Abstract
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.
Highlights
Transcriptomes are highly dynamic and vary greatly among different cell types, environmental conditions, and developmental stages
Seedlings germinated on MS medium containing 4-SU or cordycepin died after germination. 5-ethynyl uridine (5-EU) or BrU had no obvious negative effect on germination or seedling development (Supplemental Figure 1)
Our results indicate that 5-EU is taken up by the plant, is incorporated into RNAs generated from nuclear and chloroplast genes, and does not interfere with RNA processing steps such as splicing. 5-EU is effectively coupled to biotin using click chemistry, allowing for the efficient purification of labeled RNA
Summary
Transcriptomes are highly dynamic and vary greatly among different cell types, environmental conditions, and developmental stages. Plant transcriptome analysis by RNA sequencing (RNA-seq) has become an affordable tool for understanding plant development and adaptive growth, for assessing the effects of specific mutations, and for improving genome annotation. Most transcriptomic studies in plants focus on detecting steady-state transcriptomes, which are defined by the rate of RNA synthesis and degradation. The presence of long-lived and abundant RNAs in steady-state transcriptomes limits the detection of less abundant and unstable RNAs including RNA processing intermediates. Certain techniques take such challenges into account.
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