Dynamic Strain- and Stage-specific tRNA Transcription and Codon Usage in divergent Toxoplasma gondii strains. Toxoplasma gondii

tRNAs are critical linker molecules that couple mRNA to protein translation and their availability is a limiting factor for translation. tRNA transcription and the composition of the tRNA pool are both dynamically regulated in response to environmental cues. Recent work suggests that they also play an important role in regulating cell growth and differentiation. Toxoplasma gondii is a highly adaptable, early eukaryotic protozoan that is able to replicate in virtually any nucleated cell and many different host organisms, thus requiring fine-tuning of protein expression. A key feature of parasite pathogenesis is the ability to differentiate between fast growing tachyzoite and slow growing bradyzoite cyst forms, in response to stress e.g. nutrient starvation. Differentiation is accompanied by changes to the mRNA transcriptome and proteome but little is known about translational regulation. In this study, we used comparative genomics to catalogue the repertoire of tRNA genes in T. gondii and their genomic distribution. Interestingly, many tRNAs occur in regulatory regions and intronic sequences of protein-coding genes, which correlates with increased gene expression. To characterize the tRNA pool of T. gondii during stress and differentiation, we surveyed levels of precursor tRNA molecules in the tRNA population by small RNA-seq in three divergent parasite strains with varying bradyzoite differentiation competencies, cultured in normal or bradyzoite differentiation-inducing (pH shock) conditions. Almost all tRNA genes are constitutively transcribed in T. gondii tachyzoites and transcription of individual tRNA genes differs significantly between strains. The abundance of individual tRNAs in the tRNA pool reflects proteomic codon usage, suggesting that tRNA levels are tightly controlled according to the demands of translation. Together, this work suggests that modulation of the tRNA pool is a mechanism by which the protein landscape in T. gondii is regulated in different strains and developmental stages. Overall design: Three divergent Toxoplasma gondii strains were cultured in tachyzoite (pH7) or bradyzoite (pH8) growth conditions and subjected to small RNA-seq.

转运RNA（transfer RNA, tRNA）是介导信使RNA（messenger RNA, mRNA）与蛋白质翻译过程的关键衔接分子，其丰度是限制翻译进程的关键因素之一。tRNA的转录过程以及tRNA库的组成会响应环境信号发生动态调控。现有研究表明，tRNA在调控细胞生长与分化过程中同样发挥关键作用。刚地弓形虫（Toxoplasma gondii）是一种适应性极强的原始真核原生生物，几乎可在所有有核细胞以及多种不同宿主生物体内增殖，因此需要对蛋白质表达进行精细调控。该寄生虫致病过程的核心特征之一，是能够响应营养匮乏等应激信号，在快速增殖的速殖子（tachyzoite）与缓慢增殖的包囊型缓殖子（bradyzoite）之间发生分化。该分化过程伴随mRNA转录组与蛋白质组的显著改变，但目前学界对其翻译调控机制的认知仍较为有限。本研究采用比较基因组学方法，对刚地弓形虫的全套tRNA基因及其基因组分布特征进行了系统编目分析。值得注意的是，众多tRNA基因定位于蛋白质编码基因的调控区域与内含子序列中，这一分布特征与基因表达水平的升高呈显著正相关。为解析刚地弓形虫在应激与分化过程中的tRNA库特征，本研究选取三株遗传背景各异、缓殖子分化能力存在差异的寄生虫，分别在正常培养条件与缓殖子分化诱导条件（pH冲击）下进行培养，随后通过小RNA测序（small RNA-seq）检测了tRNA群体中前体tRNA分子的表达水平。刚地弓形虫速殖子中几乎所有tRNA基因均为组成型转录，且不同菌株间单个tRNA基因的转录水平存在显著差异。tRNA库中单个tRNA的丰度与蛋白质组的密码子使用偏好性高度匹配，这表明tRNA的表达水平会根据翻译过程的需求受到严格调控。综上，本研究结果表明，调控tRNA库是刚地弓形虫在不同菌株与发育阶段中调控蛋白质表达谱的关键机制之一。实验整体设计：将三株遗传背景各异的刚地弓形虫分别在速殖子培养条件（pH7）与缓殖子培养条件（pH8）下进行培养，随后进行小RNA测序（small RNA-seq）。