RESUMO
Interferons exert their biological function mainly through the activation of interferon-stimulated genes (ISGs). ISG12 (originally designated p27) belongs to a family of small, interferon alpha inducible genes of unknown function. We have determined the 5' end sequence of ISG12 cDNA from the human cell lines HeLa and AMA by RACE. Comparing this sequence to ISG12 sequences in the expressed sequence tag (EST) database revealed the presence of two alternative splice variants of ISG12 in human cells exhibiting the same open reading frame. We have sequenced the promoter region of the ISG12 gene and found ISRE, IRF1/IRF2, and STAT elements correlating to the interferon alpha inducibility of the gene. Subsequently, we have expressed human ISG12, a 12-kDa hydrophobic protein in the baculovirus expression system and with a C-terminal FLAG-tag in the human cell line 293. Recombinant ISG12 sediments in the nuclear envelope in both cell types. Finally, we have been able to demonstrate the prevalence of the ISG12 gene product in the nuclear envelope of HeLa cells treated with interferon alpha by immunocytochemical analyses. ISG12 is the first interferon induced protein found localizing to the nuclear envelope.
Assuntos
Núcleo Celular/metabolismo , Interferons/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Baculoviridae/genética , Sequência de Bases , Fracionamento Celular , Primers do DNA , DNA Complementar , Imunofluorescência , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana , Regiões Promotoras Genéticas , Proteínas/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
A full-length cDNA clone encoding the human mitochondrial tryptophanyl-tRNA synthetase (h(mt)TrpRS) has been identified. The deduced amino acid sequence shows high homology to both the mitochondrial tryptophanyl-tRNA synthetase ((mt)TrpRS) from Saccharomyces cerevisiae and to different eubacterial forms of tryptophanyl-tRNA synthetase (TrpRS). Using the baculovirus expression system, we have expressed and purified the protein with a carboxyl-terminal histidine tag. The purified His-tagged h(mt)TrpRS catalyzes Trp-dependent exchange of PP(i) in the PP(i)-ATP exchange assay. Expression of h(mt)TrpRS in both human and insect cells leads to high levels of h(mt)TrpRS localizing to the mitochondria, and in insect cells the first 18 amino acids constitute the mitochondrial localization signal sequence. Until now the human cytoplasmic tryptophanyl-tRNA synthetase (hTrpRS) was thought to function as the h(mt)TrpRS, possibly in the form of a splice variant. However, no mitochondrial localization signal sequence was ever detected and the present identification of a different (mt)TrpRS almost certainly rules out that possibility. The h(mt)TrpRS shows kinetic properties similar to human mitochondrial phenylalanyl-tRNA synthetase (h(mt)PheRS), and h(mt)TrpRS is not induced by interferon-gamma as is hTrpRS.