A yeast-optimized single-cell transcriptomics platform elucidates how mycophenolic acid and guanine alter global mRNA levels

The ability to measure the number of gene-specific mRNA molecules in individual mammalian cells has transformed the transcriptomics field. Among the key technologies enabling single-cell mRNA sequencing has been Droplet Sequencing (Drop-Seq). While this method works efficiently for mammalian cells, its direct application to yeast cells has been problematic due to cell-type specific differences such as size, doublet formation rate, and cell wall. Here we introduce YeastDropSeq, a single-cell RNA sequencing method for the study of transcriptomics in yeast. We modified and optimized the original Drop-Seq method to address the issues that emerged from smaller cell sizes and the presence of a cell wall in yeast. As proof-of-principle application of the YeastDropSeq, we investigated the transcriptomic effects of mycophenolic acid (MPA), a lifespan-extending compound that decreases de novo GMP synthesis. We compared transcript levels between cells treated with MPA and cells treated with DMSO and/or guanine, MPA’s epistatic agent. We discovered that isogenic populations of yeast cells contain transcriptionally distinct subpopulations and that the subpopulation structures were maintained despite the different treatment conditions. We found that cells treated with MPA experience an upregulation of genes coding for proteins involved in DNA replication stress-response, antioxidation, pre-RNA processing, and translation initiation. Conversely, a downregulation of mRNA expression was observed for genes encoding translation initiation and elongation factors, the 40S and 60S ribosomal subunits, and for genes involved in metal transport and mitochondrial function. YeastDropSeq will accelerate biological discovery by facilitating droplet-based transcriptomics of yeast cells.精准检测单个哺乳动物细胞内基因特异性mRNA分子数量的技术，彻底革新了转录组学领域。液滴测序（Droplet Sequencing, Drop-Seq）正是支撑单细胞mRNA测序技术发展的核心手段之一。尽管该方法在哺乳动物细胞中应用高效，但直接用于酵母细胞时却存在诸多问题，这源于两类细胞在尺寸、双联细胞形成率以及细胞壁等方面的细胞类型特异性差异。为此我们开发了YeastDropSeq——一款专为酵母转录组学研究设计的单细胞RNA测序方法。我们对原始的液滴测序方法进行了改造与优化，以解决酵母细胞尺寸更小、存在细胞壁所带来的一系列应用难题。作为YeastDropSeq的原理验证应用，我们探究了麦考酚酸（mycophenolic acid, MPA）——一种可延长寿命、抑制从头GMP合成的化合物——对酵母细胞的转录组学影响。我们比较了MPA处理组细胞、二甲基亚砜（dimethyl sulfoxide, DMSO）处理组细胞，以及MPA的上位性试剂鸟嘌呤（guanine）联合处理组细胞的转录水平差异。研究发现，遗传背景一致的酵母细胞群体中存在转录特征显著分化的亚群，且无论施加何种处理条件，这些细胞亚群的结构均得以保留。我们还观察到，经MPA处理的酵母细胞中，参与DNA复制应激响应、抗氧化、前体RNA加工以及翻译起始过程的蛋白编码基因均出现表达上调；与之相反，编码翻译起始与延伸因子、40S及60S核糖体亚基的基因，以及参与金属转运与线粒体功能的基因，其mRNA表达水平均呈现下调趋势。YeastDropSeq将通过助力基于液滴技术的酵母细胞转录组学研究，加速生物学新发现的进程。