RESUMO
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.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , RNA de Plantas/genética , Coloração e Rotulagem , Transcriptoma/genética , Meia-Vida , MicroRNAs/genética , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Plântula/genética , Uridina/metabolismoRESUMO
Intron splicing increases proteome complexity, promotes RNA stability, and enhances transcription. However, introns and the concomitant need for splicing extend the time required for gene expression and can cause an undesirable delay in the activation of genes. Here, we show that the plant microRNA processing factor SERRATE (SE) plays an unexpected and pivotal role in the regulation of intronless genes. Arabidopsis SE associated with more than 1000, mainly intronless, genes in a transcription-dependent manner. Chromatin-bound SE liaised with paused and elongating polymerase II complexes and promoted their association with intronless target genes. Our results indicate that stress-responsive genes contain no or few introns, which negatively affects their expression strength, but that some genes circumvent this limitation via a novel SE-dependent transcriptional activation mechanism. Transcriptome analysis of a Drosophila mutant defective in ARS2, the metazoan homologue of SE, suggests that SE/ARS2 function in regulating intronless genes might be conserved across kingdoms.