Structure and regulation of tRNA ligases involved in stress response

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RNA ligases seal RNA breaks and are key components of RNA processing pathways in response to cellular stress signals, including viral infections, the unfolded protein response in eukaryotes, and antibiotic stress in bacteria. Site-specific cleavage of intron-containing pre-tRNAs and pre-rRNAs generates 2ʹ,3ʹ-cyclic phosphate (cPRNA) and 5ʹ-OH termini. Three modes of ligating these termini have been described: (i) healing-sealing or 5ʹ-3ʹ ligation (mediated by viral T4 Pnkp-Rnl1 and yeast and plant-specific Trl1-Tpt1 enzymes), (ii) direct ligation or 3ʹ-5ʹ ligation (mediated by RtcB in archaea and metazoa) and (iii) possibly 2ʹ-5ʹ ligation (by LigT). LigT is a 2H family phosphoesterase that irreversibly ligates cPRNA/5ʹ-OH ends via 2ʹ-5ʹ linkage. However, Escherichia coli LigT was shown to preferentially exhibit cyclic phosphodiesterase (CPDase) activity and its biological function remains unclear. Incremental evidence suggests that RtcB plays a direct role during stress response in metazoan and bacteria. Functions of archaeal RtcB homologs beyond splicing of intron-containing-RNAs are not known and the archaeal stress response has not been characterized in detail. In this proposal, I aim to take advantage of the well-established genetic systems of euryarchaeal (Haloferax volcanii) and crenarchaeal (Sulfolobus acidocaldarius) model organisms to delineate the function of RtcB in the archaeal stress response. Here, comparative transcriptomes of the RtcB depletion strains and interactomes of the in-genome tagged RtcB will pinpoint its function in response to oxidative, DNA damage stress. I will customize a transcriptomics pipeline to decipher archaeal stress response components focusing on RNA processing pathways. Furthermore, I will use biochemical and structural approaches to investigate mechanisms of RtcB regulation by post-translational modifications and define the role of LigT in regulating of RtcB function. In this context, I will analyze the biologically relevant function of LigT and will explore two hypothetical pathways that would rely on LigT activity. Archaea being the closest prokaryotic relative of eukaryotes and having conserved tRNA processing enzymes, we envision deciphering the evolutionary relevance of the metazoan tRNA ligase complex. In conclusion, I aim to combine interdisciplinary approaches to characterize the archaeal stress response and to define the impact of RtcB in this process.