Legionella effectors reveal novel signaling function for the ribosome quality control (RQC) pathway

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Abstract/Project Summary Protein synthesis is a key metabolic step in cells that is subjected to perturbations from internal stimuli or external agents that may sometimes lead to the pausing of translating ribosomes on messenger RNAs, a phenomenon defined as ribosome stalling. Interestingly, the gram-negative bacterium Legionella pneumophila (L.p.) manipulates several critical host processes such as protein synthesis and membrane transport to establish its replicative niche in an endoplasmic reticulum (ER)-like vacuole. L.p. achieves this by injecting effector proteins via its type IV secretion system, Dot/Icm. Understanding how bacteria sabotage host processes and manipulate them to their own advantage provides invaluable insights into disease and mammalian cell biology. Recent studies have highlighted that L.p secretes toxins into infected cells that can inhibit protein synthesis and induce ribosome stalling. Cells have evolved a conserved mechanism called ribosome-associated quality control (RQC) that aids in the sensing and resolution of stalled ribosomes and the maintenance of protein homeostasis. The consequences of ribosome stalling stress to cells however remain poorly understood. By challenging the cellular translation apparatus with infections of L.p. or sub-optimal doses of translation elongation inhibitors, we have determined that the sensing of stalled ribosomes in cells activates a transcriptional and translational program that leads to the upregulation of the activating transcription factor 3 (ATF3). We have determined that this reactionary program is independent from other canonical stress response pathways such as the unfolded protein response (UPR) or the integrated stress response (ISR). Strikingly, ATF3 is regulated by L.p. derived proteins SidI and SusF, a toxin-antitoxin couple. The targets of SidI and SusF in cells are undefined. Furthermore, the upregulation of ATF3 was dependent on multiple components of the RQC pathway. Moreover, our observations indicate that ATF3 messengers are selectively translated even under conditions where nascent protein synthesis is suppressed. Concomitant to the activation of the ribosome stall induced response, L.p. also recruits a pool of translationally active ribosomes to its replicative niche within cells. This proposal aims to utilize the molecular tools derived from L.p. to understand the mechanisms that link RQC to the activation of ATF3 in the nucleus. We expect that the results from the proposed set of experiments will shed light onto fundamental mechanisms that maintain the homeostasis of protein synthesis in cells and generate new tools to study and dissect the RQC pathway. We have assembled an exceptionally strong team of experts and compelling preliminary data that highlight our ability to accomplish our goals.