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- EMDB-14384: Mammalian Dicer in the dicing state with pre-miR-15a substrate -

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Basic information

Entry
Database: EMDB / ID: EMD-14384
TitleMammalian Dicer in the dicing state with pre-miR-15a substrate
Map data
Sample
  • Complex: Dicing state of mouse oocyte dicer with pre-miR-15a
    • Complex: 59-nt precursor of miR-15a
      • RNA: 59-nt precursor of miR-15a
    • Complex: Isoform 2 of Endoribonuclease Dicer
      • Protein or peptide: Isoform 2 of Endoribonuclease Dicer
Function / homology
Function and homology information


regulation of muscle cell apoptotic process / myoblast differentiation involved in skeletal muscle regeneration / 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 / positive regulation of endothelial cell-matrix adhesion via fibronectin ...regulation of muscle cell apoptotic process / myoblast differentiation involved in skeletal muscle regeneration / 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 / positive regulation of endothelial cell-matrix adhesion via fibronectin / regulation of enamel mineralization / olfactory bulb interneuron differentiation / positive regulation of establishment of endothelial barrier / positive regulation of hepatic stellate cell proliferation / trophectodermal cell proliferation / regulation of miRNA metabolic process / regulation of odontogenesis of dentin-containing tooth / spermatogonial cell division / regulation of RNA metabolic process / peripheral nervous system myelin formation / regulation of epithelial cell differentiation / regulation of regulatory T cell differentiation / regulation of Notch signaling pathway / spinal cord motor neuron differentiation / global gene silencing by mRNA cleavage / epidermis morphogenesis / negative regulation of Schwann cell proliferation / reproductive structure development / ribonuclease III / positive regulation of myelination / apoptotic DNA fragmentation / inner ear receptor cell development / nerve development / RISC-loading complex / meiotic spindle organization / positive regulation of Schwann cell differentiation / deoxyribonuclease I activity / RISC complex assembly / regulatory ncRNA-mediated post-transcriptional gene silencing / intestinal epithelial cell development / ribonuclease III activity / pericentric heterochromatin formation / miRNA processing / regulation of stem cell differentiation / pre-miRNA processing / siRNA processing / regulation of viral genome replication / mRNA stabilization / digestive tract development / embryonic hindlimb morphogenesis / cartilage development / RISC complex / cardiac muscle cell development / embryonic limb morphogenesis / miRNA binding / positive regulation of vascular endothelial cell proliferation / regulation of myelination / regulation of neuron differentiation / negative regulation of glial cell proliferation / hair follicle morphogenesis / stem cell population maintenance / branching morphogenesis of an epithelial tube / positive regulation of miRNA metabolic process / endoplasmic reticulum-Golgi intermediate compartment / regulation of neurogenesis / hair follicle development / positive regulation of collagen biosynthetic process / spindle assembly / postsynaptic density, intracellular component / RNA processing / spleen development / positive regulation of endothelial cell migration / helicase activity / neuron projection morphogenesis / post-embryonic development / regulation of protein phosphorylation / lung development / multicellular organism growth / cerebral cortex development / rRNA processing / gene expression / growth cone / regulation of inflammatory response / regulation of gene expression / angiogenesis / defense response to virus / cell population proliferation / regulation of cell cycle / axon / glutamatergic synapse / dendrite / positive regulation of gene expression / perinuclear region of cytoplasm / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / DNA binding / RNA binding / ATP binding / nucleus / metal ion binding
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 / Dicer dimerisation domain superfamily ...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 / Dicer dimerisation domain superfamily / 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
Endoribonuclease Dicer
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.21 Å
AuthorsZanova M / Zapletal D / Kubicek K / Stefl R / Pinkas M / Novacek J
Funding support Czech Republic, 2 items
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, 2022-
Header (metadata) releaseNov 16, 2022-
Map releaseNov 16, 2022-
UpdateNov 16, 2022-
Current statusNov 16, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14384.png

Downloads & links

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Map

FileDownload / File: emd_14384.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 317.952 Å		0.83 Å/pix.
x 384 pix.
= 317.952 Å		0.83 Å/pix.
x 384 pix.
= 317.952 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.112
Minimum - Maximum-0.14119336 - 0.29840082
Average (Standard dev.)-0.00051640836 (±0.0085361665)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 317.952 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_14384_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_14384_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Dicing state of mouse oocyte dicer with pre-miR-15a

EntireName: Dicing state of mouse oocyte dicer with pre-miR-15a
Components
  • Complex: Dicing state of mouse oocyte dicer with pre-miR-15a
    • Complex: 59-nt precursor of miR-15a
      • RNA: 59-nt precursor of miR-15a
    • Complex: Isoform 2 of Endoribonuclease Dicer
      • Protein or peptide: Isoform 2 of Endoribonuclease Dicer

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Supramolecule #1: Dicing state of mouse oocyte dicer with pre-miR-15a

SupramoleculeName: Dicing state of mouse oocyte dicer with pre-miR-15a / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: 59-nt precursor of miR-15a

SupramoleculeName: 59-nt precursor of miR-15a / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Mus musculus (house mouse)

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Supramolecule #3: Isoform 2 of Endoribonuclease Dicer

SupramoleculeName: Isoform 2 of Endoribonuclease Dicer / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM

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Macromolecule #1: 59-nt precursor of miR-15a

MacromoleculeName: 59-nt precursor of miR-15a / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 19.017279 KDa
SequenceString:
UAGCAGCACA UAAUGGUUUG UGGAUGUUGA AAAGGUGCAG GCCAUACUGU GCUGCCUCA

