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

Informazioni relative al progetto

3D-Xist

ID dell’accordo di sovvenzione: 797230

DOI

10.3030/797230

Progetto chiuso

Data della firma CE 27 Marzo 2018

Data di avvio 1 Maggio 2018

Data di completamento 30 Aprile 2020

Finanziato da

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Costo totale

€ 183 454,80

Contributo UE

€ 183 454,80

Coordinato da

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)

Periodo di rendicontazione: 2018-05-01 al 2020-04-30

Sintesi del contesto e degli obiettivi generali del progetto

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.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

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.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

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