Genomewide localization of Exon‐Junction‐Complex (EJC) in Drosophila

The Exon Junction Complex (EJC) assembles upstream of exon‐exon junction during pre‐mRNA splicing and is involved in splicing, mRNA export, translation and Nonsense Meditated mRNA Decay (NMD). We have recently shown that EJC, assembled only on a subset of intron‐containing 3′UTRs, triggerrf NMD in Drosophila. This prompted us to identify globally the EJC‐bound RNAs and to locate precisely these complexes throughout the expressed RNAs in drosophila S2 cells. S2 cells stably expressing tagged version of EJC core components, eIF4AIII, Y14, Mago and Barentz, were established. Poly‐A containing RNPs associated with EJCs were purified by immunopurification (IP) and a poly‐dT capture. Footprints of EJC complexes were identified by nuclease treatment followed by another immunopurification against a second EJC component. Identity of EJC‐protected RNA fragments were characterized by high‐throughput sequencing. EJCs are found on more than 80% of the expressed transcriptome, stereotypically bound to a region 24 nt upstream of 5′ spliced site with no obvious sequence preference. Surprisingly, ~30% of spliced RNAs were not associated with any EJC. In addition, several multi‐intron transcripts show differential assembly of EJC at different junctions supporting the notion that EJCs assemble only on a subset of spliced junction and is a highly regulated process. Stowers Institute for Medical Research.