Cyril Gueydan

TIA‐1 is a translational silencer that selectively regulates the expression of TNF‐α

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‐α) transcripts. To determine the functional significance 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‐α transcripts, macrophages lacking TIA‐1 produce significantly more TNF‐α protein than wild‐type controls. The half‐life of TNF‐α 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 significantly increases the proportion of TNF‐α 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 1β, granulocyte–macrophage colony‐stimulating factor or interferon γ, indicating that its effects are, at least partially, transcript specific. 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.

Intracellular NAD levels regulate tumor necrosis factor protein synthesis in a sirtuin-dependent manner

Tumor necrosis factor (TNF) synthesis is known to play a major part in numerous inflammatory disorders, and multiple transcriptional and post-transcriptional regulatory mechanisms have therefore evolved to dampen the production of this key proinflammatory cytokine. The high expression of nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in the nicotinamide-dependent NAD biosynthetic pathway, in cells of the immune system has led us to examine the potential relationship between NAD metabolism and inflammation. We show here that intracellular NAD concentration promotes TNF synthesis by activated immune cells. Using a positive screen, we have identified Sirt6, a member of the sirtuin family, as the NAD-dependent enzyme able to regulate TNF production by acting at a post-transcriptional step. These studies reveal a previously undescribed relationship between metabolism and the inflammatory response and identify Sirt6 and the nicotinamide-dependent NAD biosynthetic pathway as novel candidates for immunointervention in an inflammatory setting.

Transcriptome-wide distribution and function of RNA hydroxymethylcytosine

Chemical modification of RNA for function Chemical modifications play an important role in modifying and regulating the function of DNA and RNA. Delatte et al. show that, in the fruit fly, many messenger RNAs (mRNAs) contain the modified base 5-hydroxymethylcytosine (5hmC). The chemical mark is added by the same enzyme that adds 5hmC to DNA. Because many mRNAs involved in neuronal development contain 5hmC, blocking the enzyme causes brain defects and is lethal. In vivo, RNA hydroxymethylation promotes mRNA translation. Science, this issue p. 282 Posttranscriptional modification of messenger RNAs (mRNAs) is prevalent in Drosophila and promotes mRNA translation. 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.

Shuttling SR proteins: more than splicing factors.

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.

Identification of the sequence determinants mediating the nucleo-cytoplasmic shuttling of TIAR and TIA-1 RNA-binding proteins

TIAR and TIA-1 are two closely related RNA-binding proteins which possess three RNA recognition motifs (RRMs) followed by an auxiliary region. These proteins are involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and regulation of mRNA translation. Here we characterize the subcellular localization of these proteins in somatic cells. We demonstrate that TIAR and TIA-1 continuously shuttle between the cytoplasm and the nucleus and belong to the class of RNA-binding proteins whose nuclear import is transcription-dependent. We identified RRM2 and the first half of the auxiliary region as important determinants for TIAR and TIA-1 nuclear accumulation. In contrast, the nuclear export of TIAR and TIA-1 is mediated by RRM3. Both RRMs contribute to TIAR and TIA-1 nuclear accumulation or export by their RNA-binding capacity. Indeed, whereas mutations of the highly conserved RNP2 or RNP1 peptides in RRM2 redistribute TIAR to the cytoplasm, similar modifications in RRM3 abolish TIAR nuclear export. Moreover, TIAR and TIA-1 nuclear accumulation is a Ran-GTP-dependent pathway, in contrast to its nuclear export which is unaffected by Ran-GTP depletion and which is independent of the major CRM1-exporting pathway. This study demonstrates the importance of TIAR and TIA-1 RNA-binding domains for their subcellular localization and provides the first evidence for distinct functions of TIAR and TIA-1 RRMs.

Mapping of a minimal AU-rich sequence required for lipopolysaccharide-induced binding of a 55-kDa protein on tumor necrosis factor-alpha mRNA.

In monocyte/macrophage cells, the translation of tumor necrosis factor-α (TNF-α) mRNA is tightly controlled. In unstimulated cells, TNF-α mRNA is translationally repressed. However, upon stimulation of the cells with various agents (e.g. lipopolysaccharides (LPS) and viruses), this repression is overcome and translation occurs. The key element in this regulation is the AU-rich sequence present in the 3′-untranslated region of TNF-α mRNA. Several groups have described the binding of proteins on AU-rich elements (AREs). We have previously reported the binding of two cytosolic protein complexes (1 and 2) to the TNF-α mRNA ARE, one of which (complex 2) is observed only following induction of TNF-α production by LPS. In this report, we have demonstrated that complex 1 involves a long fragment of the ARE, whereas the formation of the LPS-inducible complex 2 requires a minimal sequence which corresponds to the nonanucleotide UUAUUUAUU. Furthermore, we show that the RNA-binding protein involved in complex 2 has an apparent molecular mass of 55 kDa. Finally, we tested other AREs for their ability to form complex 2. We observed that the ARE derived from granulocyte/macrophage colony-stimulating factor mRNA, which does contain the nonanucleotide, is able to sustain the LPS-induced binding of the 55-kDa protein. However, c-myc mRNA, which does not contain the nonanucleotide, is unable to promote the formation of any LPS-induced complex.

