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Abstract
Exon junction complex (EJC) assembles after splicing at specific positions upstream of exon-exon junctions in mRNAs of all higher eukaryotes, affecting major regulatory events. In mammalian cell cytoplasm, EJC is essential for efficient RNA surveillance, while in Drosophila, EJC is essential for localization of oskar mRNA. Here we developed a method for isolation of protein complexes and associated RNA targets (ipaRt) to explore the EJC RNA-binding landscape in a transcriptome-wide manner in adult Drosophila. We find the EJC at canonical positions, preferably on mRNAs from genes comprising multiple splice sites and long introns. Moreover, EJC occupancy is highest at junctions adjacent to strong splice sites, CG-rich hexamers, and RNA structures. Highly occupied mRNAs tend to be maternally localized and derive from genes involved in differentiation or development. These modalities, which have notbeen reported in mammals, specify EJC assembly on a biologically coherent set of transcripts in Drosophila.
Highlights
The exon junction complex (EJC) consists of a heterotetramer core composed of eIF4AIII, Mago, Y14, and Barentsz (Btz) (Bono et al, 2006; Stroupe et al, 2006) and auxiliary factors that form the Exon junction complex (EJC) periphery (Tange et al, 2005)
The analysis revealed a strong downstream shift of EJC assembly coordinates when double-stranded RNA (dsRNA) content in region A was high and low in region B, and only a minor shift when the dsRNA content was low in region A and high in region B (Figure 6F)
We have demonstrated that dithio(bis-) succinimidylpropionate (DSP)-mediated covalent bond formation between the RNA helicase eIF4AIII and the Mago-Y14 heterodimer preserves EJCs in their ‘‘locked’’ state on mRNAs (Andersen et al, 2006; Ballut et al, 2005; Bono et al, 2006; Stroupe et al, 2006) and that efficient recovery of the bound RNAs does not require their crosslinking to eIF4AIII using UV light
Summary
The exon junction complex (EJC) consists of a heterotetramer core composed of eIF4AIII, Mago, Y14, and Barentsz (Btz) (Bono et al, 2006; Stroupe et al, 2006) and auxiliary factors that form the EJC periphery (Tange et al, 2005). The EJC is reported to play a role in translation (Chazal et al, 2013; Nott et al, 2004), nonsense-mediated decay (NMD) (Buchwald et al, 2010; Gehring et al, 2005; Melero et al, 2012; Okada-Katsuhata et al, 2012; Palacios et al, 2004; Shibuya et al, 2006; Singh et al, 2007), and RNA localization (Ghosh et al, 2012; Hachet and Ephrussi, 2001, 2004; Palacios et al, 2004; van Eeden et al, 2001). Our analysis suggests that the EJC’s bias for transcripts in Drosophila is a consequence of several modalities in the genes’ architecture, splice site number and intron
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