Mechanism of ATP turnover inhibition in the EJC

The exon junction complex (EJC) is deposited onto spliced mRNAs and is involved in many aspects of mRNA function. We have recently reconstituted and solved the crystal structure of the EJC core made of MAGOH, Y14, the most conserved portion of MLN51, and the DEAD-box ATPase eIF4AIII bound to RNA in the presence of an ATP analog. The heterodimer MAGOH/Y14 inhibits ATP turnover by eIF4AIII, thereby trapping the EJC core onto RNA, but the exact mechanism behind this remains unclear. Here, we present the crystal structure of the EJC core bound to ADP-AIF₃, the first structure of a DEAD-box helicase in the transition-mimicking state during ATP hydrolysis. It reveals a dissociative transition state geometry and suggests that the locking of the EJC onto the RNA by MAGOH/Y14 is not caused by preventing ATP hydrolysis. We further show that ATP can be hydrolyzed inside the EJC, demonstrating that MAGOH/Y14 acts by locking the conformation of the EJC, so that the release of inorganic phosphate, ADP, and RNA is prevented. Unifying features of ATP hydrolysis are revealed by comparison of our structure with the EJC–ADPNP structure and other helicases. The reconstitution of a transition state mimicking complex is not limited to the EJC and eIF4AIII as we were also able to reconstitute the complex Dbp5–RNA–ADP–AlF₃, suggesting that the use of ADP–AlF₃ may be a valuable tool for examining DEAD-box ATPases in general.     

RNA-binding protein RBM8A (Y14) and MAGOH localize to centrosome in human A549 cells

RBM8A (Y14) is carrying RNA-binding motif and forms the tight heterodimer with MAGOH. The heterodimer is known to be a member of exon junction complex on exporting mRNA and is required for mRNA metabolisms such as splicing, mRNA export and nonsense-mediated mRNA decay. Almost all RBM8A–MAGOH complexes localize in nucleoplasm and shuttle between nuclei and cytoplasm for RNA metabolism. Recently, the abnormality of G2/M transition and aberrant centrosome regulation in RBM8A- or MAGOH-deficient cells has been reported. These results prompt us to the reevaluation of the localization of RBM8A–MAGOH in human cells. Interestingly, our immunostaining experiments showed the localization of these proteins in centrosome in addition to nuclei. Furthermore, the transiently expressed eYFP-tagged RBM8A and Flag-tagged MAGOH also co-localized with centrosome signals. In addition, the proximity ligation in situ assay was performed to detect the complex formation in centrosome. Our experiments clearly showed that Myc-tagged RBM8A and Flag-tagged MAGOH formed a complex in centrosome. GFP-tagged PLK1 also co-localized with Myc-RBM8A. Our results show that RBM8A–MAGOH complex is required for M-phase progression via direct localization to centrosome rather than indirect effect.     

The exon junction core complex is locked onto RNA by inhibition of eIF4AIII ATPase activity

The multiprotein exon junction complex (EJC) is assembled on mRNAs as a consequence of splicing. EJC core components maintain a stable grip on mRNAs even as the overall EJC protein composition evolves while mRNAs travel to the cytoplasm. Here we show that recombinant EJC subunits MLN51, MAGOH and Y14, together with the DEAD-box protein eIF4AIII bound to ATP, are necessary and sufficient to form a highly stable complex on single-stranded RNA. Cross-linking and RNase protection studies indicate that this recombinant complex recapitulates the EJC core. The stable association of the recombinant EJC core with RNA is maintained by inhibition of eIF4AIII ATPase activity by MAGOH-Y14. We elucidate the modalities of EJC binding to RNA and provide the first example of how cellular machineries may use RNA helicases to clamp several proteins onto RNA in stable and sequence-independent manners.