High-throughput approaches for profiling the 59 ends of RNA degradation intermediates on a genome-wide scale are frequently applied to analyze and validate cleavage sites guided by microRNAs (miRNAs). However, the complexity of the RNA degradome other than miRNA targets is currently largely uncharacterized, and this limits the application of RNA degradome studies. We conducted a global analysis of 59-truncated mRNA ends that mapped to coding sequences (CDSs) of Arabidopsis thaliana, rice (Oryza sativa), and soybean (Glycine max). Based on this analysis, we provide multiple lines of evidence to show that the plant RNA degradome contains in vivo ribosome-protected mRNA fragments. We observed a 3-nucleotide periodicity in the position of free 59 RNA ends and a bias toward the translational frame. By examining conserved peptide upstream open reading frames (uORFs) of Arabidopsis and rice, we found a predominance of 59 termini of RNA degradation intermediates that were separated by a length equal to a ribosome-protected mRNA fragment. Through the analysis of RNA degradome data, we discovered uORFs and CDS regions potentially associated with stacked ribosomes in Arabidopsis. Furthermore, our analysis of RNA degradome data suggested that the binding of Arabidopsis ARGONAUTE7 to a noncleavable target site of miR390 might directly hinder ribosome movement. This work demonstrates an alternative use of RNA degradome data in the study of ribosome stalling.

BackgroundDegradation is essential for RNA maturation, turnover, and quality control. RNA degradome sequencing that integrates a modified 5′-rapid amplification of cDNA ends protocol with next-generation sequencing technologies is a high-throughput approach for profiling the 5′-end of uncapped RNA fragments on a genome-wide scale. The primary application of degradome sequencing has been to identify the truncated transcripts that result from endonucleolytic cleavage guided by microRNAs or small interfering RNAs. As many pathways are involved in RNA degradation, degradome data should contain other RNA species besides the cleavage remnants of small RNA targets. Nevertheless, no systematic approaches have been established to explore the hidden complexity of plant degradome.ResultsThrough analyzing Arabidopsis and rice RNA degradome data, we recovered 11 short motifs adjacent to predominant and abundant uncapped 5′-ends. Uncapped ends associated with several of these short motifs were more prevalent than those targeted by most miRNA families especially in the 3′ untranslated region of transcripts. Through genome-wide analysis, five motifs showed preferential accumulation of uncapped 5′-ends at the same position in Arabidopsis and rice. Moreover, the association of uncapped 5′-ends with a CA-repeat motif and a motif recognized by Pumilio/Fem-3 mRNA binding factor (PUF) proteins was also found in non-plant species, suggesting that common mechanisms are present across species. Based on these motifs, potential sources of RNA ends that constitute degradome data were proposed and further examined. The 5′-end of small nucleolar RNAs could be precisely captured by degradome sequencing. Position-specific enrichment of uncapped 5′-ends was seen upstream of motifs recognized by several RNA binding proteins especially for the binding site of PUF proteins. False uncapped 5′-ends produced from capped transcripts through non-specific PCR amplification were common artifacts among degradome datasets.ConclusionsThe complexity of plant RNA degradome data revealed in this study may contribute to the alternative applications of degradome in RNA research.

Exon junction complexes (EJCs) are deposited on mRNAs during splicing and displaced by ribosomes during the pioneer round of translation. Nonsense-mediated mRNA decay (NMD) degrades EJC-bound mRNA, but the lack of suitable methodology has prevented the identification of other degradation pathways. Here, we show that the RNA degradomes of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), worm (Caenorhabditis elegans), and human (Homo sapiens) cells exhibit an enrichment of 59 monophosphate (59P) ends of degradation intermediates that map to the canonical EJC region. Inhibition of 59 to 39 exoribonuclease activity and overexpression of an EJC disassembly factor in Arabidopsis reduced the accumulation of these 59P ends, supporting the notion that they are in vivo EJC footprints. Hundreds of Arabidopsis NMD targets possess evident EJC footprints, validating their degradation during the pioneer round of translation. In addition to premature termination codons, plant microRNAs can also direct the degradation of EJC-bound mRNAs. However, the production of EJC footprints from NMD but not microRNA targets requires the NMD factor SUPPRESSOR WITH MORPHOLOGICAL EFFECT ON GENITALIA PROTEIN7. Together, our results demonstrating in vivo EJC footprinting in Arabidopsis unravel the composition of the RNA degradome and provide a new avenue for studying NMD and other mechanisms targeting EJC-bound mRNAs for degradation before steady state translation.