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Certain stress conditions can induce cleavage of tRNAs around the anticodon loop via the use of the ribonuclease angiogenin. The cellular factors that regulate tRNA cleavage are not well known. In this study we used normal and eIF2α phosphorylation- deficient mouse embryonic fibroblasts and applied a microarray-based methodology to identify and compare tRNA cleavage patterns in response to hypertonic stress, oxidative stress (arsenite), and treatment with recombinant angiogenin. In all three scenarios mouse embryonic fibroblasts deficient in eIF2α phosphorylation showed a higher accumulation of tRNA fragments including those derived from initiator-tRNAMet. We have shown that tRNA cleavage is regulated by the availability of angiogenin, its substrate (tRNA), the levels of the angiogenin inhibitor RNH1, and the rates of protein synthesis. These conclusions are supported by the following findings: (i) exogenous treatment with angiogenin or knockdown of RNH1 increased tRNA cleavage; (ii) tRNA fragment accumulation was higher during oxidative stress than hypertonic stress, in agreement with a dramatic decrease of RNH1 levels during oxidative stress; and (iii) a positive correlation was observed between angiogenin- mediated tRNA cleavage and global protein synthesis rates. Identification of the stress-specific tRNA cleavage mechanisms and patterns will provide insights into the role of tRNA fragments in signaling pathways and stress-related disorders [ABSTRACT FROM AUTHOR]

When cells are subjected to nutritional stress, uncharged tRNAs accumulate and activate Gcn2p phosphorylation of eukaryotic initiation factor-2 (eIF2) and the general amino acid control pathway. The Gcn2p regulatory domain homologous to histidyl-tRNA synthetases is proposed to bind to uncharged tRNA, directly contributing to activation of Gcn2p. Here we apply a microarray technology to analyze genome-wide changes in tRNA charging in yeast upon activation of Gcn2p in response to amino acid starvation and high salinity, a stress not directly linked to nutritional deficiency. This microarray technology is applicable for all eukaryotic cells. Strains were starved for histidine, leucine, or tryptophan and shown to rapidly induce Gcn2p phosphorylation of eIF2. The relative charging level of all tRNAs was measured before and after starvation, and Gcn2p activation and the intracellular levels of the starved amino acid correlate with the observed decrease in tRNA charging. Interestingly, in some cases, tRNAs not charged with the starved amino acid became deacylated more rapidly than tRNAs charged with the starved amino acid, This increase in uncharged tRNA levels occurred although the intracellular levels for these non-starved amino acids remained unchanged. Additionally, treatment of a wild-type strain with high salinity stress showed transient changes in the charging of several different tRNAs. These results suggest that Gcn2p can be activated by many different tRNA species in the cell. These results also depict a complex cellular relationship between tRNA charging, amino acid availability, and non-nutrient stress. These relationships are best revealed by simultaneous monitoring of the charging level of all tRNAs. [ABSTRACT FROM AUTHOR]