Upper panel: Uncapped polyA-biotinylated transcript encoding luciferase was translated in RRL supplemented with U2OS extract in the presence of the indicated control RNAs or tiRNAs. (Haussecker et al., 2010; Lee et al., 2009). Stress-induced tRNA-derived ~30 and ~40 nucleotide ncRNAs (designated 5- and 3-tiRNAs, respectively) are products of secreted ribonucleases (RNY1 in yeast (Thompson and Parker, 2009b) and angiogenin in human (Fu et al., 2009; Yamasaki et al., 2009)) that cleave within the anticodon loops of mature tRNAs AM 694 AM 694 (Thompson and Parker, 2009a). The phenomenon of stress-induced tRNA cleavage has been described in nutrient-deprived (Lee and Collins, 2005), (Haiser et al., 2008), and (Garcia-Silva et al.), as well as in serum-deprived (Li et al., 2008), spore-forming (Jochl et al., 2008), phosphate-depleted (Hsieh et al., 2010), and oxidatively-stressed (Thompson et al., 2008), and (Thompson et al., 2008; Yamasaki et al., 2009). Several observations suggest that stress-induced tRNA fragments can directly inhibit protein synthesis: 1) tRNA fragments found in the phloem sap of pumpkin plants inhibit translation in wheat germ extracts (Zhang et al., 2009), 2) transfection of 5-, but not 3-, tRNA fragments inhibit global translation in human U2OS cells (Yamasaki et al., 2009), and 3) transfection of 5-, but not 3-, tRNA fragments AM 694 trigger the assembly of stress granules, cytoplasmic foci that are induced by inhibitors of translation initiation (Emara et al., 2010). The finding that angiogenin contributes AM 694 to stress-induced translational repression (Yamasaki et al., 2009) suggests that tiRNAs help to reprogram protein translation during stress. Although the mechanism by which tiRNAs inhibit protein synthesis is not known, their ability to induce the assembly of stress granules (Emara et al., 2010) suggests that they target the translation initiation machinery. Stress-induced translational repression is achieved by inhibiting the assembly of distinct pre-initiation complexes that combine to form the 48S initiation complex (Yamasaki and Anderson, 2008). Assembly of the 43S pre-initiation complex is definitely inhibited by stress-activated kinases that phosphorylate eIF2, a component of the eIF2:GTP:tRNAMet ternary complex. Assembly of the eIF4F pre-initiation complex is definitely inhibited by stress-induced inactivation of the PI3K-mTOR pathway that phosphorylates 4E-BP1 to liberate the cap-binding protein eIF4E (Sonenberg and Hinnebusch, 2009). We have found that tiRNAs contribute to the displacement of eIF4G/A from capped and uncapped mRNA and eIF4E/G/A (eIF4F) from your m7G cap. Moreover, we have implicated YB-1, a translational repressor known to displace eIF4G from RNA and eIF4E/G/A from your m7G cap (Evdokimova et al., 2001; Nekrasov et al., 2003), in this process. We propose that tiRNAs cooperate with YB-1 to re-program translation in stressed cells. RESULTS Endogenous 5-, but not 3-, tiRNAs inhibit translation We previously showed that transfection of natural 5-, but not 3-, tiRNAs inhibits global translation in U2OS Rabbit Polyclonal to GSC2 cells (Yamasaki et al., 2009). Similarly, stress-induced tRNA fragments found in the phloem sap of pumpkin vegetation (in wheat germ components (Zhang et al., 2009). We found that natural 5-, but not 3-, tiRNAs gel purified from angiogenin-treated U2OS cells significantly inhibit translation of uncapped luciferase transcripts in rabbit reticulocyte lysates (RRL) (Number 1A). Northern blotting analysis confirmed that luciferase transcripts are not degraded under these conditions (Number 1A, NB). Open in a separate window Number 1 5-tiRNAs inhibit translation of mRNA reporters luciferase mRNA (Promega) was translated in RRL in the presence of control RNA blend (Ctrl-1-2-3; derived from piRNAs or random sequences), natural 5-tiRNAs (Nat 5end) or natural 3-tiRNAs (Nat 3end). Luciferase manifestation is relative to AM 694 that in the absence of RNA (No RNA =100%). Means and standard deviations are from three self-employed experiments (*-p = 0.01C0.02 (Table S1), College students t-test, n=3). NB: Northern Blotting for the luciferase mRNA reporter. For P-values, observe Table S1. B. Synthetic tiRNAs. Uncapped luciferase.