In this project, we have applied new techniques for single-molecule imaging of mRNA translation to investigate how mRNAs are modulated during cellular stress conditions.
In one part of the project, we have investigated the role of stress granules in translational regulation. The function of stress granules is largely unclear, but it has been previously proposed that the recruitment of mRNAs into stress granules could be a mechanism to inhibit mRNA translation. In our work, we have observed that some mRNAs can undergo translation even after they are recruited to stress granules, and we further investigated how frequently this happens during cellular stress and recovery. We have also performed work to manipulate the formation of stress granules in cultured cells using optogenetic approaches. Additionally, we have identified and characterized new small-molecule compounds that inhibit the formation of stress granules, which will be a useful tool for research and for understanding the role of stress granules in pathological conditions.
In a second part of the project, we have investigated how specific sequence elements allow the preferential translation of stress-related mRNAs. A group of stress-related mRNAs undergoes preferential translation during stress conditions. These mRNAs usually contain special elements, such as upstream open reading frames (uORFs), which can recruit the translational machinery and affect mRNA translation via multiple mechanisms. How exactly these sequence elements facilitate the preferential translation of stress-related mRNAs is not completely understood. To address this, we have set out to dissect the translational control of ATF4 mRNA, a key stress-related mRNA that contains two uORFs. We have generated a number of cell lines that enable us to measure on single-molecule level how many ribosomes are recruited to the uORFs and to the main coding sequence of ATF4. This work has provided insight into how uORFs interact with the translation machinery to promote preferential translation of ATF4 mRNA during stress conditions. This work will be continued at the host institute.
The project results were presented at two international conferences and multiple local meetings. The transfer of knowledge was further accomplished by mentoring and supervision of junior researchers. Importantly, in the course of this project, we have also optimized the imaging techniques and generated new tools for analyzing the single-molecule imaging data. Some of the tools for image analysis have already been adopted by other researchers at the host institute. We have made these tools publicly available, sharing computational workflows (KNIME Hub, GitHub) and DNA constructs (Addgene). This will accelerate the adoption of these techniques by a wider scientific community and facilitate further research.
