The mechanism of insulin action is a central theme in biology and medicine. In addition to the rather rare condition of insulin deficiency caused by autoimmune destruction of pancreatic β-cells, genetic and acquired abnormalities of insulin action underlie the far more common conditions of type 2 diabetes, obesity and insulin resistance. The latter predisposes to diseases ranging from hypertension to Alzheimer disease and cancer. Hence, understanding the biochemical and cellular properties of insulin receptor signalling is arguably a priority in biomedical research. In the past decade, major progress has led to the delineation of mechanisms of glucose transport, lipid synthesis, storage and mobilization. In addition to direct effects of insulin on signalling kinases and metabolic enzymes, the discovery of mechanisms of insulin-regulated gene transcription has led to a reassessment of the general principles of insulin action. These advances will accelerate the discovery of new treatment modalities for diabetes.

Bile acids (BAs) exert pleiotropic metabolic effects, and physicochemical properties of different BAs affect their function. In rodents, insulin regulates BA composition, in part by regulating the BA 12α-hydroxylase CYP8B1. However, it is unclear whether a similar effect occurs in humans. To address this question, we examined the relationship between clamp-measured insulin sensitivity and plasma BA composition in a cohort of 200 healthy subjects and 35 type 2 diabetic (T2D) patients. In healthy subjects, insulin resistance (IR) was associated with increased 12α-hydroxylated BAs (cholic acid, deoxycholic acid, and their conjugated forms). Furthermore, ratios of 12α-hydroxylated/non–12α-hydroxylated BAs were associated with key features of IR, including higher insulin, proinsulin, glucose, glucagon, and triglyceride (TG) levels and lower HDL cholesterol. In T2D patients, BAs were nearly twofold elevated, and more hydrophobic, compared with healthy subjects, although we did not observe disproportionate increases in 12α-hydroxylated BAs. In multivariate analysis of the whole dataset, controlling for sex, age, BMI, and glucose tolerance status, higher 12α-hydroxy/non–12α-hydroxy BA ratios were associated with lower insulin sensitivity and higher plasma TGs. These findings suggest a role for 12α-hydroxylated BAs in metabolic abnormalities in the natural history of T2D and raise the possibility of developing insulin-sensitizing therapeutics based on manipulations of BA composition.

Early transfer RNA (tRNA) processing events in Saccharomyces cerevisiae are coordinated in the nucleolus, the site normally associated with ribosome biosynthesis. To test whether spatial organization of the tRNA pathway begins with nucleolar clustering of the genes, we have probed the subnuclear location of five different tRNA gene families. The results show that tRNA genes, though dispersed in the linear genome, colocalize with 5S ribosomal DNA and U14 small nucleolar RNA at the nucleolus. Nucleolar localization requires tRNA gene transcription-complex formation, because inactivation of the promoter at a single locus removes its nucleolar association. This organization of tRNA genes must profoundly affect the spatial packaging of the genome and raises the question of whether gene types might be coordinated in three dimensions to regulate transcription.

The 274 tRNA genes in Saccharomyces cerevisiae are scattered throughout the linear maps of the 16 chromosomes, but the genes are clustered at the nucleolus when compacted in the nucleus. This clustering is dependent on intact nucleolar organization and contributes to tRNA gene-mediated (tgm) silencing of RNA polymerase II transcription near tRNA genes. After examination of the localization mechanism, we find that the chromosome-condensing complex, condensin, is involved in the clustering of tRNA genes. Conditionally defective mutations in all five subunits of condensin, which we confirm is bound to active tRNA genes in the yeast genome, lead to loss of both pol II transcriptional silencing near tRNA genes and nucleolar clustering of the genes. Furthermore, we show that condensin physically associates with a subcomplex of RNA polymerase III transcription factors on the tRNA genes. Clustering of tRNA genes by condensin appears to be a separate mechanism from their nucleolar localization, as microtubule disruption releases tRNA gene clusters from the nucleolus, but does not disperse the clusters. These observations suggest a widespread role for condensin in gene organization and packaging of the interphase yeast nucleus.[Keywords: tRNA gene; condensin; microtubules; nuclear organization; nucleolus] Supplemental material is available at http://www.genesdev.org.

Maf1 is an essential and specific mediator of transcriptional repression in the RNA polymerase (pol) III system. Maf1-dependent repression occurs in response to a wide range of conditions, suggesting that the protein itself is targeted by the major nutritional and stress-signaling pathways. We show that Maf1 is a substrate for cAMP-dependent PKA in vitro and is differentially phosphorylated on PKA sites in vivo under normal versus repressing conditions. PKA activity negatively regulates Maf1 function because strains with unregulated high PKA activity block repression of pol III transcription in vivo, and strains lacking all PKA activity are hyperrepressible. Nuclear accumulation of Maf1 is required for transcriptional repression and is regulated by two nuclear localization sequences in the protein. An analysis of PKA phosphosite mutants shows that the localization of Maf1 is affected via the N-terminal nuclear localization sequence. In particular, mutations that prevent phosphorylation at PKA consensus sites promote nuclear accumulation of Maf1 without inducing repression. These results indicate that negative regulation of Maf1 by PKA is achieved by inhibiting its nuclear import and suggest that a PKA-independent activation step is required for nuclear Maf1 to function in the repression of pol III transcription. Finally, we report a previously undescribed phenotype for Maf1 in tRNA genemediated silencing of nearby RNA pol II transcription.nuclear import ͉ phosphorylation ͉ tRNA biosynthesis T he action of all three nuclear RNA polymerases (pols) in the synthesis of rRNAs, ribosomal protein mRNAs, and tRNAs is coordinately regulated to control ribosome biogenesis and cell growth in response to nutrients and many other conditions (1). In Saccharomyces cerevisiae, Maf1 has been identified as an absolute and specific effector of repression in the pol III system (2). The diversity of conditions that signal repression, combined with the essential role of Maf1 in this process, suggests that the Maf1 protein is targeted by multiple signaling pathways.Genomewide localization of the pol III transcription apparatus has shown that nutrient deprivation and entry into stationary phase causes a significant decrease in polymerase occupancy on pol III genes (3, 4). This change is Maf1-dependent and is presumably a consequence of the direct interaction of Maf1 with the polymerase (5, 6). Consistent with this view, an in vitro system that recapitulates Maf1-dependent repression identified two steps that are inhibited as follows: polymerase recruitment to existing TFIIIB-DNA complexes and de novo assembly of the initiation factor TFIIIB onto DNA (5). In the latter step, Maf1 is thought to target the activity of TFIIIB via a direct interaction with one of its subunits, Brf1 (2, 5). However, the mechanism by which Maf1 inhibits TFIIIB-DNA assembly and transcription is not yet known.Maf1 is a phylogenetically conserved and structurally novel protein that lacks homology to any motifs of known function (6). However, three conserved doma...