MDA5 is an essential intracellular sensor for several viruses, including picornaviruses, and elicits antiviral interferon (IFN) responses by recognizing viral dsRNAs. MDA5 has been implicated in autoimmunity. However, the mechanisms of how MDA5 contributes to autoimmunity remain unclear. Here we provide direct evidence that dysregulation of MDA5 caused autoimmune disorders. We established a mutant mouse line bearing MDA5 mutation by ENU mutagenesis, which spontaneously developed lupus-like autoimmune symptoms without viral infection. Inflammation was dependent on an adaptor molecule, MAVS indicating the importance of MDA5-signaling. In addition, intercrossing the mutant mice with type I IFN receptor-deficient mice ameliorated clinical manifestations. This MDA5 mutant could activate signaling in the absence of its ligand but was paradoxically defective for ligand- and virus-induced signaling, suggesting that the mutation induces a conformational change in MDA5. These findings provide insight into the association between disorders of the innate immune system and autoimmunity.

Cytoplasmic aminoacyl-tRNA synthetases of higher eukaryotes acquired extra peptides in the course of their evolution. It has been thought that these appendices are related to the occurrence of the multiprotein complex consisting of at least eight different tRNA synthetase polypeptides. This complex is believed to be a signature feature of metazoans. In this study, we used multiple sequence alignments to infer the locations of the peptide appendices from human cytoplasmic tRNA synthetases found in the multisynthetase complex. The selected peptide appendices ranged from 22 aa of aspartyl-tRNA synthetase to 267 aa of methionyltRNA synthetase. We then made genetic constructions to investigate interactions between all 64 combinations of these peptides that were individually fused to nonsynthetase test proteins. The analyses identified 11 (10 heterologous and 1 homologous) interactions. The six peptide-dependent interactions paralleled what had been detected by crosslinking methods applied to the isolated multisynthetase complex. Thus, small peptide appendices seem to link together different synthetases into a complex. In addition, five interacting pairs that had not been detected previously were suggested from the observed peptide-dependent complexes.

The nucleotide 73 (N73) "discriminator" base in the acceptor stem is a key element for efficient and specific aminoacylation of tRNAs and of microhelix substrates derived from tRNA acceptor stems. This nucleotide was possibly one of the first to be used for differentiating among groups of early RNA substrates by tRNA synthetases. In contrast to many other synthetases, we report here that the class II human lysyl-tRNA synthetase is relatively insensitive to the nature of N73. We cloned, sequenced, and expressed the enzyme, which is a close homologue of the class II yeast aspartyl-tRNA synthetase whose co-crystal structure (with tRNAAsp) is known. The latter enzyme has a strong requirement for G73, which interacts with 4 of the 14 residues within the "motif 2" loop of the enzyme. Even though eukaryotic lysine tRNAs also encode G73, the motif 2 loop sequence of lysyl-tRNA synthetase differs at multiple positions from that of the aspartate enzyme. Indeed, the recombinant human lysine enzyme shows little preference for G, and even charges human tRNA transcripts encoding the A73 found in E. coli lysine tRNAs. Moreover, while the lysine enzyme is the only one in E. coli to be encoded by two separate genes, a double mutant that disables both genes is complemented by a cDNA expressing the human protein. Thus, the sequence of the loop of motif 2 of human lysyl-tRNA synthetase specifies a structural variation that accommodates nucleotide degeneracy at position 73. This sequence might be used as a starting point for obtaining highly specific interactions with any given N73 by simple amino acid replacements.

Endothelial monocyte activating polypeptide II (EMA-PII) is a cytokine that is specifically induced by apoptosis.Its precursor (pro-EMAPII) has been suggested to be identical to p43, which is associated with the multi-tRNA synthetase complex. Herein, we have demonstrated that the N-terminal domain of pro-EMAPII interacts with the Nterminal extension of human cytoplasmic arginyl-tRNA synthetase (RRS) using genetic and immunoprecipitation analyses. Aminoacylation activity of RRS was enhanced about 2.5-fold by the interaction with pro-EMAPII but not with its N-or C-terminal domains alone. The N-terminal extension of RRS was not required for enzyme activity but did mediate activity stimulation by pro-EMAPII. Pro-EMAPII reduced the apparent K m of RRS to tRNA, whereas the k cat value remained unchanged. Therefore, the precursor of EMAPII is a multi-functional protein that assists aminoacylation in normal cells and releases the functional cytokine upon apoptosis.Aminoacyl-tRNA synthetases (ARSs) 1 catalyze ligation of their cognate amino acids to specific tRNAs. Although basic architecture of the core domain is well conserved among ARSs, unique peptide extensions have been found in the N-or Cterminal ends of metazoan enzymes (1-3). Although these extensions have been thought to be involved in heterologous molecular interactions, their functional significance is not yet understood.A macromolecular protein complex consisting of at least nine different ARSs has been found in higher eukaryotes (1-3). This multi-ARS complex also contains three nonsynthetase components, p18, p38, and p43 whose functions are not clear (4 -7). Among these nonsynthetase components, p43 has been proposed to be a precursor of a tumor-specific cytokine, endothelial monocyte-activating polypeptide II (EMAPII) based on over 80% sequence identity between the two proteins (6). EMAPII was originally identified in the culture medium of murine fibrosarcoma cells induced by methylcholanthrene A (8). It triggers an acute inflammatory response (9, 10) and is involved in development-related apoptosis (11).The precursor for EMAPII (pro-EMAPII) is processed at the Asp residue of ASTD/S sequence to release the C-terminal cytokine domain of 23 kDa (11). Its C-terminal domain shares homology with the C-terminal parts of methionyl-tRNA synthetases of prokaryotes, archaea and nematode, and also a yeast protein, Arc1p/G4p, which interacts with methionyl-and glutamyl-tRNA synthetases. The N-terminal domain of pro-EMAPII does not show homology to any known proteins, and its function has not been understood.EMAPII is expressed in a wide range of cell lines and normal tissues (12) and its mRNA level is unchanged during apoptosis (11) although its production is induced by apoptosis. The present work was designed to address whether pro-EMAPII is identical to p43 and to understand its function in the normal cell. The results showed that pro-EMAPII is associated with the N-terminal extension of human arginyl-tRNA synthetase (RRS), facilitating aminoacylation reaction. EXPE...

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Pparγ2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Pparγ2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis.