N et al., 2002). Uridine modified tRNAs have an enhanced ability to “wobble” and read G-ending codons, forming a functionally redundant decoding program (Johansson et al., 2008). Having said that, only a handful of biological roles for these APC Purity & Documentation modifications are recognized. Uridine mcm5 modifications enable the translation of AGA and AGG codons during DNA damage (Begley et al., 2007), influence specific telomeric gene silencing or DNA harm responses (Chen et al., 2011b), and function in exocytosis (Esberg et al., 2006). These roles can’t totally clarify why these modifications are ubiquitous, or how they’re advantageous to cells. Interestingly, studies in yeast link these tRNA modifications to nutrient-dependent responses. Both modifications consume metabolites derived from sulfur metabolism, mostly S-adenosylmethionine (SAM) (Kalhor and Clarke, 2003; Nau, 1976), and cysteine (Leidel et al., 2009; Noma et al., 2009). These modifications appear to be downstream in the TORC1 pathway, as yeast lacking these modifications are hypersensitive to rapamycin (Fichtner et al., 2003; Goehring et al., 2003b; Leidel et al., 2009; Nakai et al., 2008), and interactions might be detected among Uba4p and Kog1/TORC1 (Laxman and Tu, 2011). These modification pathways also play vital roles in nutrient stress-dependent dimorphic foraging yeast behavior (Abdullah and Cullen, 2009; Goehring et al., 2003b; Laxman and Tu, 2011). We reasoned that deciphering the interplay involving these modifications, nutrient availability and cellular metabolism would reveal a functional logic to their biological significance. Herein, we show that tRNA uridine thiolation abundance reflects sulfur-containing amino acid availability, and functions to regulate translational capacity and amino acid homeostasis. Uridine thiolation represents a key BRD7 custom synthesis mechanism by which translation and development are regulated synchronously with metabolism. These findings have considerable implications for our understanding of cellular amino acid-sensing mechanisms, and using the accompanying manuscript (Sutter et al., 2013), show how sulfur-containing amino acids serve as sentinel metabolites for cell development handle.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCell. Author manuscript; obtainable in PMC 2014 July 18.Laxman et al.PageRESULTStRNA uridine thiolation amounts reflect intracellular sulfur amino acid availabilityNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptWe have been intrigued by connections involving tRNA uridine modification pathways and nutrients, specially considering that mutants of tRNA uridine-modifying enzymes had been hypersensitive to rapamycin (Figure S1A). We first tested no matter whether tRNA uridine modification amounts changed in response to different nutrient environments. To qualitatively assay tRNA uridine thiolation, tRNAs were resolved on urea-PAGE gels containing the sulfur-coordinating mercury agent APM (Nakai et al., 2008) (Supplemental Data). We confirmed that the enzyme Uba4p is needed for all tRNA thiolation (Figure S1B). Even though the majority of tRNALys (UUU), tRNAGlu (UUC) and tRNAGln (UUG) were thiolated in cells expanding either in YPD (wealthy medium) or below continuous glucose-limitation, a fraction of those tRNAs remained unthiolated (Figure S1B), suggesting that this modification was not constitutive, and may well transform in abundance beneath particular circumstances. We then developed targeted LC-MS/MS techniques to quantitatively measure amounts of thiolated, m.