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 inducedtranslational 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.

Seryl-tRNA Synthetases in Translation and Beyond

“…[17,100,101] Previous studies have suggested a link between the tRNA aminoacylation and highmolecular-weight cellular complexes such as the cytoskeleton or ribosomes. [102][103][104] In all three domains of life, aaRSs form a variety of complexes with each other and with the non-enzymatic factors, [17,100] which may promote association of aaRSs with the ribosome. [102][103][104] However, the structural basis of these interactions and potential mechanistic implications are not well understood.…”

“…[102][103][104] In all three domains of life, aaRSs form a variety of complexes with each other and with the non-enzymatic factors, [17,100] which may promote association of aaRSs with the ribosome. [102][103][104] However, the structural basis of these interactions and potential mechanistic implications are not well understood. Bacterial-type SerRS rarely interacts with other proteins and only a few interacting partners have been identified to date.…”

Reply to Goodman et al.: Imaging protein synthesis with puromycin and the subcellular localization of puromycin–polypeptide conjugates

“…Once formed on the ribosome, a puromycin conjugate dissociates from it and is free to diffuse rapidly, just like a completed polypeptide chain. The letter by Goodman et al (5) cites the work by David et al (6) as evidence that polypeptide-puromycin conjugates colocalize with ribosomes on the endoplasmic reticulum. This reading of the work by David et al (6) ignores the critical fact that the experiment was performed in the presence of cycloheximide (6), which blocks the dissociation of polypeptide-puromycin conjugates from ribosomes.…”

“…The letter by Goodman et al (5) cites the work by David et al (6) as evidence that polypeptide-puromycin conjugates colocalize with ribosomes on the endoplasmic reticulum. This reading of the work by David et al (6) ignores the critical fact that the experiment was performed in the presence of cycloheximide (6), which blocks the dissociation of polypeptide-puromycin conjugates from ribosomes. Furthermore, the work by David et al (6) used a permeabilization step before fixation to remove soluble polypeptide-puromycin conjugates (6).…”

“…This reading of the work by David et al (6) ignores the critical fact that the experiment was performed in the presence of cycloheximide (6), which blocks the dissociation of polypeptide-puromycin conjugates from ribosomes. Furthermore, the work by David et al (6) used a permeabilization step before fixation to remove soluble polypeptide-puromycin conjugates (6). Thus, the staining pattern observed in the work by David et al (6) was the pattern of puromycin conjugates that are prevented from leaving the ribosome, which is very different from the studies that used puromycin without cycloheximide (2, 3); thus, this finding cannot be taken to reflect the normal distribution of puromycin conjugates in a cell.…”