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.

In monocyte/macrophages, the translation of tumor necrosis factor ␣ (TNF-␣) mRNA is tightly regulated. In unstimulated cells, translation of TNF-␣ mRNA is blocked. Upon stimulation with lipopolysaccharides, this repression is overcome, and the mRNA becomes efficiently translated. The key element in this regulation is the AU-rich element (ARE). We have previously reported the binding of two cytosolic protein complexes to the TNF-␣ mRNA ARE. One of these complexes (complex 1) forms with extracts of both unstimulated and lipopolysaccharide-stimulated macrophages and requires a large fragment of the ARE containing clustered AUUUA pentamers. The other complex (complex 2) is only detected after cell activation, binds to a minimal UUAUUUAUU nonamer, and is composed of a 55-kDa protein. Here, we report the identification of the RNAbinding protein TIAR as a protein involved in complex 1. The RNA sequence bound by TIAR and the cytoplasmic localization of this protein in macrophages argue for an involvement of TIAR in TNF mRNA posttranscriptional regulation. Tumor necrosis factor-␣ (TNF-␣)1 is a cytokine predominantly produced by macrophages but also by lymphocytes, NK cells, astrocytes, and other cell types. The most powerful inducers of TNF-␣ production by macrophages are the lipopolysaccharides (LPS), which are membrane components released by Gram-negative bacteria in the course of infection (1). It is now well established that the induction of TNF-␣ production upon stimulation of macrophages by LPS results from both an enhancement of TNF-␣ gene transcription and a translational derepression of the mRNA. In unstimulated macrophages, TNF-␣ mRNA translation is blocked. Upon stimulation with LPS, this repression is overcome, and TNF-␣ mRNA becomes efficiently translated (2). The key element involved in this regulation is the AU-rich element (ARE) located in the 3Ј-untranslated region (-UTR) of TNF-␣ mRNA (3). This 70-nucleotide-long sequence is composed of several repeats of the AUUUA pentamer. The physiological importance of TNF-␣ mRNA translational control is demonstrated by the fact that the expression of a TNF-␣ transgene lacking its 3Ј-UTR in mouse leads to severe inflammatory disorders (4).Similar AREs are found in the 3Ј-UTR of a growing number of mRNAs encoding cytokines, protooncogenes, or other transiently expressed proteins (5). These sequences have also been shown to regulate mRNA stability (6).In former studies, we reported that TNF-␣ mRNA ARE can form two complexes with proteins present in cytosolic macrophage extracts. One of these complexes (complex 1) forms with extracts of both unstimulated and LPS-stimulated macrophages and requires a large fragment of the ARE containing clustered AUUUA pentamers. The other complex (complex 2) is only detected after cell activation, binds to a minimal UUAUUUAUU nonamer, and is composed of a 55-kDa protein (7,8).To identify the proteins involved in both complexes, we designed a cloning strategy based on the differential screening of a macrophage cDNA expression library wi...

Tumour necrosis factor (TNF)-α mRNA contains an AU-rich element (ARE) in its 3′ untranslated region (3′UTR), which determines its half-life and translational efficiency. In unstimulated macrophages, TNF-α mRNA is repressed translationally, and becomes efficiently translated upon cell activation. Gel retardation experiments and screening of a macrophage cDNA expression library with the TNF-α ARE allowed the identification of TIA-1-related protein (TIAR), T-cell intracellular antigen-1 (TIA-1) and tristetraprolin (TTP) as TNF-α ARE-binding proteins. Whereas TIAR and TIA-1 bind the TNF-α ARE independently of the activation state of macrophages, the TTP-ARE complex is detectable upon stimulation with lipopolysaccharide (LPS). Moreover, treatment of LPS-induced macrophage extracts with phosphatase significantly abrogates TTP binding to the TNF-α ARE, indicating that TTP phosphorylation is required for ARE binding. Carballo, Lai and Blackshear [(1998) Science 281, 1001–1005] showed that TTP was a TNF-α mRNA destabilizer. In contrast, TIA-1, and most probably TIAR, acts as a TNF-α mRNA translational silencer. A two-hybrid screening with TIAR and TIA-1 revealed the capacity of these proteins to interact with other RNA-binding proteins. Interestingly, TIAR and TIA-1 are not engaged in the same interaction, indicating for the first time that TIAR and TIA-1 can be functionally distinct. These findings also suggest that ARE-binding proteins interact with RNA as multimeric complexes, which might define their function and their sequence specificity.

Serine-arginine (SR) proteins commonly designate a family of eukaryotic RNA binding proteins containing a protein domain composed of several repeats of the arginine-serine dipeptide, termed the arginine-serine (RS) domain. This protein family is involved in essential nuclear processes such as constitutive and alternative splicing of mRNA precursors. Besides participating in crucial activities in the nuclear compartment, several SR proteins are able to shuttle between the nucleus and the cytoplasm and to exert regulatory functions in the latter compartment. This review aims at discussing the properties of shuttling SR proteins with particular emphasis on their nucleo-cytoplasmic traffic and their cytoplasmic functions. Indeed, recent findings have unravelled the complex regulation of SR protein nucleo-cytoplasmic distribution and the diversity of cytoplasmic mechanisms in which these proteins are involved. SR proteins: definition and general featuresSerine-arginine (SR) proteins were first described as a family of co-purifying proteins capable of restoring splicing in S100 splicing-deficient extracts. These factors are structurally highly related as they are composed of a carboxy-terminal domain enriched with the arginine-serine (RS) dipeptide, preceded by at least one RNA binding domain of the RNA recognition motif (RRM) type [1]. These proteins play an essential role in constitutive and alternative splicing of mRNA precursors [2]. In the last 10 years, however, other functions have been unravelled for these proteins. Indeed, SR protein prototypes such as SRSF1 (ASF ⁄ SF2) and SRSF2 (SC35) were reported to stimulate transcriptional elongation (for a review see [3]). Certain SR proteins participate in mRNA translation (see [4] and below). Finally, a recent study indicates that SRSF1 promotes microRNA processing by facilitating Drosha-mediated cleavage [5]. Altogether, these findings highlight the broader roles of SR proteins in gene expression.Genome-wide searches for proteins containing RS domains revealed several other 'non-classical' SR proteins, termed SR-like proteins because of differences in the structure of the RS domain and ⁄ or lack of a prototypical RRM. While a large subset of SR-like proteins is involved in pre-mRNA processing, several members of this protein family are associated with other cellular functions [4,6].'Classical' SR proteins exist in plants [7], metazoans [1] and in some unicellular eukaryotes, such as the fission yeast Schizosaccharomyces pombe [8,9]. However, they are not present in all eukaryotes and are Abbreviations ARE, AU-rich element; NMD, nonsense-mediated decay; PIAS1, protein inhibitor of activated STAT-1; RRM, RNA recognition motif;