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

Project Information

3D-Xist

Grant agreement ID: 797230

DOI

10.3030/797230

Project closed

EC signature date 27 March 2018

Start date 1 May 2018

End date 30 April 2020

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 183 454,80

EU contribution

€ 183 454,80

Coordinated by

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)

Reporting period: 2018-05-01 to 2020-04-30

Summary of the context and overall objectives of the project

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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

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