title: p23H implicated as cis/trans regulator of AlaXp-directed editing for mammalian cell homeostasis.
abstract: The toxicity of mistranslation of serine for alanine appears to be universal, and is prevented in part by the editing activities of alanyl-tRNA synthetases (AlaRSs), which remove serine from mischarged tRNA(Ala). The problem of serine mistranslation is so acute that free-standing, genome-encoded fragments of the editing domain of AlaRSs are found throughout evolution. These AlaXps are thought to provide functional redundancy of editing. Indeed, archaeal versions rescue the conditional lethality of bacterial cells harboring an editing-inactive AlaRS. In mammals, AlaXps are encoded by a gene that fuses coding sequences of a homolog of the HSP90 cochaperone p23 (p23(H)) to those of AlaXp, to create p23(H)AlaXp. Not known is whether this fusion protein, or various potential splice variants, are expressed as editing-proficient proteins in mammalian cells. Here we show that both p23(H)AlaXp and AlaXp alternative splice variants can be detected as proteins in mammalian cells. The variant that ablated p23(H) and encoded just AlaXp was active in vitro. In contrast, neither the p23(H)AlaXp fusion protein, nor the mixture of free p23(H) with AlaXp, was active. Further experiments in a mammalian cell-based system showed that RNAi-directed suppression of sequences encoding AlaXp led to a serine-sensitive increase in the accumulation of misfolded proteins. The results demonstrate the dependence of mammalian cell homeostasis on AlaXp, and implicate p23(H) as a cis- and trans-acting regulator of its activity.
authors.affiliation: The Skaggs Institute for Chemical Biology and Department of Molecular Biology, Scripps Research Institute, BCC-379, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Mir Hussain
authors.initials: MH
authors.lastname: Nawaz
authors.forename: Eve
authors.initials: E
authors.lastname: Merriman
authors.forename: Xiang-Lei
authors.initials: XL
authors.lastname: Yang
authors.forename: Paul
authors.initials: P
authors.lastname: Schimmel
mesh_terms: D000412:Alanine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D006706:Homeostasis / Q000502:physiology*; D019746:Intramolecular Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D000071876:Prostaglandin-E Synthases; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D034622:RNA Interference; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D020033:Protein Isoforms; D012346:RNA, Transfer, Amino Acyl; D012694:Serine; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases; D000412:Alanine-tRNA Ligase