Table1_Dynamics of alternative polyadenylation in single root cells of Arabidopsis thaliana.xlsx

IntroductionSingle-cell RNA-seq (scRNA-seq) technologies have been widely used to reveal the diversity and complexity of cells, and pioneering studies on scRNA-seq in plants began to emerge since 2019. However, existing studies on plants utilized scRNA-seq focused only on the gene expression regulation. As an essential post-transcriptional mechanism for regulating gene expression, alternative polyadenylation (APA) generates diverse mRNA isoforms with distinct 3’ ends through the selective use of different polyadenylation sites in a gene. APA plays important roles in regulating multiple developmental processes in plants, such as flowering time and stress response.
MethodsIn this study, we developed a pipeline to identify and integrate APA sites from different scRNA-seq data and analyze APA dynamics in single cells. First, high-confidence poly(A) sites in single root cells were identified and quantified. Second, three kinds of APA markers were identified for exploring APA dynamics in single cells, including differentially expressed poly(A) sites based on APA site expression, APA markers based on APA usages, and APA switching genes based on 3′ UTR (untranslated region) length change. Moreover, cell type annotations of single root cells were refined by integrating both the APA information and the gene expression profile.
ResultsWe comprehensively compiled a single-cell APA atlas from five scRNA-seq studies, covering over 150,000 cells spanning four major tissue branches, twelve cell types, and three developmental stages. Moreover, we quantified the dynamic APA usages in single cells and identified APA markers across tissues and cell types. Further, we integrated complementary information of gene expression and APA profiles to annotate cell types and reveal subtle differences between cell types.
DiscussionThis study reveals that APA provides an additional layer of information for determining cell identity and provides a landscape of APA dynamics during Arabidopsis root development.

引言
单细胞RNA测序（single-cell RNA-seq, scRNA-seq）技术已被广泛用于揭示细胞的多样性与复杂性，植物领域的scRNA-seq开创性研究自2019年起逐渐涌现。然而，现有植物scRNA-seq研究仅聚焦于基因表达调控。作为调控基因表达的关键转录后机制，可变多聚腺苷酸化（alternative polyadenylation, APA）通过选择性使用基因内不同的多聚腺苷酸化位点，产生具有不同3'末端的多样mRNA异构体。APA在调控植物诸多发育过程（如开花时间与胁迫响应）中发挥重要作用。
方法
本研究开发了一套分析流程，用于从不同scRNA-seq数据中识别并整合APA位点，并解析单细胞中的APA动态变化。首先，我们对单细胞根细胞中的高可信度多聚腺苷酸化（poly(A)）位点进行了识别与定量。其次，我们识别出三类用于探究单细胞APA动态变化的APA标记物，包括基于APA位点表达量的差异表达poly(A)位点、基于APA使用情况的APA标记物，以及基于3'非翻译区（3′ untranslated region, UTR）长度变化的APA转换基因。此外，通过整合APA信息与基因表达谱，我们对单细胞根细胞的细胞类型注释进行了优化。
结果
本研究从5项scRNA-seq研究中系统性整合构建了单细胞APA图谱，涵盖超过15万个细胞，涉及4个主要组织分支、12种细胞类型以及3个发育阶段。此外，我们对单细胞中的动态APA使用情况进行了定量，并鉴定出跨组织与细胞类型的APA标记物。进一步地，我们整合基因表达与APA图谱的互补信息以注释细胞类型，并揭示细胞类型间的细微差异。
讨论
本研究表明，APA可为细胞身份鉴定提供额外维度的信息，并揭示了拟南芥根发育过程中的APA动态变化全景。