p23 ^H 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.

A Remodeled Hsp90 Molecular Chaperone Ensemble with the Novel Cochaperone Aarsd1 Is Required for Muscle Differentiation

“…Based on our own extensive RT-PCR and immunoblotting experiments ( Fig. 1C and 2), we conclude that a short form, here denoted Aarsd1S, is the protein product of transcript 4 and corresponds to what was previously referred to as tsp23 (28) or p23 H (29). The CS domain is encoded by exons 1 to 4, and in the case of Aarsd1S, the coding sequence extends (40 amino acids out of a total of 131) through exon 5 and into the very beginning of exon 8.…”

“…Antibodies used for the specific recognition of Hsp90␣ were rabbit polyclonal antibody PA3-013 (Affinity BioReagents) and mouse monoclonal antibody (MAb) EMD-17D7 (Calbiochem). MAb H90-10 against Hsp90␤ and MAb JJ10 against p23 were kindly provided by David O. Toft (Mayo Clinic, Rochester, NY), and the polyclonal antiserum that recognizes the three main Aarsd1 isoforms was kindly provided by Paul Schimmel (Scripps Research Institute) (29). Myosin heavy chain was detected with MAb BA-G5 (Abcam), Cdk4 was detected with MAb Ab-6 (Thermo Scientific), and Klf15 was detected with rabbit polyclonal antibody Ag4690 (Proteintech).…”

“…H domain, present in some splice isoforms, inhibits this editing activity both in cis and in trans (29). Based on these findings, it seemed possible that this unusual p23 relative, or set of relatives, replaces p23 in muscle and contributes to the fundamental remodeling of the Hsp90 ensemble, conceivably to adapt it to muscle-specific functions.…”

A Multiple Aminoacyl-tRNA Synthetase Complex That Enhances tRNA-Aminoacylation in African Trypanosomes

“…In addition, bioinformatic analysis identified the T. brucei Aarsd1 (TbAarsd1), the homolog of mouse Aarsd1, although it was not found in the MARS complex. TbAarsd1 may be involved in preventing misaminoacylation, similar to its homologs (68,69). The distribution of these domains in separate proteins that may or may not be stably associated with the MARS complex or be part of the aaRS enzyme in different species likely reflects their different needs for aminoacylation quality control.…”

Transfer RNA: A dancer between charging and mis-charging for protein biosynthesis

“…AlaX, which could be divided into two types based on the presence or absence of C-Ala domain, is encoded in the three domains of life [93]. In mammals, it is expressed as a fused protein with the HSP90 cochaperone p23 homolog in some tissues [99]. The active site for hydrolysis shares homology with the editing domains of AlaRS and ThrRS.…”