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Decapped mRNAs during Arabidopsis Early Flower Development

ABSTRACT: The composition of the transcriptome is regulated by both mRNA synthesis and degradation. One route for mRNA decay is through 5’ decapping, which can be initiated by decapping enzymes and small RNAs. Although decapped RNAs are an important intermediate for mRNA decay, their identity and abundance have never been studied on a large scale. Here we present an experimental method for transcriptome-wide profiling of decapped mRNAs. We applied the method to study the prevalence of decapped transcripts during the early stages of Arabidopsis thaliana flower development. Decapped transcripts were identified for the majority of expressed genes, although at different levels. By comparing decapped RNA levels with steady-state overall transcript levels, our study provides evidence for widespread decapping-mediated mRNA degradation control in numerous biological processes and for genes of varied molecular functions, implying that mRNA decapping is a dynamically regulated process. Sequence analyses identified structural features of transcripts and cis-elements that were associated with levels of decapping.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE12043 | GEO | 2008/10/10

SECONDARY ACCESSION(S): PRJNA113369

REPOSITORIES: GEO

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Decapped mRNAs during Arabidopsis Early Flower Development

Project description:The composition of the transcriptome is regulated by both mRNA synthesis and degradation. One route for mRNA decay is through 5’ decapping, which can be initiated by decapping enzymes and small RNAs. Although decapped RNAs are an important intermediate for mRNA decay, their identity and abundance have never been studied on a large scale. Here we present an experimental method for transcriptome-wide profiling of decapped mRNAs. We applied the method to study the prevalence of decapped transcripts during the early stages of Arabidopsis thaliana flower development. Decapped transcripts were identified for the majority of expressed genes, although at different levels. By comparing decapped RNA levels with steady-state overall transcript levels, our study provides evidence for widespread decapping-mediated mRNA degradation control in numerous biological processes and for genes of varied molecular functions, implying that mRNA decapping is a dynamically regulated process. Sequence analyses identified structural features of transcripts and cis-elements that were associated with levels of decapping. Four sets of biologically independent tissue samples were collect 0,1,2,3,4, and 5 days after activation of the AP1-GR fusion protein. Decapped mRNA and total mRNA of each time point from each set were co-hybridized to micoarrays. Dyes used for labeling the RNA populations derived from the individual samples were switched in the replicate experiments to reduce dye-related artefacts.

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Project description:Eukaryotic RNAs with premature termination codons (PTCs) are eliminated by nonsense-mediated decay (NMD). While human nonsense RNA degradation can be initiated either by an endonucleolytic cleavage event near the PTC or through decapping, the individual contribution of these activities on endogenous substrates has remained unresolved. Here we unambiguously establish that SMG6-catalyzed endonucleolysis is the primary initiating step in human nonsense RNA decay. We also show that both protein-coding and ‘non-coding’ genes hosting snoRNAs in their introns produce considerable amounts of NMD-sensitive splice variants, suggesting that these RNAs are merely by-products of a primary snoRNA production process. Finally, genes encoding multiple snoRNAs generally yield elevated numbers of alternative transcript isoforms, enabling the differential expression of individual snoRNAs. These findings demonstrate a hitherto unappreciated potential for decoupling of the individual expression levels of functional exon- and intron-encoded species from such composite genes. HEK293 Flp-In T-Rex cells were subjected to siRNA-mediated depletion of XRN1 and co-depletion of XRN1 with either SMG6 or UPF1. All the treated samples together with the controls were subjected to both RNA-seq and 5'-end-seq. RNA-seq was used for detecting NMD isoforms and their expression levels. 5'-end-seq was used for finding NMD decay intermediates (decapped and endocleaved molecules). CAGE was used to distinguish decapped from endocleaved RNA fragments.

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