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

Informacje na temat projektu

3D-Xist

Identyfikator umowy o grant: 797230

DOI

10.3030/797230

Projekt został zamknięty

Data podpisania przez KE 27 Marca 2018

Data rozpoczęcia 1 Maja 2018

Data zakończenia 30 Kwietnia 2020

Finansowanie w ramach

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Koszt całkowity

€ 183 454,80

Wkład UE

€ 183 454,80

Koordynowany przez

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)

Okres sprawozdawczy: 2018-05-01 do 2020-04-30

Podsumowanie kontekstu i ogólnych celów projektu

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.

Prace wykonane od początku projektu do końca okresu sprawozdawczego oraz najważniejsze dotychczasowe rezultaty

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.

Innowacyjność oraz oczekiwany potencjalny wpływ (w tym dotychczasowe znaczenie społeczno-gospodarcze i szersze implikacje społeczne projektu)

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