- EMDB-14217: Cryo-electron tomography of in vitro assembled Bruno-EGFP and osk... -
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Basic information
Entry
Database: EMDB / ID: EMD-14217
Title
Cryo-electron tomography of in vitro assembled Bruno-EGFP and oskar 3'UTR RNA condensates aged for 30 minutes followed by addition of RFP-PTB
Map data
tomogram of in vitro assembled Bruno oskar 3'UTR RNA condensates that were aged for 30 minutes followed by addition of PTB
Sample
Complex: Condensates assembled using 10 micro molar Bruno-EGFP with 100 nano molar oskar 3'UTR RNA, aged for 30min followed by addition of 2 micro molar RFP-PTB
EIPOD fellowship under Marie Sklodowska-Curie Actions COFUND
Germany
Citation
Journal: Cell / Year: 2022 Title: Liquid-to-solid phase transition of oskar ribonucleoprotein granules is essential for their function in Drosophila embryonic development. Authors: Mainak Bose / Marko Lampe / Julia Mahamid / Anne Ephrussi / Abstract: Asymmetric localization of oskar ribonucleoprotein (RNP) granules to the oocyte posterior is crucial for abdominal patterning and germline formation in the Drosophila embryo. We show that oskar RNP ...Asymmetric localization of oskar ribonucleoprotein (RNP) granules to the oocyte posterior is crucial for abdominal patterning and germline formation in the Drosophila embryo. We show that oskar RNP granules in the oocyte are condensates with solid-like physical properties. Using purified oskar RNA and scaffold proteins Bruno and Hrp48, we confirm in vitro that oskar granules undergo a liquid-to-solid phase transition. Whereas the liquid phase allows RNA incorporation, the solid phase precludes incorporation of additional RNA while allowing RNA-dependent partitioning of client proteins. Genetic modification of scaffold granule proteins or tethering the intrinsically disordered region of human fused in sarcoma (FUS) to oskar mRNA allowed modulation of granule material properties in vivo. The resulting liquid-like properties impaired oskar localization and translation with severe consequences on embryonic development. Our study reflects how physiological phase transitions shape RNA-protein condensates to regulate the localization and expression of a maternal RNA that instructs germline formation.
tomogram of in vitro assembled Bruno oskar 3'UTR RNA condensates that were aged for 30 minutes followed by addition of PTB. Tomogram was gaussian filtered with a kernel size 3.
Entire : Condensates assembled using 10 micro molar Bruno-EGFP with 100 na...
Entire
Name: Condensates assembled using 10 micro molar Bruno-EGFP with 100 nano molar oskar 3'UTR RNA, aged for 30min followed by addition of 2 micro molar RFP-PTB
Components
Complex: Condensates assembled using 10 micro molar Bruno-EGFP with 100 nano molar oskar 3'UTR RNA, aged for 30min followed by addition of 2 micro molar RFP-PTB
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Supramolecule #1: Condensates assembled using 10 micro molar Bruno-EGFP with 100 na...
Supramolecule
Name: Condensates assembled using 10 micro molar Bruno-EGFP with 100 nano molar oskar 3'UTR RNA, aged for 30min followed by addition of 2 micro molar RFP-PTB type: complex / Chimera: Yes / ID: 1 / Parent: 0
Source (natural)
Organism: Drosophila melanogaster (fruit fly)
Recombinant expression
Organism: Spodoptera (butterflies/moths)
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Experimental details
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Structure determination
Method
cryo EM
Processing
electron tomography
Aggregation state
particle
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Sample preparation
Buffer
pH: 7.5
Vitrification
Cryogen name: ETHANE-PROPANE
Details
Condensates assembled using 10 micro molar Bruno-EGFP with 100 nano molar oskar 3'UTR RNA, aged for 30min followed by addition of 2 micro molar RFP-PTB
Sectioning
Other: NO SECTIONING
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Electron microscopy
Microscope
FEI TITAN KRIOS
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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