Transcriptome analysis by RNA sequencing (RNA-seq) has become an indispensable core 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 (GRO-seq), have been applied in plants and provide information about RNA synthesis rates. Here, we demonstrate that RNA labeling with a modified, non-toxic uridine analog, 5-ethynyl uridine (5-EU), in Arabidopsis thaliana seedlings provides insight into the dynamic nature of a plant transcriptome. Pulse-labeling with 5-EU allowed the detection and analysis of nascent and unstable RNAs, of RNA processing intermediates generated by splicing, and of chloroplast RNAs. We also conducted pulse-chase experiments with 5-EU, which allowed us to determine RNA stabilities without the need for chemical inhibition of transcription using compounds such as actinomycin and cordycepin. Genome-wide analysis of RNA stabilities by 5-EU pulse-chase experiments revealed that this inhibitor-free RNA stability measurement results in RNA half-lives much shorter than those reported after chemical inhibition of transcription. In summary, our results show that the Arabidopsis nascent transcriptome contains unstable RNAs and RNA processing intermediates, and suggest that half-lives of plant RNAs are largely overestimated. Our results lay the ground for an easy and affordable nascent transcriptome analysis and inhibitor-free analysis of RNA stabilities 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核心研究工具。几乎所有的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。植物中仅应用了几种特殊的测序技术，例如全局连续测序（GRO-seq），可提供有关RNA合成速率的信息。在这里，我们证明在拟南芥幼苗中用修饰的无毒尿苷类似物5-乙炔基尿苷（5-EU）标记RNA提供了对植物转录组动态性质的了解。使用5-EU进行脉冲标记可以检测和分析新生和不稳定的RNA，剪接产生的RNA加工中间体和叶绿体RNA。我们还使用5-EU进行了脉冲追踪实验，该实验使我们无需使用化学抑制剂如放线菌素和虫草素就可以确定转录的稳定性，而无需进行化学抑制。通过5-EU脉冲追踪实验对RNA稳定性进行全基因组分析，结果表明，这种无抑制剂的RNA稳定性测量结果使RNA半衰期比化学抑制转录后报道的半衰期短得多。总而言之，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，并且表明植物RNA的半衰期被高估了。