Quantitative polyadenylation map of mouse embryonic epidermal lineages

We establish an experimental and bioinformatic pipeline using 3SEQ to quantitatively measure mRNA expression and reliably determine 3' end formation by sequencing polyadenylated transcripts. When applied to purified mouse embryonic skin stem cells direct differentiation lineages, we identify 15,651 UTRs representing 10,442 distinct mRNAs that are abundantly expressed in the skin. We determine that ~80% of UTRs are formed by using canonical A[A/U]UAAA polyadenylation signals, whereas ~20% of UTRs utilize alternative signals. We demonstrate that comparing with qPCR, our RNA-Seq approach can precisely measure mRNA fold-change and accurately determine the expression of mRNAs over four orders of magnitude. We also reported 453 out of 10,442 genes (4.3%) show differential 3' end usage between skin stem cells and their direct differentiation lineages. Among them, core components of the miRNA pathway, including Dicer, Dgcr8, Xpo5 and Ago2, show dynamic 3' UTR formation patterns, indicating a self-regulatory mechanism. Together, our quantitative analysis reveals a dynamic picture of mRNA 3' end formation in closely related somatic stem cell lineages. Perform 3SEQ in E14 epidermal basal cells and E14 epidermal suprabasal cells.本研究搭建了一套基于3SEQ的实验与生物信息学流程，通过对聚腺苷酸化转录本进行测序，实现mRNA表达的定量检测，并可靠鉴定mRNA的3'端形成情况。当将该流程应用于纯化的小鼠胚胎皮肤干细胞及其直接分化谱系时，我们共鉴定出15651个未翻译区域（UTR，Untranslated Region），对应10442种在皮肤中高表达的不同mRNA。我们发现约80%的UTR由经典的A[A/U]UAAA聚腺苷酸化信号介导形成，而约20%的UTR则采用替代性聚腺苷酸化信号。我们通过与实时定量PCR（qPCR）对比验证，证实本研究的RNA-Seq方法可精准测定mRNA的表达倍数变化，并能在四个数量级范围内准确定量mRNA的表达水平。我们还发现，在10442个基因中，有453个（占比4.3%）在皮肤干细胞与其直接分化谱系之间存在3'端使用差异。其中，microRNA通路（miRNA pathway）的核心组分，包括Dicer、Dgcr8、Xpo5及Ago2，均呈现出动态的3' UTR形成模式，提示存在一种自我调控机制。综上，本研究的定量分析揭示了紧密关联的体细胞干细胞谱系中mRNA 3'端形成的动态图景。本研究还在胚胎第14天（E14）的表皮基底细胞与表皮上层细胞中开展了3SEQ实验。