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Investigating the molecular mechanisms of translational reprogramming during cellular stress

Periodic Reporting for period 1 - mRNAstress (Investigating the molecular mechanisms of translational reprogramming during cellular stress)

Reporting period: 2020-08-01 to 2022-07-31

Human cells are frequently exposed to stress conditions such as toxins, oxidative stress, nutrient deprivation or hypoxia. These conditions can inflict damage to cells and potentially lead to cell death. To minimize the damage and adapt to the stress conditions, cells respond by reducing the rate of protein synthesis and boosting various repair pathways. The ability of cells to deal with stress is crucial for longevity of the organism, and it is now clear that defects in cellular stress responses can lead to age-related neurodegenerative disorders and other diseases. Understanding the molecular mechanisms of cellular adaptation to stress is thus crucial for addressing the healthcare challenges faced by ageing populations.

More specifically, cells have evolved an adaptive signalling pathway termed the integrated stress response (ISR), which is activated by a broad range of stress signals. The activation of this pathway has three major outcomes in cells: First, global inhibition of mRNA translation leads to reduced protein synthesis and allows cells to conserve energy and nutrients. Second, a small group of stress-related mRNAs are preferentially translated, producing a specific set of proteins that are required for recovery from stress. Third, clustering of mRNA into sub-cellular structures termed stress granules, whose function is largely unclear.

The main goal of this project is to unravel the mechanisms that govern the regulation of mRNA translation during the cellular stress response. To do this, we investigate how specific sequence elements allow the preferential translation of stress-related mRNAs, and how stress granules affect mRNA translation.
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.
This project has provided important insight into the translational regulation during cellular stress, and the relationship between stress granules and translational regulation. Stress granules have been proposed as a potential therapeutic target in neurodegenerative disorders (e.g. amyotrophic lateral sclerosis) and certain types of cancer. Understanding the function of stress granules, and being able to modulate their formation, will be important for guiding the efforts towards a new class of therapeutics that modulate RNA-protein granules.
Overview of the integrated stress response pathway
Investigating the role of stress granules in translational regulation
Strategy of using single-molecule imaging to measure uORF-mediated translation