Post-transcriptional modification of the tRNA anticodon loop is critical for translation. Yeast Trm7 is required for 29-O-methylation of C 32 and N 34 of tRNA Phe , tRNA Trp , and tRNA Leu(UAA) to form Cm 32 and Nm 34 , and trm7-D mutants have severe growth and translation defects, but the reasons for these defects are not known. We show here that overproduction of tRNA Phe suppresses the growth defect of trm7-D mutants, suggesting that the crucial biological role of Trm7 is the modification of tRNA Phe . We also provide in vivo and in vitro evidence that Trm7 interacts with ORF YMR259c (now named Trm732) for 29-O-methylation of C 32 , and with Rtt10 (named Trm734) for 29-O-methylation of N 34 of substrate tRNAs and provide evidence for a complex circuitry of anticodon loop modification of tRNA Phe , in which formation of Cm 32 and Gm 34 drives modification of m 1 G 37 (1-methylguanosine) to yW (wyebutosine). Further genetic analysis shows that the slow growth of trm7-D mutants is due to the lack of both Cm 32 and Nm 34 , and the accompanying loss of yW, because trm732-D trm734-D mutants phenocopy trm7-D mutants, whereas each single mutant is healthy; nonetheless, TRM732 and TRM734 each have distinct roles, since mutations in these genes have different genetic interactions with trm1-D mutants, which lack m 2,2 G 26 in their tRNAs. We speculate that 29-O-methylation of the anticodon loop may be important throughout eukaryotes because of the widespread conservation of Trm7, Trm732, and Trm734 proteins, and the corresponding modifications, and because the putative human TRM7 ortholog FTSJ1 is implicated in nonsyndromic X-linked mental retardation.

No abstract

Central melanocortin 4 receptors (MC4Rs) stimulate energy expenditure and inhibit food intake. MC4Rs activate the G protein Gα, but whether Gα mediates all MC4R actions has not been established. Individuals with Albright hereditary osteodystrophy (AHO), who have heterozygous Gα-inactivating mutations, only develop obesity when the Gα mutation is present on the maternal allele because of tissue-specific genomic imprinting. Furthermore, evidence in mice implicates Gα imprinting within the central nervous system (CNS) in this disorder. In this study, we examined the effects of Gα in MC4R-expressing cells on metabolic regulation. Mice with homozygous Gα deficiency in MC4R-expressing cells (MC4RGsKO) developed significant obesity with increased food intake and decreased energy expenditure, along with impaired insulin sensitivity and cold-induced thermogenesis. Moreover, the ability of the MC4R agonist melanotan-II (MTII) to stimulate energy expenditure and to inhibit food intake was impaired in MC4RGsKO mice. MTII failed to stimulate the secretion of the anorexigenic hormone peptide YY (PYY) from enteroendocrine L cells, a physiological response mediated by MC4R-Gα signaling, even though baseline PYY levels were elevated in these mice. In Gα heterozygotes, mild obesity and reduced energy expenditure were present only in mice with a Gα deletion on the maternal allele in MC4R-expressing cells, whereas food intake was unaffected. These results demonstrate that Gα signaling in MC4R-expressing cells is required for controlling energy balance, thermogenesis, and peripheral glucose metabolism. They further indicate that Gα imprinting in MC4R-expressing cells contributes to obesity in Gα knockout mice and probably in individuals with Albright hereditary osteodystrophy as well.

No abstract

In both mice and patients with Albright hereditary osteodystrophy, heterozygous inactivating mutations of Gsα, a ubiquitously expressed G protein that mediates receptor-stimulated intracellular cAMP production, lead to obesity and insulin resistance but only when the mutation is present on the maternal allele. This parent-of-origin effect in mice was shown to be due to Gsα imprinting in one or more brain regions. The ventromedial hypothalamus (VMH) is involved in the regulation of energy and glucose homeostasis, but the role of Gsα in VMH on metabolic regulation is unknown. To examine this, we created VMH-specific Gsα-deficient mice by mating Gsα-floxed mice with SF1-cre mice. Heterozygotes with Gsα mutation on either the maternal or paternal allele had a normal metabolic phenotype, and there was no molecular evidence of Gsα imprinting, indicating that the parent-of-origin metabolic effects associated with Gsα mutations is not due to Gsα deficiency in VMH SF1 neurons. Homozygous VMH Gsα knockout mice (VMHGsKO) showed no changes in body weight on either a regular or high-fat diet. However, glucose metabolism (fasting glucose, glucose tolerance, insulin sensitivity) was significantly improved in male VMHGsKO mice, with the difference more dramatic on the high-fat diet. In addition, male VMHGsKO mice on the high-fat diet showed a greater anorexigenic effect and increased VMH signal transducer and activator of transcription-3 phosphorylation in response to leptin. These results indicate that VMH Gsα/cyclic AMP signaling regulates glucose homeostasis and alters leptin sensitivity in mice, particularly in the setting of excess caloric intake.