contributed equally to this work TIA-1 and TIAR are related proteins that bind to an AU-rich element (ARE) in the 3¢ untranslated region of tumor necrosis factor alpha (TNF-a) transcripts. To determine the functional signi®cance of this interaction, we used homologous recombination to produce mutant mice lacking TIA-1. Although lipopolysaccharide (LPS)-stimulated macrophages derived from wild-type and TIA-1 ±/± mice express similar amounts of TNF-a transcripts, macrophages lacking TIA-1 produce signi®cantly more TNF-a protein than wild-type controls. The half-life of TNF-a transcripts is similar in wild-type and TIA-1 ±/± macrophages, indicating that TIA-1 does not regulate transcript stability. Rather, the absence of TIA-1 signi®cantly increases the proportion of TNF-a transcripts that associate with polysomes, suggesting that TIA-1 normally functions as a translational silencer. TIA-1 does not appear to regulate the production of interleukin 1b, granulocyte±macrophage colony-stimulating factor or interferon g, indicating that its effects are, at least partially, transcript speci®c. Mice lacking TIA-1 are hypersensitive to the toxic effects of LPS, indicating that this translational control pathway may regulate the organismal response to microbial stress.

#Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.

Uncontrolled TNF-α synthesis is known to play an important role in numerous inflammatory disorders, and multiple transcriptional and post-transcriptional regulatory mechanisms have therefore evolved to dampen the production of this important pro-inflammatory cytokine. By examining the anti-inflammatory properties of the vitamin B3 constituent nicotinamide, we have uncovered a novel regulatory pathway controlling TNF-α production. Exogenous nicotinamide inhibits TNF-α secretion through modulation of mRNA translation efficiency. Moreover, the capacity to produce TNF-α appears to be directly correlated with intracellular NAD levels, suggesting that a NAD-dependent biological event that can be inhibited by nicotinamide controls TNF-α synthesis in cells of the immune system. Sirtuins represent NAD-dependent deacetylases involved in regulation of gene expression in both mammals and yeasts, and are known to be inhibited by nicotinamide. We demonstrate herein that similarly to nicotinamide, structurally unrelated sirtuin inhibitors downregulate TNF-α secretion with minimal effect on TNF-α gene transcription. By over-expressing individual sirtuin members in cell lines transiently expressing TNF-α, we have identified SIRT6 as a sirtuin member able to upregulate TNF-α synthesis in vitro. In agreement with this finding, bone-marrow derived dendritic cells from SIRT6 KO mice display reduced TNF-α synthesis in response to CpG. Collectively, these data delineate a novel relationship between metabolism and the inflammatory response, by uncovering the role of SIRT6 in the control of TNF-α secretion.

No abstract

. Chem. 258, 4331 (1983)]. The enzyme was measured in 4M urea extracts of skin samples [H. M. Kagan and K. A. Sullivan, Methods Enzymol. 82A, 637 (1982)] by an enzyme-linked immunoassay. The net accumulation oflysyl oxidase in the extracellular matrix of dorsal skin (in micrograms per gram ± SEM) was reduced from 500± 123 to 146 ± 55 by week 8 (P < 0.01; for two replicates, n = 4 to 6 each). The lysyl oxidase antibody preparation and the enzyme-linked inumunoassay were done as described by H. Bode and H. Stegeman [J. Immunol. Methods 72,421 (1984) Carroll and B. D. Stallar [J. Biol. Chem. 258, 24 (1983)]. A preliminary report on the characterization of lysyl oxidase and its quantitation by enzyme-linked inmmunosorbent assay (ELISA) has been published by D. Tinker, N. Romero, and R. B.] and S. B.