell cycle / nuclear body / positive regulation of gene expression / perinuclear region of cytoplasm / glutamatergic synapse / enzyme binding / negative regulation of transcription by RNA polymerase II
Similarity search - Function
RISC-loading complex subunit TRBP2 / TRBP2 , first double-stranded RNA binding domain / TRBP2 , second double-stranded RNA binding domain / TRBP2 , third double-stranded RNA binding domain / Ribonuclease III domain / Ribonuclease iii, N-terminal Endonuclease Domain; Chain A / Dicer, double-stranded RNA-binding domain / : / Dicer, partner-binding domain / Dicer, dsRNA-binding domain ...RISC-loading complex subunit TRBP2 / TRBP2 , first double-stranded RNA binding domain / TRBP2 , second double-stranded RNA binding domain / TRBP2 , third double-stranded RNA binding domain / Ribonuclease III domain / Ribonuclease iii, N-terminal Endonuclease Domain; Chain A / 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 / 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 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RISC-loading complex subunit TARBP2 / Endoribonuclease Dicer
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.81 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 1.0Dec 28, 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
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
D: RISC-loading complex subunit TARBP2 isoform 1


Theoretical massNumber of molelcules
Total (without water)285,9343
Polymers285,9343
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: immunoprecipitation, Pull-down experiment
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3720 Å2
ΔGint-20 kcal/mol
Surface area77630 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
#3: Protein RISC-loading complex subunit TARBP2 isoform 1 / Protamine-1 RNA-binding protein / PRM-1 RNA-binding protein / TAR RNA-binding protein 2


Mass: 39991.059 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Tarbp2, Prbp / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P97473

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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
1Pre-dicing state of mouse somatic dicer with pre-mir-15a and TARBP2COMPLEXall0MULTIPLE SOURCES
2Pre-dicing state of mouse somatic dicer with TARBP2COMPLEX#1, #31RECOMBINANT
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
52Spodoptera frugiperda (fall armyworm)7108
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

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1Topazparticle selection
4cryoSPARCv3.3.1CTF correction
7UCSF Chimera1.15model fittingFit to Map
11cryoSPARCv3.3.1classification
12cryoSPARCv3.3.13D reconstructionafterwards processing in DeepEMhancer
13Coot0.9.6model refinementReal Space Refine Zone
14PHENIX1.19.2-4158model refinementReal-space refinement
15ISOLDE1.1.0model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2219694
3D reconstructionResolution: 3.81 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 437518 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 189.2 / Protocol: FLEXIBLE FIT / Space: REAL
Target criteria: Ramachandran Plot, Rotamer Analysis, Density Fit Analysis, Correlation coefficient
Details: We used our previous deposition 7YYM as an initial model source. TARBP2 initial coordinates were predicted by AlphaFold. Initial local fitting was done using Chimera and then Coot's Real ...Details: We used our previous deposition 7YYM as an initial model source. TARBP2 initial coordinates were predicted by AlphaFold. Initial local fitting was done using Chimera and then Coot's Real Space Refine Zone. PHENIX Real-space refinement was used for flexible fitting. 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: 7YYM / Initial refinement model-ID: 1 / PDB-ID: 7YYM

7yym

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412551
ELECTRON MICROSCOPYf_angle_d0.84817268
ELECTRON MICROSCOPYf_dihedral_angle_d13.0194898
ELECTRON MICROSCOPYf_chiral_restr0.0532015
ELECTRON MICROSCOPYf_plane_restr0.0152000

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