An alternative transcriptome shapes cell fate transitions in yeast (TIF-seq). An alternative transcriptome shapes cell fate transitions in yeast (TIF-seq)

Alternative mRNA isoforms and long noncoding RNAs (lncRNA) make up a large fraction of the transcriptome and play key functions in cell-fate programming. These transcripts often initiate upstream of coding gene promoters from alternative transcription start sites (TSS) where they can regulate gene expression in cis through transcription-coupled chromatin alterations. How, when and where transcription of alternative cis-acting RNAs regulates local gene expression remains poorly understood. Here, we use a high-resolution quantitative approach to study alternative TSS and transcript end site (TES) usage during three different cell fate transitions in yeast: entry into gametogenesis, commitment to meiotic divisions and return to vegetative growth. We propose that an alternative transcriptome of mRNA isoforms and lncRNAs shapes local gene expression during cell fate transitions. Hence, changes in the types and proportions of different RNAs transcribed at a locus are important inputs for gene expression at distinct stages of development. Overall design: Transcript Isoform sequencing (TIF) analysis of Saccharomyces cerevisiae SK1 strains. All strains used in this study harbor the IME1gene controlled by a copper inducible promoter (pCUP1), the NDT80 gene controlled by a galactose inducible promoter (pGAL) and a constitutively expressed Gal4 transcription factor fused to the hormone binding domain of the estrogen receptor (Gal4-ER). For the sporulation time course, cells were grown overnight in YPD medium, diluted, and shifted to sporulation media (SPO). IME1 was induced after 2h in Spo by adding 50 μM CuSO4. Time points for each cell fate transition were pooled in equal proportions for library construction and sequencing. Two independent replicates were sequenced.. Details for library preparation are described below.

可变mRNA异构体与长链非编码RNA（long noncoding RNA，lncRNA）占转录组的重要组分，并在细胞命运编程中发挥核心调控功能。这类转录本通常起始于编码基因启动子上游的可变转录起始位点（transcription start site，TSS），并可通过转录偶联的染色质重塑以顺式作用方式调控基因表达。目前，可变顺式作用RNA的转录如何、何时以及在何处调控局部基因表达，仍有待深入阐释。本研究采用高分辨率定量分析方法，探究酿酒酵母（Saccharomyces cerevisiae）中三类不同细胞命运转变过程中的可变TSS与转录终止位点（transcript end site，TES）使用模式，这三类转变分别为配子发生启动、减数分裂分化命运确立，以及营养生长回归。我们提出，由mRNA异构体与lncRNA构成的可变转录组，可在细胞命运转变过程中塑造局部基因表达模式。因此，某一基因座上转录的不同RNA的类型与比例变化，是发育不同阶段基因表达的关键调控输入因素。整体实验设计：对酿酒酵母SK1菌株开展转录异构体测序（Transcript Isoform sequencing，TIF）分析。本研究使用的所有菌株均携带三类调控元件：由铜诱导型启动子（pCUP1）调控的IME1基因、由半乳糖诱导型启动子（pGAL）调控的NDT80基因，以及与雌激素受体激素结合域融合的组成型表达Gal4转录因子（Gal4-ER）。针对孢子形成时间进程实验：细胞先在YPD培养基中过夜培养，经稀释后转接至孢子形成培养基（SPO）；在SPO培养基中培养2小时后，通过添加50 μM硫酸铜（CuSO4）诱导IME1基因表达。各细胞命运转变的时间点样本按等比例混合，用于文库构建与高通量测序。本研究设置两个独立生物学重复并完成测序。文库制备的详细步骤详见下文。