3D structure of the long non-coding RNA Xist by complementary cryo-electron tomography and single particle cryo-electron microscopy

Informations projet

3D-Xist

N° de convention de subvention: 797230

DOI

10.3030/797230

Projet clôturé

Date de signature de la CE 27 Mars 2018

Date de début 1 Mai 2018

Date de fin 30 Avril 2020

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 183 454,80

Contribution de l’UE

€ 183 454,80

Coordonné par

THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD
United Kingdom

Periodic Reporting for period 1 - 3D-Xist (3D structure of the long non-coding RNA Xist by complementary cryo-electron tomography and single particle cryo-electron microscopy)

Période du rapport: 2018-05-01 au 2020-04-30

Résumé du contexte et des objectifs généraux du projet

Recent studies have highlighted a pivotal role for long non-coding RNAs (lncRNAs) in normal physiology and disease. As a key example, Xist lncRNA is crucial during the early development of female mammals and is involved in many diseases. To date, cellular and developmental studies have advanced our knowledge of the function of Xist, but a detailed understanding of molecular mechanism is lacking.

Therefore, the overarching aim of my proposed research has been to directly visualise Xist ribonucleoprotein particles (RNPs) in near-native cells. To this end, I have formulated the following objectives: 1) culturing and preparing fully vitrified mammalian cells expressing Xist RNAs optimised for cryo electron tomography (ET); 2) identifying region in which Xist RNPs are located in the vitrified cell; 3) targeted thinning of cryo-fixed mammalian cell nucleus containing the Xist RNP domain; 4) collecting tilt-series on the thinned lamellae; 5) reconstructing cryo-electron tomogram.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

Overview of the results from this project includes:
• Preparation of vitrified mammalian cells on TEM grids
• Identification of Xist domain using cryogenic fluorescence imaging
• Preparation of stable lamellae on the cell
• Collecting cryo-tilt-series data of the lamellae
• Reconstructing cryo-tomogram of the mammalian cell

The progress has been presented and communicated in international scientific meetings.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

This project has initiated and established a workflow of in situ structural biology in mouse embryonic stem cells (mESCs). mESCs have served as an important model to study early embryonic development in mammals. This research provides a new avenue to study mammalian embryonic development using in situ structural biology methods.

cryo electron tomogram