As noted in this earlier essay, the poly(A) tail collaborates with the 5′-end of the mRNA (the so-called cap) to promote both mRNA translation and stability. Accordingly, decapping is a good hallmark for mRNA turnover. In a recent issue of The Plant Cell, Jiao et al. describe an approach to study uncapped mRNAs on a global basis. Briefly, these authors take advantage of the fact that an uncapped mRNA has a 5′ phosphate group, and thus can be a substrate for RNA ligase. By attaching an RNA adapter to the uncapped mRNAs using this enzyme, and then purifying and amplifying DNA products derived from these, the authors were able to prepare probes for microarray studies. Thus, they were able to assess uncapped mRNA abundance on a genome-wide basis. As a test for this approach, they studied decapping genome-wide during the early stages of flowering using a mutant arrested for flower development at a specific stage, but carrying a chemically-inducible transgene that could trigger flowering by providing for some of the functionality missing in the mutant. This group found that a sizable portion of mRNAs that could be detected on the microarrays (approaching 40%) were either “over-capped” or “under-capped”; that is to say, the relative abundances of uncapped mRNAs differed from the total levels of the corresponding transcripts. They also found a number of transcripts (some 300 or so) whose capping status changed during flowering. All told, as stated by the authors, this system should be useful for exploring regulated mRNA turnover, and for identifying correlations between mRNA sequence/structure and stability.
The abstract of the paper:
The composition of the transcriptome is determined by a balance between mRNA synthesis and degradation. An important route for mRNA degradation produces uncapped mRNAs, and this decay process can be initiated by decapping enzymes, endonucleases, and small RNAs. Although uncapped mRNAs are an important intermediate for mRNA decay, their identity and abundance have never been studied on a large scale until recently. Here, we present an experimental method for transcriptome-wide profiling of uncapped mRNAs that can be used in any eukaryotic system. We applied the method to study the prevalence of uncapped transcripts during the early stages of Arabidopsis thaliana flower development. Uncapped transcripts were identified for the majority of expressed genes, although at different levels. By comparing uncapped RNA levels with steady state overall transcript levels, our study provides evidence for widespread mRNA degradation control in numerous biological processes involving genes of varied molecular functions, implying that uncapped mRNA levels are dynamically regulated. Sequence analyses identified structural features of transcripts and cis-elements that were associated with different levels of uncapping. These transcriptome-wide profiles of uncapped mRNAs will aid in illuminating new regulatory mechanisms of eukaryotic transcriptional networks.