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

Información del proyecto

3D-Xist

Identificador del acuerdo de subvención: 797230

DOI

10.3030/797230

Proyecto cerrado

Fecha de la firma de la CE 27 Marzo 2018

Fecha de inicio 1 Mayo 2018

Fecha de finalización 30 Abril 2020

Financiado con arreglo a

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coste total

€ 183 454,80

Aportación de la UE

€ 183 454,80

Coordinado por

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)

Período documentado: 2018-05-01 hasta 2020-04-30

Resumen del contexto y de los objetivos generales del proyecto

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.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

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.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

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