Proteins Associated with the Exon Junction Complex Also Control the Alternative Splicing of Apoptotic Regulators

Abstract

Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x S splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.

The RNA-binding profile of Acinus, a peripheral component of the exon junction complex, reveals its role in splicing regulation

“…In agreement with its presumed role in pre-mRNA splicing, Acinus has also been identified as a component of functional spliceosomes (Rappsilber et al 2002;Zhou et al 2002). Additionally, Acinus regulates the splicing of a subset of genes involved in apoptosis in human cells (Michelle et al 2012) and has been proposed to couple Retinoic acid mediated transcription and splicing . Interestingly, Acinus, alongside the EJC, is required for definition and excision of neighboring introns in Drosophila (Hayashi et al 2014;Malone et al 2014).…”

“…In mammalian cells, knockdown of EJC core proteins results in widespread alternative splicing changes, with similar changes in splicing observed upon knockdown of different EJC core components, yet displaying differences with down-regulation of other splicing factors that are not components of the EJC . A role for EJC components in the regulation of splicing of apoptotic regulators has also been reported (Michelle et al 2012). EJCs have been shown to multimerize with one another and form higher-order complexes containing in particular numerous SR proteins, leading to a model whereby EJCs and SR proteins could work in cooperation to promote mRNA packaging and compaction, affecting pre-mRNA splicing (Singh et al 2012).…”

Transcriptome-wide modulation of splicing by the Exon Junction Complex

“…The splicing changes depend on EJC core integrity A previous study showed that individual EJC components seem to preferentially associate with the Bcl-x premRNA to regulate its alternative splicing, probably independently of EJC core integrity [32]. Therefore, we have examined whether the EJC-dependent splicing events identified in this study require EJC core.…”

“…Several studies before have indicated that the EJC protein can regulate splicing in different species [27][28][29]31,32]. However, these studies only focused on a specific set of candidate genes.…”

EJC core component MLN51 interacts with eIF3 and activates translation

“…Once clamped onto mRNA, the tetrameric core acts as a platform to recruit multiple factors conferring different functions to the EJC as mRNA progresses from splicing to translation. Notably, the EJC contributes in regulating the splicing of specific transcripts (8)(9)(10) and participates in mRNA transport (11). The EJC also plays a major role in nonsense-mediated mRNA decay (NMD) that triggers the decay of aberrant mRNAs containing a premature termination codon (12).…”