Abstract
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 5' monophosphate (5'P) ends of degradation intermediates that map to the canonical EJC region. Inhibition of 5' to 3' exoribonuclease activity and overexpression of an EJC disassembly factor in Arabidopsis reduced the accumulation of these 5'P 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.
Highlights
Many eukaryotic pre-mRNAs undergo splicing, in which intervening sequences are excised and the exon junction complex (EJC) is deposited in a region 20-24 nt upstream of the exon-exon junction (Le Hir et al, 2000)
If a termination codon situated more than 50-55 nt upstream of an exon-exon junction is encountered by a ribosome during the pioneer round of translation, it is generally recognized as a premature termination codon (PTC) and promotes nonsense-mediated mRNA decay (NMD) (Nagy and Maquat, 1998)
Many studies have demonstrated that the EJCs downstream of a termination codon are crucial for NMD, NMD can occur in an EJC-independent manner
Summary
Many eukaryotic pre-mRNAs undergo splicing, in which intervening sequences (introns) are excised and the exon junction complex (EJC) is deposited in a region 20-24 nt upstream of the exon-exon junction (Le Hir et al, 2000). High-throughput approaches for profiling 5 ′ monophosphate ends of RNA degradation intermediates (hereafter referred to as 5′P ends) such as Degradome-Seq (Addo-Quaye et al, 2008), Parallel Analysis of RNA Ends (PARE) (German et al, 2008), Genome-wide Mapping of Uncapped and Cleaved Transcripts (GMUCT) (Gregory et al., 2008), and 5′P sequencing (Pelechano et al, 2015), provide a more reliable way to detect the direct targets of RNA degradation pathways These approaches have been applied for transcriptome-wide identification of endonucleolytic cleavage events directed by microRNA (miRNA) in diverse plant species and catalyzed by SMG6 in human (Homo sapiens) cells as well as the substrates of exoribonuclease 4 (XRN4), which catalyzes 5′-3′ RNA decay in Arabidopsis (Arabidopsis thaliana). We observed EJC footprints in some miRNA targets in addition to NMD targets, demonstrating that RNA degradome data can be applied to the study of mRNA degradation before steady-state translation
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call