3´tsRNAs are fully charged. All experiments were performed in triplicate and representative northern blots are shown.

(A) Under normal growth conditions, 3´tsRNAs are aminoacylated. HeLa total RNA extracted under conditions with pH less than 5.0 was either acetylated or deacylated, ethanol precipitated, and exonuclease T digested. A 20 nt size marker was loaded in each gel and total RNA without any treatment was used for comparison in the right lane. DNA probes antisense to tsRNA^Leu, tsRNA^Ala, and tsRNA^Gly were used in a northern blot (top left panel and bottom panel). The hsa-miR-223-3p miRCURY LNA miRNA (Qiagen) detection probe was used for quantifying this miRNA (top right panel). (B) Leucinol is an analog of leucine and an inhibitor of LARS1. (C) The tsRNA^Leu is fully charged in the presence of leucinol. Total RNA isolated from HeLa cells grown with or without leucinol (2 mM) were extracted and examined for their charging status. The full-length tRNA^Leu was ~50% charged under this condition (top) while the tsRNA^Leu is fully charged (bottom). (D) Quantification of tRNA^Leu and tsRNA^Leu in the presence or absence of leucinol. Values were normalized to control cells. Data are means ± standard deviations (s.d.), n = 3 independent experiments, **, P < 0.01, as determined by Student’s one-tailed t-test.