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RNA binding targets aminoacyl-tRNA synthetases to translating ribosomes.

ABSTRACT: Here, we examine tRNA-aminoacyl synthetase (ARS) localization in protein synthesis. Proteomics reveals that ten of the twenty cytosolic ARSs associate with ribosomes in sucrose gradients: phenylalanyl-RS (FRS), and the 9 ARSs that form the multi-ARS complex (MSC). Using the ribopuromycylation method (RPM) for localizing intracellular translation, we show that FRS and the MSC, and to a lesser extent other ARSs, localize to translating ribosomes, most strikingly when translation is restricted to poxvirus or alphavirus factories in infected cells. Immunoproximity fluorescence indicates close proximity between MSC and the ribosome. Stress induced-translational shutdown recruits the MSC to stress-granules, a depot for mRNA and translation components. MSC binding to mRNA provides a facile explanation for its delivery to translating ribosomes and stress granules. These findings, along with the abundance of the MSC (9 × 10(6) copies per cell, roughly equimolar with ribosomes), is consistent with the idea that MSC specificity, recently reported to vary with cellular stress (Netzer, N., Goodenbour, J. M., David, A., Dittmar, K. A., Jones, R. B., Schneider, J. R., Boone, D., Eves, E. M., Rosner, M. R., Gibbs, J. S., Embry, A., Dolan, B., Das, S., Hickman, H. D., Berglund, P., Bennink, J. R., Yewdell, J. W., and Pan, T. (2009) Nature 462, 522-526) can be modulated at the level of individual mRNAs to modify decoding of specific gene products.

SUBMITTER: David A

PROVIDER: S-EPMC3121505 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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RNA binding targets aminoacyl-tRNA synthetases to translating ribosomes.

David Alexandre A Netzer Nir N Strader Michael Brad MB Das Suman R SR Chen Cai Yun CY Gibbs James J Pierre Philippe P Bennink Jack R JR Yewdell Jonathan W JW

The Journal of biological chemistry 20110401 23

Here, we examine tRNA-aminoacyl synthetase (ARS) localization in protein synthesis. Proteomics reveals that ten of the twenty cytosolic ARSs associate with ribosomes in sucrose gradients: phenylalanyl-RS (FRS), and the 9 ARSs that form the multi-ARS complex (MSC). Using the ribopuromycylation method (RPM) for localizing intracellular translation, we show that FRS and the MSC, and to a lesser extent other ARSs, localize to translating ribosomes, most strikingly when translation is restricted to p ...[more]

PMID: 21460219

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Project description:BackgroundTreatment of parasitic diseases has been challenging due to evolution of drug resistant parasites, and thus there is need to identify new class of drugs and drug targets. Protein translation is important for survival of malarial parasite, Plasmodium, and the pathway is present in all of its life cycle stages. Aminoacyl tRNA synthetases are primary enzymes in protein translation as they catalyse amino acid addition to the cognate tRNA. This study sought to understand differences between Plasmodium and human aminoacyl tRNA synthetases through bioinformatics analysis.MethodsPlasmodium berghei, Plasmodium falciparum, Plasmodium fragile, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Plasmodium yoelii and human aminoacyl tRNA synthetase sequences were retrieved from UniProt database and grouped into 20 families based on amino acid specificity. These families were further divided into two classes. Both families and classes were analysed. Motif discovery was carried out using the MEME software, sequence identity calculation was done using an in-house Python script, multiple sequence alignments were performed using PROMALS3D and TCOFFEE tools, and phylogenetic tree calculations were performed using MEGA vs 7.0 tool. Possible alternative binding sites were predicted using FTMap webserver and SiteMap tool.ResultsMotif discovery revealed Plasmodium-specific motifs while phylogenetic tree calculations showed that Plasmodium proteins have different evolutionary history to the human homologues. Human aaRSs sequences showed low sequence identity (below 40%) compared to Plasmodium sequences. Prediction of alternative binding sites revealed potential druggable sites in PfArgRS, PfMetRS and PfProRS at regions that are weakly conserved when compared to the human homologues. Multiple sequence analysis, motif discovery, pairwise sequence identity calculations and phylogenetic tree analysis showed significant differences between parasite and human aaRSs proteins despite functional and structural conservation. These differences may provide a basis for further exploration of Plasmodium aminoacyl tRNA synthetases as potential drug targets.ConclusionThis study showed that, despite, functional and structural conservation, Plasmodium aaRSs have key differences from the human homologues. These differences in Plasmodium aaRSs can be targeted to develop anti-malarial drugs with less toxicity to the host.

Project description:BACKGROUND:A century ago, pantheras were abundant across Asia. Illegal hunting and trading along with loss of habitat have resulted in the designation of Panthera as a genus of endangered species. In addition to the onslaught from humans, pantheras are also susceptible to outbreaks of several infectious diseases, including babesiosis. The latter is a hemoprotozoan disease whose causative agents are the eukaryotic parasites of the apicomplexan genus Babesia. Babesiosis affects a varied range of animals including humans (Homo sapiens), bovines (e.g. Bos taurus), pantheras (e.g. Panthera tigris, P. leo, P. pardus) and equines. Babesia spp. are transmitted by the tick vector Ixodes scapularis or ticks of domestic animals, namely Rhipicephalus (Boophilus) microplus and R. (B.) decoloratus. At the level of protein translation within these organisms, the conserved aminoacyl tRNA synthetase (aaRS) family offers an opportunity to identify the sequence and structural differences in the host (Panthera) and parasites (Babesia spp.) in order to exploit these for drug targeting Babesia spp. METHODS:Using computational tools we investigated the genomes of Babesia spp. and Panthera tigris so as to annotate their aaRSs. The sequences were analysed and their subcellular localizations were predicted using Target P1.1, SignalP 3.0, TMHMM v.2.0 and Deeploc 1.0 web servers. Structure-based analysis of the aaRSs from P. tigris and its protozoan pathogens Babesia spp. was performed using Phyre2 and chimera. RESULTS:We identified 33 (B. bovis), 34 (B. microti), 33 (B. bigemina) and 33 (P. tigris) aaRSs in these respective organisms. Poor sequence identity (~ 20-50%) between aaRSs from Babesia spp. and P. tigris was observed and this merits future experiments to validate new drug targets against Babesia spp. CONCLUSIONS:Overall this work provides a foundation for experimental investigation of druggable aaRSs from Babesia sp. in an effort to control Babesiosis in Panthera.

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RNA binding targets aminoacyl-tRNA synthetases to translating ribosomes.

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RNA binding targets aminoacyl-tRNA synthetases to translating ribosomes.

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