Summary Transfer RNA (tRNA) is traditionally viewed as an adaptor molecule that helps ribosomes synthesize proteins by decoding nucleotide triplets linking mRNA sequence to amino acid sequence of protein. Recent findings demonstrate that tRNAs also serve as a major source of small non-coding RNAs, so called tRNA-derived fragments (tRFs). The emerging concept in molecular biology is that these tRFs perform regulatory functions, although relatively little is known about their precise roles in cell physiology. Work from our and other laboratories has shown that in response to a variety of abiotic stresses (e.g. oxidative or nutrient stress), the vertebrate-specific ribonuclease (RNase) angiogenin (ANG) is activated to target the anticodon loops of tRNAs to produce a specific subclass of tRFs, known as tRNA-derived stress-induced RNAs (tiRNAs). Since their discovery in 2009, tiRNAs have been found to play roles in stress adaptation, cell survival and apoptosis. One of the relatively well studied roles of selected subset of tiRNAs (derived from tRNAAla and tRNACys) is inhibition of translation initiation via interference with functions of the cap-binding complex eIF4F (Ivanov et al. Mol Cell 2011). This proposal will further investigate functions of novel, previously unexplored, tiRNAs in regulation of translation. Our preliminary data suggest that alternative mechanisms of translation modulation exist that are different from the mechanisms used by tiRNAAla/Cys. In Aim1, we will dissect the molecular mechanisms of tiRNA- mediated regulation of translation. Our preliminary data strongly support a model in which multiple non- overlapping mechanisms are used by specific tiRNAs to mediate both inhibition and stimulation of mRNA translation. In Aim 2, we will identify and characterize mRNA targets of tiRNAs by employing candidate and unbiased proteomic and ribosome profiling approaches. Our preliminary data suggest that specific tiRNAs target specific pool of mRNAs thus reprogramming cellular translation. Our proposal is highly innovative and will broadly impact RNA biology. Successful completion of the proposed studies will result in characterization of novel translational control mechanisms acting during stress. Our studies are also important from a therapeutic point of view because multiple pathophysiological conditions are linked to changes in tiRNA production. Mutations affecting the RNase activity of ANG are found in patients with the neurodegenerative diseases Amyotrophic Lateral Sclerosis and Parkinson’s disease. Also, ANG is over- expressed in multiple cancers and its expression correlates with misbalanced tiRNA production, indicating a necessity to understand the function of tiRNAs. Finally, this proposal will generate many resources for the entire RNA/tRNA community and we will ensure that these resources are available for future use.

Project Narrative The tRNA cleavage pathway is an emerging mechanism of gene expression regulation based on the production of various tRNA-derived fragments. This mechanism is critical not only for survival under adverse conditions but also implicated in many human diseases such as cancer and neurodegenerative diseases. Here we propose to systematically characterize biogenesis, functions and molecular mechanisms of tRNA-derived molecules.