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- PDB-6x46: NMR solution structure of Asterix/Gtsf1 from mouse (CHHC zinc fin... -

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Basic information

Entry
Database: PDB / ID: 6x46
TitleNMR solution structure of Asterix/Gtsf1 from mouse (CHHC zinc finger domains)
ComponentsGametocyte-specific factor 1
KeywordsRNA BINDING PROTEIN / CHHC zinc finger / RNA binding
Function / homology
Function and homology information


spermatogenesis / cell differentiation / metal ion binding / cytoplasm
Similarity search - Function
Gametocyte-specific factor 1, vertebrate / TRM13/UPF0224 family, U11-48K-like CHHC zinc finger domain / U11-48K-like CHHC zinc finger / Zinc finger CHHC U11-48K-type profile. / Zinc finger C2H2 superfamily
Similarity search - Domain/homology
Gametocyte-specific factor 1
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodSOLUTION NMR / DGSA-distance geometry simulated annealing
AuthorsIpsaro, J.J. / O'Brien, P.A. / Bhattacharya, S. / Palmer III, A.G. / Joshua-Tor, L.
Funding support United States, 7items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM097888 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM050291 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM130398 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM008281-28 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)S10RR026540 United States
National Institutes of Health/Office of the DirectorS10OD016432 United States
CitationJournal: Cell Rep / Year: 2021
Title: Asterix/Gtsf1 links tRNAs and piRNA silencing of retrotransposons.
Authors: Jonathan J Ipsaro / Paul A O'Brien / Shibani Bhattacharya / Arthur G Palmer / Leemor Joshua-Tor /
Abstract: The Piwi-interacting RNA (piRNA) pathway safeguards genomic integrity by silencing transposable elements (transposons) in the germline. While Piwi is the central piRNA factor, others including ...The Piwi-interacting RNA (piRNA) pathway safeguards genomic integrity by silencing transposable elements (transposons) in the germline. While Piwi is the central piRNA factor, others including Asterix/Gtsf1 have also been demonstrated to be critical for effective silencing. Here, using enhanced crosslinking and immunoprecipitation (eCLIP) with a custom informatic pipeline, we show that Asterix/Gtsf1 specifically binds tRNAs in cellular contexts. We determined the structure of mouse Gtsf1 by NMR spectroscopy and identified the RNA-binding interface on the protein's first zinc finger, which was corroborated by biochemical analysis as well as cryo-EM structures of Gtsf1 in complex with co-purifying tRNA. Consistent with the known dependence of long terminal repeat (LTR) retrotransposons on tRNA primers, we demonstrate that LTR retrotransposons are, in fact, preferentially de-repressed in Asterix mutants. Together, these findings link Asterix/Gtsf1, tRNAs, and LTR retrotransposon silencing and suggest that Asterix exploits tRNA dependence to identify transposon transcripts and promote piRNA silencing.
History
DepositionMay 22, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 3, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 14, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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 / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Gametocyte-specific factor 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)14,3483
Polymers14,2181
Non-polymers1312
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, The elution profile of the purified protein most closely matches with that expected of a monomer.
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 500structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Gametocyte-specific factor 1 / Protein FAM112B


Mass: 14217.607 Da / Num. of mol.: 1 / Mutation: C28S, C76S, C100S, C103S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Gtsf1, Cue110, Fam112b / Plasmid: pFL / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / Tissue (production host): ovary / References: UniProt: Q9DAN6
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic72D 1H-15N HSQC
123isotropic72D 1H-13C HSQC
132isotropic53D HNCA
142isotropic93D HN(CO)CA
152isotropic93D HNCO
162isotropic73D HN(CA)CO
172isotropic73D HN(CA)CB
182isotropic93D HN(COCA)CB
192isotropic73D (H)CCH-TOCSY
1102isotropic63D HBHA(CO)NH
1112isotropic63D H(CCO)NH
1122isotropic63D (H)C(CCO)NH
1131isotropic73D 1H-15N NOESY
1143isotropic73D 1H-13C NOESY
1153isotropic73D 1H-13C NOESY aromatic

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution10.5 mM [U-15N] Gtsf1, 90% H2O/10% D2O15N_sample90% H2O/10% D2O
solution20.8 mM [U-13C; U-15N] Gtsf1, 90% H2O/10% D2O13C_15N_sample90% H2O/10% D2O
solution30.8 mM [U-13C; U-15N] Gtsf1, 100% D2O13C_15N_D2O_sample100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.5 mMGtsf1[U-15N]1
0.8 mMGtsf1[U-13C; U-15N]2
0.8 mMGtsf1[U-13C; U-15N]3
Sample conditionsDetails: Buffer composition was 50 mM MES, pH 6.5, 200 mM NaCl, 5 mM TCEP, 2:1 stoichiometric ZnCl2, 4:1 stoichiometric MgCl2, and 0.02% azide. 0.1 mM DSS was included in samples for internal ...Details: Buffer composition was 50 mM MES, pH 6.5, 200 mM NaCl, 5 mM TCEP, 2:1 stoichiometric ZnCl2, 4:1 stoichiometric MgCl2, and 0.02% azide. 0.1 mM DSS was included in samples for internal referencing of 1H chemical shifts.
Ionic strength: 200 mM NaCl mM / Label: conditions / pH: 6.5 / Pressure: 1 atm / Temperature: 298 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker DRXBrukerDRX6005
Bruker AVANCE IIIBrukerAVANCE III7006
Bruker AVANCEBrukerAVANCE8007
Bruker AVANCEBrukerAVANCE9009

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Processing

NMR software
NameVersionDeveloperClassification
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer, Baxprocessing
SparkyLee, Tonelli, Markleypeak picking
SparkyLee, Tonelli, Markleychemical shift assignment
ARIA2.3Linge, Habeck, Rieping, Nilgesstructure calculation
ARIA2.3Linge, Habeck, Rieping, Nilgesrefinement
CNS1.2Brungerrefinement
RefinementMethod: DGSA-distance geometry simulated annealing / Software ordinal: 6
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 500 / Conformers submitted total number: 20

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