N et al., 2002). Uridine modified tRNAs have an enhanced ability to “wobble” and read G-ending codons, forming a functionally redundant decoding technique (Johansson et al., 2008). Having said that, only a handful of biological roles for these modifications are known. Uridine mcm5 modifications allow the translation of AGA and AGG codons during DNA harm (Begley et al., 2007), influence certain telomeric gene silencing or DNA CMV web damage responses (Chen et al., 2011b), and function in exocytosis (Esberg et al., 2006). These roles can’t totally explain why these modifications are Caspase 9 Formulation ubiquitous, or how they may be advantageous to cells. Interestingly, studies in yeast link these tRNA modifications to nutrient-dependent responses. Both modifications consume metabolites derived from sulfur metabolism, primarily 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 of 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 is usually detected involving 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 amongst these modifications, nutrient availability and cellular metabolism would reveal a functional logic to their biological value. 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 important mechanism by which translation and development are regulated synchronously with metabolism. These findings have substantial implications for our understanding of cellular amino acid-sensing mechanisms, and together with the accompanying manuscript (Sutter et al., 2013), show how sulfur-containing amino acids serve as sentinel metabolites for cell growth manage.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCell. Author manuscript; accessible 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 were intrigued by connections between tRNA uridine modification pathways and nutrients, particularly because mutants of tRNA uridine-modifying enzymes have been hypersensitive to rapamycin (Figure S1A). We very first tested no matter whether tRNA uridine modification amounts changed in response to unique nutrient environments. To qualitatively assay tRNA uridine thiolation, tRNAs have been resolved on urea-PAGE gels containing the sulfur-coordinating mercury agent APM (Nakai et al., 2008) (Supplemental Information and facts). We confirmed that the enzyme Uba4p is needed for all tRNA thiolation (Figure S1B). Whilst the majority of tRNALys (UUU), tRNAGlu (UUC) and tRNAGln (UUG) had been thiolated in cells developing either in YPD (wealthy medium) or under continuous glucose-limitation, a fraction of those tRNAs remained unthiolated (Figure S1B), suggesting that this modification was not constitutive, and may possibly transform in abundance below certain situations. We then developed targeted LC-MS/MS solutions to quantitatively measure amounts of thiolated, m.
