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- EMDB-14854: Mammalian Dicer in the "dicing state" with pre-miR-15a substrate -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-14854
TitleMammalian Dicer in the "dicing state" with pre-miR-15a substrate
Map data
Sample
  • Complex: Dicing state of mouse somatic dicer with pre-mir-15a
    • Complex: Dicing state of mouse somatic dicer
      • Protein or peptide: Endoribonuclease Dicer
    • Complex: Pre-mir-15a
      • RNA: 59-nt precursor of miR-15a
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 / positive regulation of establishment of endothelial barrier / olfactory bulb interneuron differentiation / 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: 5.91 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
DepositionApr 27, 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_14854.png

Downloads & links

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Map

FileDownload / File: emd_14854.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.835 Å
Density
Contour LevelBy AUTHOR: 0.124
Minimum - Maximum-0.18801418 - 0.4673915
Average (Standard dev.)-0.00011640316 (±0.0143638095)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 320.63998 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened version of main EM map

Fileemd_14854_additional_1.map
AnnotationSharpened version of main EM map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_14854_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_14854_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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

EntireName: Dicing state of mouse somatic dicer with pre-mir-15a
Components
  • Complex: Dicing state of mouse somatic dicer with pre-mir-15a
    • Complex: 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: Dicing state of mouse somatic dicer with pre-mir-15a

SupramoleculeName: Dicing state of mouse somatic dicer with pre-mir-15a / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Details: TARBP2 present in the sample but its density was not observed in final 3D EM map

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Supramolecule #2: Dicing state of mouse somatic dicer

SupramoleculeName: Dicing state of mouse somatic dicer / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Supramolecule #3: Pre-mir-15a

SupramoleculeName: Pre-mir-15a / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: 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: 226.925656 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
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 CVEALLGCYL 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 IFESLAGAIY MDSGMSLEVV WQVYYPMMQP LIEKFSANVP RSPVRELLEM EPETAKFSPA ERT YDGKVR VTVEVVGKGK FKGVGRSYRI AKSAAARRAL RSLKANQPQV PNSGRGENLY FQGASDYKDH DGDYKDHDGS HHHH HHHH

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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: Mus musculus (house mouse)
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

Concentration0.2 mg/mL
BufferpH: 8
Component:
ConcentrationName
50.0 mMTris
100.0 mMSodium Chloride
1.0 mMDithiotreitol
2.0 mMCalcium 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 K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Number real images: 48253 / Average electron dose: 60.198 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: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2219694
CTF correctionSoftware - Name: cryoSPARC (ver. v3.3.1)
Final reconstructionNumber classes used: 27 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.91 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3.1) / Number images used: 64038
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 50 / Avg.num./class: 2600 / Software - Name: cryoSPARC (ver. v3.3.1)

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Atomic model buiding 1

Initial model
PDB IDChain

7yyn

chain_id: A

7yyn

chain_id: B
DetailsWe 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 520
Target criteria: Ramachandran Plot, Rotamer Analysis, Density Fit Analysis, Correlation coefficient
Output model

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

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