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- EMDB-14383: Mammalian Dicer in the "pre-dicing state" with pre-miR-15a substrate -

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Basic information

Entry
Database: EMDB / ID: EMD-14383
TitleMammalian Dicer in the "pre-dicing state" with pre-miR-15a substrate
Map data
Sample
  • Complex: Pre-dicing state of mouse somatic dicer with pre-mir-15a
    • Complex: Pre-dicing state of mouse somatic dicer
      • Protein or peptide: Endoribonuclease Dicer
    • Complex: pre-mir-15a
      • RNA: 59-nt precursor of miR-15a
Keywordsendoribonuclease / dsRNA / complex / gene silencing / post-transcriptional / catalytic complex / cytoplasm / RNA BINDING PROTEIN
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 / olfactory bulb interneuron differentiation / regulation of RNA metabolic process / regulation of enamel mineralization / positive regulation of establishment of endothelial barrier / positive regulation of hepatic stellate cell proliferation / trophectodermal cell proliferation / regulation of odontogenesis of dentin-containing tooth / epidermis morphogenesis / spermatogonial cell division / regulation of miRNA metabolic process / peripheral nervous system myelin formation / reproductive structure development / regulation of epithelial cell differentiation / global gene silencing by mRNA cleavage / regulation of Notch signaling pathway / spinal cord motor neuron differentiation / regulation of regulatory T cell differentiation / negative regulation of Schwann cell proliferation / apoptotic DNA fragmentation / positive regulation of myelination / inner ear receptor cell development / ribonuclease III / meiotic spindle organization / deoxyribonuclease I activity / positive regulation of Schwann cell differentiation / nerve development / RISC-loading complex / RISC complex assembly / regulatory ncRNA-mediated post-transcriptional gene silencing / intestinal epithelial cell development / ribonuclease III activity / miRNA processing / pericentric heterochromatin formation / pre-miRNA processing / siRNA processing / digestive tract development / regulation of stem cell differentiation / regulation of viral genome replication / RISC complex / mRNA stabilization / embryonic hindlimb morphogenesis / cartilage development / cardiac muscle cell development / embryonic limb morphogenesis / miRNA binding / positive regulation of miRNA metabolic process / positive regulation of vascular endothelial cell proliferation / regulation of neuron differentiation / hair follicle morphogenesis / regulation of myelination / negative regulation of glial cell proliferation / branching morphogenesis of an epithelial tube / stem cell population maintenance / endoplasmic reticulum-Golgi intermediate compartment / regulation of neurogenesis / postsynaptic density, intracellular component / hair follicle development / positive regulation of collagen biosynthetic process / spindle assembly / RNA processing / spleen development / positive regulation of endothelial cell migration / post-embryonic development / neuron projection morphogenesis / helicase activity / lung development / regulation of protein phosphorylation / multicellular organism growth / cerebral cortex development / rRNA processing / regulation of inflammatory response / gene expression / growth cone / regulation of gene expression / defense response to virus / angiogenesis / 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 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
Endoribonuclease Dicer
Similarity search - Component
Biological speciesMus musculus (house mouse) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.19 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-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14383.png

Downloads & links

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Map

FileDownload / File: emd_14383.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
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: 0.113
Minimum - Maximum-0.019333707 - 1.7928202
Average (Standard dev.)0.0012280807 (±0.025896171)
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: half map B

Fileemd_14383_half_map_1.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_14383_half_map_2.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Pre-dicing state of mouse somatic dicer with pre-mir-15a

EntireName: Pre-dicing state of mouse somatic dicer with pre-mir-15a
Components
  • Complex: Pre-dicing state of mouse somatic dicer with pre-mir-15a
    • Complex: Pre-dicing state of mouse somatic dicer
      • Protein or peptide: Endoribonuclease Dicer
    • Complex: pre-mir-15a
      • RNA: 59-nt precursor of miR-15a

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Supramolecule #1: Pre-dicing state of mouse somatic dicer with pre-mir-15a

SupramoleculeName: Pre-dicing state of mouse somatic dicer with pre-mir-15a
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Pre-dicing state of mouse somatic dicer

SupramoleculeName: Pre-dicing state of mouse somatic dicer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: pre-mir-15a

SupramoleculeName: pre-mir-15a / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Endoribonuclease Dicer

