Transcriptome analysis by RNA sequencing (RNA-seq) has become an indispensable research tool in modern plant biology. Virtually all RNA-seq studies provide a snapshot of the steady state transcriptome, which contains valuable information about RNA populations at a given time but lacks information about the dynamics of RNA synthesis and degradation. Only a few specialized sequencing techniques, such as global run-on sequencing, have been used to provide information about RNA synthesis rates in plants. Here, we demonstrate that RNA labeling with the modified, nontoxic uridine analog 5-ethynyl uridine (5-EU) in Arabidopsis (Arabidopsis thaliana) seedlings provides insight into plant transcriptome dynamics. Pulse labeling with 5-EU revealed nascent and unstable RNAs, RNA processing intermediates generated by splicing, and chloroplast RNAs. Pulse-chase experiments with 5-EU allowed us to determine RNA stabilities without the need for chemical transcription inhibitors such as actinomycin and cordycepin. Inhibitor-free, genome-wide analysis of polyadenylated RNA stability via 5-EU pulse-chase experiments revealed RNAs with shorter half-lives than those reported after chemical inhibition of transcription. In summary, our results indicate that the Arabidopsis nascent transcriptome contains unstable RNAs and RNA processing intermediates and suggest that polyadenylated RNAs have low stability in plants. Our technique lays the foundation for easy, affordable, nascent transcriptome analysis and inhibitor-free analysis of RNA stability in plants.

通过RNA测序（RNA-seq）进行转录组分析已成为现代植物生物学中必不可少的研究工具。几乎所有RNA-seq研究都提供了稳态转录组的快照，其中包含给定时间有关RNA种群的有价值的信息，但缺少有关RNA合成和降解动力学的信息。仅使用了一些专门的测序技术（例如全局连续测序）来提供有关植物中RNA合成速率的信息。在这里，我们证明了在拟南芥（Arabidopsis thaliana）中用修饰的无毒尿苷类似物5-乙炔基尿苷（5-EU）标记RNA）幼苗提供了对植物转录组动力学的深入了解。用5-EU进行脉冲标记显示出新生的和不稳定的RNA，剪接产生的RNA处理中间体以及叶绿体RNA。使用5-EU进行的脉冲追踪实验使我们无需化学转录抑制剂（如放线菌素和虫草素）即可确定RNA的稳定性。通过5-EU脉冲追踪实验对多聚腺苷酸RNA稳定性进行无抑制剂的全基因组分析，发现半衰期比化学抑制转录后报道的半衰期短。总之，我们的结果表明拟南芥新生的转录组包含不稳定的RNA和RNA加工中间体，并表明聚腺苷酸化RNA在植物中的稳定性很低。我们的技术奠定了轻松，负担得起的基础，