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- PDB-7slp: Cryo-EM structure of 7SK core RNP with linear RNA -

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Basic information

Entry
Database: PDB / ID: 7slp
TitleCryo-EM structure of 7SK core RNP with linear RNA
Components
  • 7SK snRNA methylphosphate capping enzyme
  • La-related protein 7
  • Linear 7SK RNA
KeywordsRNA BINDING PROTEIN/RNA / non-coding RNA / La-related protein / methylphosphate capping enzyme / transcription regulation / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


RNA 5'-gamma-phosphate methyltransferase activity / U6 2'-O-snRNA methylation / snRNA metabolic process / snRNA modification / 7SK snRNP / positive regulation of snRNA transcription by RNA polymerase II / 7SK snRNA binding / snRNA binding / box C/D sno(s)RNA 3'-end processing / positive regulation of protein localization to Cajal body ...RNA 5'-gamma-phosphate methyltransferase activity / U6 2'-O-snRNA methylation / snRNA metabolic process / snRNA modification / 7SK snRNP / positive regulation of snRNA transcription by RNA polymerase II / 7SK snRNA binding / snRNA binding / box C/D sno(s)RNA 3'-end processing / positive regulation of protein localization to Cajal body / RNA methyltransferase activity / RNA methylation / : / negative regulation of viral transcription / S-adenosylmethionine-dependent methyltransferase activity / O-methyltransferase activity / regulation of mRNA splicing, via spliceosome / negative regulation of transcription elongation by RNA polymerase II / positive regulation of G1/S transition of mitotic cell cycle / U6 snRNA binding / Transferases; Transferring one-carbon groups; Methyltransferases / RNA splicing / mRNA processing / spermatogenesis / cell differentiation / ribonucleoprotein complex / mRNA binding / negative regulation of transcription by RNA polymerase II / RNA binding / nucleoplasm / nucleus / cytosol
Similarity search - Function
LARP7, RNA recognition motif 1 / LARP7, RNA recognition motif 2 / La-related protein 7, La domain / RNA methyltransferase bin3, C-terminal / Bin3-type S-adenosyl-L-methionine binding domain / RNA methyltransferase Bin3-like / Bicoid-interacting protein 3 (Bin3) / Bin3-type S-adenosyl-L-methionine (SAM) domain profile. / RNA binding motif / Lupus La protein ...LARP7, RNA recognition motif 1 / LARP7, RNA recognition motif 2 / La-related protein 7, La domain / RNA methyltransferase bin3, C-terminal / Bin3-type S-adenosyl-L-methionine binding domain / RNA methyltransferase Bin3-like / Bicoid-interacting protein 3 (Bin3) / Bin3-type S-adenosyl-L-methionine (SAM) domain profile. / RNA binding motif / Lupus La protein / xRRM domain profile. / La protein, xRRM domain / La domain containing protein / La domain / Domain in the RNA-binding Lupus La protein; unknown function / La-type HTH domain / La-type HTH domain profile. / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Winged helix DNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily / Winged helix-like DNA-binding domain superfamily / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
S-ADENOSYL-L-HOMOCYSTEINE / RNA / RNA (> 10) / La-related protein 7 / 7SK snRNA methylphosphate capping enzyme
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsYang, Y. / Liu, S. / Zhou, Z.H. / Feigon, J.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131901 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI155170 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM071940 United States
American Heart Association20POST35210850 United States
CitationJournal: Mol Cell / Year: 2022
Title: Structural basis of RNA conformational switching in the transcriptional regulator 7SK RNP.
Authors: Yuan Yang / Shiheng Liu / Sylvain Egloff / Catherine D Eichhorn / Tanya Hadjian / James Zhen / Tamás Kiss / Z Hong Zhou / Juli Feigon /
Abstract: 7SK non-coding RNA (7SK) negatively regulates RNA polymerase II (RNA Pol II) elongation by inhibiting positive transcription elongation factor b (P-TEFb), and its ribonucleoprotein complex (RNP) is ...7SK non-coding RNA (7SK) negatively regulates RNA polymerase II (RNA Pol II) elongation by inhibiting positive transcription elongation factor b (P-TEFb), and its ribonucleoprotein complex (RNP) is hijacked by HIV-1 for viral transcription and replication. Methylphosphate capping enzyme (MePCE) and La-related protein 7 (Larp7) constitutively associate with 7SK to form a core RNP, while P-TEFb and other proteins dynamically assemble to form different complexes. Here, we present the cryo-EM structures of 7SK core RNP formed with two 7SK conformations, circular and linear, and uncover a common RNA-dependent MePCE-Larp7 complex. Together with NMR, biochemical, and cellular data, these structures reveal the mechanism of MePCE catalytic inactivation in the core RNP, unexpected interactions between Larp7 and RNA that facilitate a role as an RNP chaperone, and that MePCE-7SK-Larp7 core RNP serves as a scaffold for switching between different 7SK conformations essential for RNP assembly and regulation of P-TEFb sequestration and release.
History
DepositionOct 24, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 30, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 6, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2May 18, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 7SK snRNA methylphosphate capping enzyme
B: La-related protein 7
R: Linear 7SK RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)116,7134
Polymers116,3283
Non-polymers3841
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein 7SK snRNA methylphosphate capping enzyme / MePCE / Bicoid-interacting protein 3 homolog / Bin3 homolog


Mass: 35122.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MEPCE, BCDIN3 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: Q7L2J0, Transferases; Transferring one-carbon groups; Methyltransferases
#2: Protein La-related protein 7 / La ribonucleoprotein domain family member 7 / hLARP7 / P-TEFb-interaction protein for 7SK stability / PIP7S


Mass: 55109.129 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LARP7, HDCMA18P / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q4G0J3
#3: RNA chain Linear 7SK RNA


Mass: 26096.230 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: Chemical ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE / S-Adenosyl-L-homocysteine


Type: L-peptide linking / Mass: 384.411 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H20N6O5S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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
17SK linear core RNP with MePCE,Larp7 and linear 7SK RNACOMPLEX#1-#30MULTIPLE SOURCES
27SK snRNA methylphosphate capping enzyme, La-related protein 7COMPLEX#1-#21RECOMBINANT
3RNACOMPLEX#31SYNTHETIC
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K

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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 FIELDBright-field microscopy / Nominal magnification: 130000 X / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18rc3_3805: / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
10cryoSPARCinitial Euler assignment
11RELIONfinal Euler assignment
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 159349 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0085372
ELECTRON MICROSCOPYf_angle_d1.167450
ELECTRON MICROSCOPYf_dihedral_angle_d23.7741071
ELECTRON MICROSCOPYf_chiral_restr0.06867
ELECTRON MICROSCOPYf_plane_restr0.009809

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