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Macromolecule #2: Isoform 2 of Endoribonuclease Dicer

MacromoleculeName: Isoform 2 of Endoribonuclease Dicer / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: ribonuclease III
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 200.040938 KDa
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM
SequenceString: MVWSHPQFEK GGGSGGGSGG SAWSHPQFEK GDYPYDVPDY AGTENLYFQG LVDSRDTEVY TSQPCEIVVD CGPFTDRSGL YERLLMELE AALDFINDCN VAVHSKERDS TLISKQILSD CRAVLVVLGP WCADKVAGMM VRELQKYIKH EQEELHRKFL L FTDTLLRK ...String:
MVWSHPQFEK GGGSGGGSGG SAWSHPQFEK GDYPYDVPDY AGTENLYFQG LVDSRDTEVY TSQPCEIVVD CGPFTDRSGL YERLLMELE AALDFINDCN VAVHSKERDS TLISKQILSD CRAVLVVLGP WCADKVAGMM VRELQKYIKH EQEELHRKFL L FTDTLLRK IHALCEEYFS PASLDLKYVT PKVMKLLEIL RKYKPYERQQ FESVEWYNNR NQDNYVSWSD SEDDDDDEEI EE KEKPETN FPSPFTNILC GIIFVERRYT AVVLNRLIKE AGKQDPELAY ISSNFITGHG IGKNQPRSKQ MEAEFRKQEE VLR KFRAHE TNLLIATSVV EEGVDIPKCN LVVRFDLPTE YRSYVQSKGR ARAPISNYVM LADTDKIKSF EEDLKTYKAI EKIL RNKCS KSADGAEADV HAGVDDEDAF PPYVLRPDDG GPRVTINTAI GHINRYCARL PSDPFTHLAP KCRTRELPDG TFYST LYLP INSPLRASIV GPPMDSVRLA ERVVALICCE KLHKIGELDE HLMPVGKETV KYEEELDLHD EEETSVPGRP GSTKRR QCY PKAIPECLRE SYPKPDQPCY LYVIGMVLTT PLPDELNFRR RKLYPPEDTT RCFGILTAKP IPQIPHFPVY TRSGEVT IS IELKKSGFTL SQQMLELITR LHQYIFSHIL RLEKPALEFK PTGAESAYCV LPLNVVNDSG TLDIDFKFME DIEKSEAR I GIPSTKYSKE TPFVFKLEDY QDAVIIPRYR NFDQPHRFYV ADVYTDLTPL SKFPSPEYET FAEYYKTKYN LDLTNLNQP LLDVDHTSSR LNLLTPRHLN QKGKALPLSS AEKRKAKWES LQNKQILVPE LCAIHPIPAS LWRKAVCLPS ILYRLHCLLT AEELRAQTA SDAGVGVRSL PVDFRYPNLD FGWKKSIDSK SFISSCNSSL AESDNYCKHS TTVVPEHAAH QGATRPSLEN H DQMSVNCK RLPAESPAKL QSEVSTDLTA INGLSYNKNL ANGSYDLVNR DFCQGNQLNY FKQEIPVQPT TSYPIQNLYN YE NQPKPSN ECPLLSNTYL DGNANTSTSD GSPAVSTMPA MMNAVKALKD RMDSEQSPSV GYSSRTLGPN PGLILQALTL SNA SDGFNL ERLEMLGDSF LKHAITTYLF CTYPDAHEGR LSYMRSKKVS NCNLYRLGKK KGLPSRMVVS IFDPPVNWLP PGYV VNQDK SNSEKWEKDE MTKDCLLANG KLGEACEEEE DLTWRAPKEE AEDEDDFLEY DQEHIQFIDS MLMGSGAFVR KISLS PFSA SDSAYEWKMP KKASLGSMPF ASGLEDFDYS SWDAMCYLDP SKAVEEDDFV VGFWNPSEEN CGVDTGKQSI SYDLHT EQC IADKSIADCV AALLGCYLTS CGERAAQLFL CSLGLKVLPV IKRTSREKAL DPAQENGSSQ QKSLSGSCAS PVGPRSS AG KDLEYGCLKI PPRCMFDHPD AEKTLNHLIS GFETFEKKIN YRFKNKAYLL QAFTHASYHY NTITDCYQRL EFLGDAIL D YLITKHLYED PRQHSPGVLT DLRSALVNNT IFASLAVKYD YHKYFKAVSP ELFHVIDDFV KFQLEKNEMQ GMDSELRRS EEDEEKEEDI EVPKAMGDIF ASLAGAIYMD SGMSLEVVWQ VYYPMMQPLI EKFSANVPRS PVRELLEMEP ETAKFSPAER TYDGKVRVT VEVVGKGKFK GVGRSYRIAK SAAARRALRS LKANQPQVPN SGRGENLYFQ GASDYKDHDG DYKDHDGSHH H HHHHH

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.20 mg/mL
BufferpH: 8
Component:
ConcentrationName
50.0 mMTris
100.0 mMSodium Chloride
1.0 mMDithiotreitol
2.0 mMMagnesium Chloride

Details: The buffer was always fresh in RNAse-free manner
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Described in STAR methods.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 9956 / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 230362
CTF correctionSoftware - Name: cryoSPARC (ver. v3.3.1)
Final reconstructionNumber classes used: 36 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.21 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3.1) / Number images used: 84697
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 50 / Avg.num./class: 3477 / Software - Name: cryoSPARC (ver. v3.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsInitial 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
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 451.4 / Target criteria: Correlation coefficient
Output model

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

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