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- PDB-9asm: Human Drosha and DGCR8 in complex with Pri-let-7f1 -

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Basic information

Entry
Database: PDB / ID: 9asm
TitleHuman Drosha and DGCR8 in complex with Pri-let-7f1
Components
  • Isoform 4 of Drosha
  • Microprocessor complex subunit DGCR8
  • Pri-let-7f1
KeywordsRNA BINDING PROTEIN/RNA / RNAi / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


positive regulation of pre-miRNA processing / microprocessor complex / regulation of miRNA metabolic process / protein-RNA adaptor activity / DEAD/H-box RNA helicase binding / primary miRNA binding / Transcriptional Regulation by MECP2 / ribonuclease III / regulation of regulatory T cell differentiation / primary miRNA processing ...positive regulation of pre-miRNA processing / microprocessor complex / regulation of miRNA metabolic process / protein-RNA adaptor activity / DEAD/H-box RNA helicase binding / primary miRNA binding / Transcriptional Regulation by MECP2 / ribonuclease III / regulation of regulatory T cell differentiation / primary miRNA processing / miRNA metabolic process / regulation of stem cell proliferation / ribonuclease III activity / pre-miRNA processing / MicroRNA (miRNA) biogenesis / SMAD binding / R-SMAD binding / lipopolysaccharide binding / rRNA processing / double-stranded RNA binding / site of double-strand break / regulation of inflammatory response / protein-macromolecule adaptor activity / defense response to Gram-negative bacterium / defense response to Gram-positive bacterium / postsynaptic density / nuclear body / heme binding / DNA damage response / positive regulation of gene expression / nucleolus / glutamatergic synapse / protein homodimerization activity / RNA binding / nucleoplasm / metal ion binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
RNase III, double-stranded RNA binding domain, animal / Microprocessor complex subunit DGCR8 / Ribonuclease-III-like / Ribonuclease III / Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain / Double-stranded RNA binding motif ...RNase III, double-stranded RNA binding domain, animal / Microprocessor complex subunit DGCR8 / Ribonuclease-III-like / Ribonuclease III / Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain / 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 / WW domain superfamily / WW/rsp5/WWP domain profile. / Domain with 2 conserved Trp (W) residues / WW domain
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RNA (> 100) / Microprocessor complex subunit DGCR8 / Ribonuclease 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsGarg, A. / Joshua-Tor, L.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 GM114147 United States
CitationJournal: Mol Cell / Year: 2024
Title: The structural landscape of Microprocessor-mediated processing of pri-let-7 miRNAs.
Authors: Ankur Garg / Renfu Shang / Todor Cvetanovic / Eric C Lai / Leemor Joshua-Tor /
Abstract: MicroRNA (miRNA) biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri- ...MicroRNA (miRNA) biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri-miRNA sequence motifs affect MP recognition, fidelity, and efficiency. Here, we performed cryoelectron microscopy (cryo-EM) and biochemical studies of several let-7 family pri-miRNAs in complex with human MP. We show that MP has the structural plasticity to accommodate a range of pri-miRNAs. These structures revealed key features of the 5' UG sequence motif, more comprehensively represented as the "flipped U with paired N" (fUN) motif. Our analysis explains how cleavage of class-II pri-let-7 members harboring a bulged nucleotide generates a non-canonical precursor with a 1-nt 3' overhang. Finally, the MP-SRSF3-pri-let-7f1 structure reveals how SRSF3 contributes to MP fidelity by interacting with the CNNC motif and Drosha's Piwi/Argonaute/Zwille (PAZ)-like domain. Overall, this study sheds light on the mechanisms for flexible recognition, accurate cleavage, and regulated processing of different pri-miRNAs by MP.
History
DepositionFeb 26, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Isoform 4 of Drosha
B: Microprocessor complex subunit DGCR8
C: Microprocessor complex subunit DGCR8
E: Pri-let-7f1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)372,0688
Polymers371,8584
Non-polymers2114
Water362
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 3 molecules ABC

#1: Protein Isoform 4 of Drosha / Ribonuclease 3 / Ribonuclease III / RNase III / p241


Mass: 155574.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DROSHA, RN3, RNASE3L, RNASEN / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9NRR4, ribonuclease III
#2: Protein Microprocessor complex subunit DGCR8 / DiGeorge syndrome critical region 8


Mass: 86171.203 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DGCR8, C22orf12, DGCRK6, LP4941 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8WYQ5

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RNA chain , 1 types, 1 molecules E

#3: RNA chain Pri-let-7f1


Mass: 43940.816 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) / References: GenBank: 15212042

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Non-polymers , 3 types, 6 molecules

#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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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
1RNAi_protein_f1COMPLEX#1-#30MULTIPLE SOURCES
2DR/DGCOMPLEX#1-#21RECOMBINANT
3RNA_f1COMPLEX#31RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera frugiperda (fall armyworm)7108
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: 2200 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 77.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Warp1.0.9particle selection
2PHENIX1.20_4459:model refinement
5Warp1.0.9CTF correction
10cryoSPARC3.0.2initial Euler assignment
11cryoSPARC3.0.2final Euler assignment
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 378162 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312103
ELECTRON MICROSCOPYf_angle_d0.48916799
ELECTRON MICROSCOPYf_dihedral_angle_d12.4342422
ELECTRON MICROSCOPYf_chiral_restr0.0361902
ELECTRON MICROSCOPYf_plane_restr0.0041820

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