Comprehensive mapping of exon junction complex binding sites reveals universal EJC deposition in Drosophila

The exon junction complex (EJC) is involved in most steps of the mRNA life cycle, ranging from splicing to nonsense-mediated mRNA decay (NMD). It is assembled by the splicing machinery onto mRNA in a sequence-independent manner. A fundamental open question is whether the EJC is deposited onto all exon-exon junctions or only on a subset of them. Several previous studies have made observations supportive of the latter, yet these have been limited by methodological constraints. In this study, we sought to overcome these limitations via the integration of two different approaches for transcriptome-wide mapping of EJCs. Our results revealed that nearly all, if not all, internal exons consistently harbor an EJC in Drosophila, demonstrating that EJC presence is an inherent consequence of the splicing reaction. Furthermore, our study underscores the limitations of eCLIP methods in fully elucidating the landscape of RBP binding sites. Our findings highlight how highly specific (low false positive) methodologies can lead to erroneous interpretations due to partial sensitivity (high false negatives). This study contributes to our understanding of EJC deposition and its association with pre-mRNA splicing. The universal presence of EJC on internal exons underscores its significance in ensuring proper mRNA processing. Additionally, our observations highlight the need to consider both specificity and sensitivity in RBP mapping methodologies.