dCas9 · Dissection of the mammalian transcription termination mechanism by CRISPRi technology.
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2017-08-01 → 2019-07-31
- EU contribution
- €183,455
- Participants
- 1
- Scheme
- MSCA-IF-EF-ST
Lines connect the coordinator with its partners.
Results in brief
Dissection of the mammalian transcription termination mechanism by CRISPRi technology.
Proteins in the cell are made according to the mRNA template, synthesised by DNA-dependent RNA polymerase according to the DNA template in what is called transcription process. However the mRNA template is suitable for protein synthesis only if RNA polymerase starts and stops this synthesis, or transcription, at very specific points. While DNA of the “simpler” organisms, such as bacteria, contains well-defined nucleotide sequence signals, recognised by polymerase as start and stop signs, principles of human DNA “punctuation” are less clear. One of the proposed stop mechanisms in human genes depends on polymerase slowdown. In the dCAS9 project we directly tested if such a slowdown can work as a stop trigger for genes in the cells. To check this hypothesis, we employed a modified bacterial dCas9 protein, which binds to a specific nucleotide sequence on the DNA. This protein was positioned on the way of transcribing polymerase as a sort of roadblock. We demonstrated that such an obstacle is sufficient to induce polymerase slowdown and eventually transcription termination in a way similar to native pause-induced termination type. Importantly, this happens only after polymerase passes certain “end of mRNA” signal. We also demonstrated, that this dCAS9 system can to some extent rescue genes in cases when termination is altered by external factors, such as osmotic stress. This work allowed to check the influence of an isolated factor on termination process for the first time and expands our knowledge about its mechanism.
Data: CORDIS, © European Union
Project objective
RNA polymerase II (RNAPII) transcribes protein-coding genes in eukaryotes. The proper RNAPII transcription termination is crucial for generation of functional mRNAs, and then proteins. Termination, or stop of RNA synthesis, may occur before or after mRNA 3’-end cleavage and polyadenylation. In the last decade many details have been acquired about mRNA maturation, but still little is known about the termination per se. In this proposal I will redress this problem.One of the proposed transcription termination triggers is RNAPII elongation complex pausing downstream of mRNA maturation signal. I will test this hypothesis, using sequence-specific protein binding in the living cells.In the proposed project I will combine cutting-edge CRISPRi/dCas9 technology with classical molecular biology, high-throughput sequencing and computational biology. This approach will allow me to address my research objectives: (1) to elucidate the role of elongation complex pausing in transcription termination and (2) to characterise the induced pausing in terms of stress-resistance. Also by implementation of this project I will develop a bioengineering tool to induce elongation complex pausing.This timely approach, taking advantage of innovative technologies, will allow me to elucidate previously unknown details of RNAPII transcription termination in mammals and expand our knowledge about this process. Importantly, my approach will allow studies on the chromosomal genes in the living cells, making the results unique and valuable. Implementation of the proposed project will enhance my proficiency by improving and diversifying my skills in experimental and computational science as well as boosting my transferable skills, such as scientific communication and writing, project management and networking. This altogether will improve my competitiveness and potential on the way to independent career. Thus the proposal corresponds well with the aims of the Work Programme.
Original text from CORDIS.
Participants
- THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD · OxfordCoordinatorUnited Kingdom
Links
Data: CORDIS, © European Union