Comprehensive characterization of mRNAs associated with yeast cytosolic aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases (aaRSs) are a conserved family of enzymes with an essential role in protein synthesis: ligating amino acids to cognate tRNA molecules for translation. In addition to their role in tRNA charging, aaRSs have acquired non-canonical functions, including post-transcriptional regulation of mRNA expression. Yet, the extent and mechanisms of these post-transcriptional functions are largely unknown. Herein, we performed a comprehensive transcriptome analysis to define the mRNAs that are associated with almost all aaRSs present in <i>S. cerevisiae</i> cytosol. Nineteen (out of twenty) isogenic strains of GFP-tagged cytosolic aaRSs were subjected to immunoprecipitation with anti-GFP beads along with an untagged control. mRNAs associated with each aaRS were then identified by RNA-seq. The extent of mRNA association varied significantly between aaRSs, from MetRS in which none appeared to be statistically significant, to PheRS that binds hundreds of different mRNAs. Interestingly, many target mRNAs are bound by multiple aaRSs, suggesting co-regulation by this family of enzymes. Gene Ontology analyses for aaRSs with a considerable number of target mRNAs discovered an enrichment for pathways of amino acid metabolism and of ribosome biosynthesis. Furthermore, sequence and structure motif analysis revealed for some aaRSs an enrichment for motifs that resemble the anticodon stem loop of cognate tRNAs. These data suggest that aaRSs coordinate mRNA expression in response to amino acid availability and may utilize RNA elements that mimic their canonical tRNA binding partners.

氨酰-tRNA合成酶（Aminoacyl-tRNA synthetases，aaRSs）是一类保守的酶家族，在蛋白质合成中发挥核心作用：将氨基酸连接至同源tRNA分子以完成翻译过程。除了参与tRNA氨酰化修饰之外，aaRSs还演化出非经典功能，包括对mRNA表达的转录后调控。然而，这类转录后功能的覆盖范围与作用机制仍在很大程度上尚不明确。本研究通过全面的转录组分析，鉴定出酿酒酵母（Saccharomyces cerevisiae，S. cerevisiae）胞质中几乎所有aaRSs所结合的mRNA。我们将20株胞质aaRSs的GFP标记同基因菌株中的19株，与未标记的对照菌株一同采用抗GFP磁珠进行免疫共沉淀；随后通过RNA测序（RNA-seq）鉴定出与每种aaRS相结合的mRNA。不同aaRSs与mRNA的结合程度差异显著：从MetRS未检测到具有统计学意义的结合mRNA，到PheRS可结合数百种不同的mRNA。有趣的是，诸多靶mRNA可被多种aaRSs结合，这提示该酶家族存在协同调控作用。对靶mRNA数量较多的aaRSs进行基因本体（Gene Ontology，GO）富集分析发现，其靶基因显著富集于氨基酸代谢与核糖体生物合成通路。此外，序列与结构基序分析显示，部分aaRSs所识别的结合基序与对应tRNA的反密码子茎环结构高度相似。上述结果表明，aaRSs可响应氨基酸的可获得性协调mRNA表达，并可能通过模拟其经典结合伴侣tRNA的RNA元件来发挥调控功能。