Life Sciences & Biotechnology
Title : | Live cell imaging of RNA aggregation in nucleotide repeat expansion disorders |
Area of research : | Life Sciences & Biotechnology |
Focus area : | Molecular Biology, Biomedical Imaging |
Principal Investigator : | Dr. Sourav Kumar Dey, Indian Institute Of Technology (Indian School Of Mines) Dhanbad, Jharkhand |
Timeline Start Year : | 2024 |
Timeline End Year : | 2026 |
Contact info : | souravdey@iitism.ac.in |
Equipments : | Non-refrigerated microcentrifuge
Power Supply Unit For Gel Electrophoresis
Thermal Cycler
Vertical polyacrylamide gel electrophoresis and blotting system
Refrigerated Microcentrifuge
Micropipettes |
Details
Executive Summary : | Nucleotide repeat expansion disorders, such as Huntington disease and ALS, are genetically inherited neurological disorders with mutations in tri- or hexa-nucleotide repeat sequences with high G/C content. These diseases manifest when the number of repeats is above a certain threshold value. Transcription of these diseased genes creates RNAs that can phase separate in vitro and form aberrant RNA foci in cells. Understanding how these repeat-containing toxic RNAs undergo phase separation could help create a direct link between cellular toxicity and nuclear foci formation. To image phase-separated nuclear RNA foci in live cells, the MS2-MCP-based RNA imaging system was used, which utilized an MCP-GFP fusion protein. However, the presence of high excess proteins can theoretically affect the phase separation behavior of these toxic RNAs, which are thought to be initiated by RNA-RNA interactions. Therefore, an RNA-based imaging system without any protein would be the ideal choice for imaging these nuclear RNA foci.
The project aims to develop Squash-based mRNA imaging tags for live cell imaging of toxic RNA foci. In vitro studies will be conducted to confirm that the introduction of Squash-based imaging tags does not alter the phase separation of these repeat-containing RNAs. A Tet-On inducible expression system will be used to introduce the Squash-tagged toxic mRNAs in cell lines, and the physicochemical properties of the toxic RNA foci due to various types of repeat sequences related to different diseases will be studied. Live cell imaging of toxic RNA foci based on patient-derived lymphoblastoid cell lines (LCLs) will also be developed. |
Total Budget (INR): | 29,53,500 |
Organizations involved