MacromoleculeName: Endoribonuclease Dicer / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: ribonuclease III
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 226.809594 KDa
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM
SequenceString: MVWSHPQFEK GGGSGGGSGG SAWSHPQFEK GDYPYDVPDY AGTENLYFQG LVDMKSPALQ PLSMAGLQLM TPASSPMGPF FGLPWQQEA IHDNIYTPRK YQVELLEAAL DHNTIVCLNT GSGKTFIAVL LTKELAHQIR GDLNPHAKRT VFLVNSANQV A QQVSAVRT ...String:
MVWSHPQFEK GGGSGGGSGG SAWSHPQFEK GDYPYDVPDY AGTENLYFQG LVDMKSPALQ PLSMAGLQLM TPASSPMGPF FGLPWQQEA IHDNIYTPRK YQVELLEAAL DHNTIVCLNT GSGKTFIAVL LTKELAHQIR GDLNPHAKRT VFLVNSANQV A QQVSAVRT HSDLKVGEYS DLEVNASWTK ERWSQEFTKH QVLIMTCYVA LTVLKNGYLS LSDINLLVFD ECHLAILDHP YR EIMKLCE SCPSCPRILG LTASILNGKC DPEELEEKIQ KLERILRSDA ETATDLVVLD RYTSQPCEIV VDCGPFTDRS GLY ERLLME LEAALDFIND CNVAVHSKER DSTLISKQIL SDCRAVLVVL GPWCADKVAG MMVRELQKYI KHEQEELHRK FLLF TDTLL RKIHALCEEY FSPASLDLKY VTPKVMKLLE ILRKYKPYER QQFESVEWYN NRNQDNYVSW SDSEDDDDDE EIEEK EKPE TNFPSPFTNI LCGIIFVERR YTAVVLNRLI KEAGKQDPEL AYISSNFITG HGIGKNQPRS KQMEAEFRKQ EEVLRK FRA HETNLLIATS VVEEGVDIPK CNLVVRFDLP TEYRSYVQSK GRARAPISNY VMLADTDKIK SFEEDLKTYK AIEKILR NK CSKSADGAEA DVHAGVDDED AFPPYVLRPD DGGPRVTINT AIGHINRYCA RLPSDPFTHL APKCRTRELP DGTFYSTL Y LPINSPLRAS IVGPPMDSVR LAERVVALIC CEKLHKIGEL DEHLMPVGKE TVKYEEELDL HDEEETSVPG RPGSTKRRQ CYPKAIPECL RESYPKPDQP CYLYVIGMVL TTPLPDELNF RRRKLYPPED TTRCFGILTA KPIPQIPHFP VYTRSGEVTI SIELKKSGF TLSQQMLELI TRLHQYIFSH ILRLEKPALE FKPTGAESAY CVLPLNVVND SGTLDIDFKF MEDIEKSEAR I GIPSTKYS KETPFVFKLE DYQDAVIIPR YRNFDQPHRF YVADVYTDLT PLSKFPSPEY ETFAEYYKTK YNLDLTNLNQ PL LDVDHTS SRLNLLTPRH LNQKGKALPL SSAEKRKAKW ESLQNKQILV PELCAIHPIP ASLWRKAVCL PSILYRLHCL LTA EELRAQ TASDAGVGVR SLPVDFRYPN LDFGWKKSID SKSFISSCNS SLAESDNYCK HSTTVVPEHA AHQGATRPSL ENHD QMSVN CKRLPAESPA KLQSEVSTDL TAINGLSYNK NLANGSYDLV NRDFCQGNQL NYFKQEIPVQ PTTSYPIQNL YNYEN QPKP SNECPLLSNT YLDGNANTST SDGSPAVSTM PAMMNAVKAL KDRMDSEQSP SVGYSSRTLG PNPGLILQAL TLSNAS DGF NLERLEMLGD SFLKHAITTY LFCTYPDAHE GRLSYMRSKK VSNCNLYRLG KKKGLPSRMV VSIFDPPVNW LPPGYVV NQ DKSNSEKWEK DEMTKDCLLA NGKLGEACEE EEDLTWRAPK EEAEDEDDFL EYDQEHIQFI DSMLMGSGAF VRKISLSP F SASDSAYEWK MPKKASLGSM PFASGLEDFD YSSWDAMCYL DPSKAVEEDD FVVGFWNPSE ENCGVDTGKQ SISYDLHTE QCIADKSIAD CVAALLGCYL TSCGERAAQL FLCSLGLKVL PVIKRTSREK ALDPAQENGS SQQKSLSGSC ASPVGPRSSA GKDLEYGCL KIPPRCMFDH PDAEKTLNHL ISGFETFEKK INYRFKNKAY LLQAFTHASY HYNTITDCYQ RLEFLGDAIL D YLITKHLY EDPRQHSPGV LTDLRSALVN NTIFASLAVK YDYHKYFKAV SPELFHVIDD FVKFQLEKNE MQGMDSELRR SE EDEEKEE DIEVPKAMGD IFASLAGAIY MDSGMSLEVV WQVYYPMMQP LIEKFSANVP RSPVRELLEM EPETAKFSPA ERT YDGKVR VTVEVVGKGK FKGVGRSYRI AKSAAARRAL RSLKANQPQV PNSGRGENLY FQGASDYKDH DGDYKDHDGS HHHH HHHH

UniProtKB: Endoribonuclease Dicer

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

MacromoleculeName: 59-nt precursor of miR-15a / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 19.017279 KDa
SequenceString:
UAGCAGCACA UAAUGGUUUG UGGAUGUUGA AAAGGUGCAG GCCAUACUGU GCUGCCUCA

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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 mMMagensium Chloride

Details: The buffer was always prepared 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: 16601 / 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: 1212966
Startup modelType of model: NONE
Final reconstructionNumber classes used: 103 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.19 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3.1) / Number images used: 154665
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 160 / Avg.num./class: 1020 / Software - Name: cryoSPARC (ver. v3.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsEndoribonuclease mouse Dicer from AlphaFold database was used as initial model. Initial local fitting was done using Chimera and then Coot's Real Space Refine Zone.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 134.8
Target criteria: Ramachandran Plot, Rotamer Analysis, Density Analysis
Output model

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

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Atomic model buiding 2

DetailsPHENIX Real-space refinement was used for flexible fitting.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 134.8 / Target criteria: Correlation coefficient
Output model

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

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Atomic model buiding 3

DetailsISOLDE was used for flexible fitting with torsion restraints defined for polypeptide and distance restraints for polyribonucleotide chain.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 134.8
Target criteria: Ramachandran Plot, Rotamer Analysis, Clash Score
Output model

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

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