Tristetraprolin regulation of interleukin 23 mRNA stability prevents a spontaneous inflammatory disease

Tristetraprolin deficiency results in enhanced IL-23 via dysregulated mRNA decay that leads to an inflammatory syndrome characterized by cachexia, myeloid hyperplasia, dermatitis, and erosive arthritis.

Methylprednisolone differentially regulates IL-10 and tumour necrosis factor (TNF) production during murine endotoxaemia

IL‐10 is an endogenous antiinflammatory cytokine that inhibits TNF biosynthesis and protects mice  from lipopolysaccharide (LPS)‐induced lethality. As synthetic glucocorticoids are widely used as antiinflammatory agents, we analysed the effects of methylprednisolone administration on IL‐10 biosynthesis during murine endotoxaemia. We found that low doses of methylprednisolone (2–10 mg/kg) markedly inhibited TNF production but did not affect serum levels of IL‐10, while a high methylprednisolone dose (50 mg/kg) increased LPS‐induced IL‐10 levels. In parallel, we observed that LPS‐induced IL‐10 production is TNF‐independent in this experimental setting. Experiments conducted in vitro indicated that methylprednisolone (from 0.01 to 100 μg/ml) also increased the biosynthesis of IL‐10 by LPS‐activated mouse peritoneal macrophages. We conclude that methylprednisolone differentially regulates IL‐10 and TNF production induced by LPS both in vivo and in vitro at the macrophage level.

Transportin-1 and Transportin-2: protein nuclear import and beyond.

Nearly 20 years after its identification as a new β‐karyopherin mediating the nuclear import of the RNA‐binding protein hnRNP A1, Transportin‐1 is still commonly overlooked in comparison with its best known cousin, Importin‐β. Transportin‐1 is nonetheless a considerable player in nucleo‐cytoplasmic transport. Over the past few years, significant progress has been made in the characterization of the nuclear localization signals (NLSs) that Transportin‐1 recognizes, thereby providing the molecular basis of its diversified repertoire of cargoes. The recent discovery that mutations in the Transportin‐dependent NLS of FUS cause mislocalization of this protein and result in amyotrophic lateral sclerosis illustrates the importance of Transportin‐dependent import for human health. Besides, new functions of Transportin‐1 are emerging in processes other than nuclear import. Here, we summarize what is known about Transportin‐1 and the related β‐karyopherin Transportin‐2.

PARP12, an Interferon-stimulated Gene Involved in the Control of Protein Translation and Inflammation

Background: The individual role of members of the poly(ADP-ribose) polymerase family is unclear. Results: PARP12 displays a dual subcellular localization and effector function, controlling both protein translation and NF-κB signaling. Conclusion: PARP12 mediates two important effector mechanisms linked to the establishment of an anti-viral state. Significance: ADP-ribosylation may play an important role in the innate control of microbial infections. Transcriptome analyses have recently identified PARP12, a member of a large family of ADP-ribosyl transferases, as an interferon-induced gene (ISG), whose function remains incompletely characterized. We demonstrate herein that PARP12 is a genuine ISG, whose expressed protein displays at least two distinct subcellular locations and related functions. Upon ectopic expression or exposure to oxidative stress, PARP12 is recruited to stress-granules (SGs), known sites of mRNA translational arrest. Accordingly, PARP12 was found to block mRNA translation, possibly upon association to the translational machinery. Both the N-terminal domain (containing an RNA-binding domain characterized by the presence of five CCCH-type Zn-fingers) and integrity of the catalytic domain are required for this suppressive function. In contrast, stimulation with LPS leads to the localization of PARP12 to p62/SQSTM1 (an adaptor protein involved in innate signaling and autophagy) containing structures, unrelated to SGs. Deletion of the N-terminal domain promotes the association of the protein to p62/SQSTM1, suggesting that the RNA-binding domain is responsible for the subcellular localization of PARP12. Association to p62/SQSTM1 was found to correlate with increased NF-κB signaling, suggesting a role for PARP12 in inflammation. Collectively, these observations suggest that PARP12 can alternate between two distinct subcellular compartments associated to two distinct cellular functions. The present work therefore identifies PARP12 as an ISG with a potential role in cellular defenses against viral infections.