In the first phase of the project, we aimed to identify a particular type of small non-coding RNAs previously suggested to play a key role in DNA double-strand break repair (Work package 1). For that, we relied on the latest sequencing technology (small RNA next-generation sequencing) allowing us to read and identify all small RNA molecules present in cells. Toward this goal, we first set up a pipeline for bioinformatics analysis and processing of large datasets generated by sequencing instruments. We performed this part of the project in collaboration with the School of Mathematics at the University. The collaboration materialized into a master’s research project creating the opportunity for a graduate student to participate in the project and train in our laboratory. Overall, we induced DNA double-strand breaks at specific locations in the genome of cells and we analysed all small RNAs produced from these locations.
The second phase of the project consisted in taking a different approach to detect directly RNA molecules using microscopy (Work package 2). For that, we used a technique for detection of individual RNA molecules (single molecule) inside cells by hybridizing RNAs to fluorescent probes (fluorescence in situ hybridization, FISH). We set up an improved variant of this technique recently developed in France at the Centre National de la Recherche Scientifique, which is known as smiFISH (single-molecule inexpensive FISH). Using this method, we observed individual RNA molecules.
We suspected that the RNA molecules of interest are likely to interact with proteins, in particular with enzymes capable of unfolding RNA molecules, which are known as RNA helicases. In the final phase of the project, we used assays for measuring specific DNA repair activities inside cells to address the role of these RNA helicases in DNA double-strand break repair (Work package 3).
Surprisingly, our results diverged from previously published reports on small non-coding RNAs and DNA repair. Our data indicates that small RNA molecules may not have as general role in DNA repair as it had been anticipated. In the same time, we were able to show that RNA helicases are involved in DNA double-strand break repair through a mechanism which is still not completely clear.
We presented our results to a small part of the community at a workshop on the role of RNAs and proteins in DNA double-strand repair, which was organized in France, 2017. This meeting gave birth to new collaborations and revealed a great complexity of RNA-dependent DNA repair mechanisms. Future work will be necessary to fully understand how RNA molecules function in DNA repair.
