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.     

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.     

Mutational analysis of human eIF4AIII identifies regions necessary for exon junction complex formation and nonsense-mediated mRNA decay

The exon junction complex (EJC) is deposited on mRNAs by the process of pre-mRNA splicing and is a key effector of downstream mRNA metabolism. We previously demonstrated that human eIF4AIII, which is essential for nonsense-mediated mRNA decay (NMD), constitutes at least part of the RNA-binding platform anchoring other EJC components to the spliced mRNA. To determine the regions of eIF4AIII that are functionally important for EJC formation, for binding to other EJC components, and for NMD, we now report results of an extensive mutational analysis of human eIF4AIII. Using GFP-, GST- or Flag-fusions of eIF4AIII versions containing site-specific mutations or truncations, we analyzed subcellular localizations, protein-protein interactions, and EJC formation in vivo and in vitro. We also tested whether mutant proteins could rescue NMD inhibition resulting from RNAi depletion of endogenous eIF4AIII. Motifs Ia and VI, which are conserved among the eIF4A family of RNA helicases (DEAD-box proteins), are crucial for EJC formation and NMD, as is one eIF4AIII-specific region. An additional eIF4AIII-specific motif forms part of the binding site for MLN51, another EJC core component. Mutations in the canonical Walker A and B motifs that eliminate RNA-dependent ATP hydrolysis by eIF4AIII in vitro are of no detectable consequence for EJC formation and NMD activation. Implications of these findings are discussed in the context of other recent results and a new structural model for human eIF4AIII based on the known crystal structure of Saccharomyces cerevisiae eIF4AI.     

eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay

The exon junction complex (EJC), a set of proteins deposited on mRNAs as a consequence of pre-mRNA splicing, is a key effector of downstream mRNA metabolism. We have identified eIF4AIII, a member of the eukaryotic translation initiation factor 4A family of RNA helicases (also known as DExH/D box proteins), as a novel EJC core component. Crosslinking and antibody inhibition studies suggest that eIF4AIII constitutes at least part of the platform anchoring other EJC components to spliced mRNAs. A nucleocytoplasmic shuttling protein, eIF4AIII associates in vitro and in vivo with two other EJC core factors, Y14 and Magoh. In mammalian cells, eIF4AIII is essential for nonsense-mediated mRNA decay (NMD). Finally, a model is proposed by which eIF4AIII represents a new functional class of DExH/D box proteins that act as RNA clamps or 'place holders' for the sequence-independent attachment of additional factors to RNAs.     

Exon junction complex enhances translation of spliced mRNAs at multiple steps

Translation of spliced mRNAs is enhanced by exon junction complex (EJC), which is deposited on mRNAs as a result of splicing. Although this phenomenon itself is well known, the underlying molecular mechanism remains poorly understood. Here we show, using siRNAs against Y14 and eIF4AIII and spliced or intronless constructs that contain different types of internal ribosome entry sites (IRESes), that Y14 and eIF4AIII increase translation of spliced mRNAs before and after formation of the 80S ribosome complex, respectively. These results suggest that EJC modulates translation of spliced mRNA at multiple steps.     

The three-dimensional arcitecture of the EJC core

The exon junction complex (EJC) is a macromolecular complex deposited at splice junctions on mRNAs as a consequence of splicing. At the core of the EJC are four proteins: eIF4AIII, a member of the DExH/D-box family of NTP-dependent RNA binding proteins, Y14, Magoh, and MLN51. These proteins form a stable heterotetramer that remains bound to the mRNA throughout many different cellular environments. We have determined the three-dimensional (3D) structure of this EJC core using negative-stain random-conical tilt electron microscopy. This structure represents the first structure of a DExH/D-box protein in complex with its binding partners. The EJC core is a four-lobed complex with a central channel and dimensions consistent with its known RNA footprint of about ten nucleotides. Using known X-ray crystallographic structures and a model of three of the four components, we propose a model for complex assembly on RNA and explain how Y14:Magoh may influence eIF4AIII's RNA binding.     

The EJC factor eIF4AIII modulates synaptic strength and neuronal protein expression

Proper neuronal function and several forms of synaptic plasticity are highly dependent on precise control of mRNA translation, particularly in dendrites. We find that eIF4AIII, a core exon junction complex (EJC) component loaded onto mRNAs by pre-mRNA splicing, is associated with neuronal mRNA granules and dendritic mRNAs. eIF4AIII knockdown markedly increases both synaptic strength and GLUR1 AMPA receptor abundance at synapses. eIF4AIII depletion also increases ARC, a protein required for maintenance of long-term potentiation; arc mRNA, one of the most abundant in dendrites, is a natural target for nonsense-mediated decay (NMD). Numerous new NMD candidates, some with potential to affect synaptic activity, were also identified computationally. Two models are presented for how translation-dependent decay pathways such as NMD might advantageously function as critical brakes for protein synthesis in cells such as neurons that are highly dependent on spatially and temporally restricted protein expression.     

Structural insights into the exon junction complex

In higher eukaryotes, the exon junction complex is loaded onto spliced mRNAs at a precise position upstream of exon junctions, where it remains during nuclear export and cytoplasmic localisation until it is removed during the first translation round. The exon junction core complex consists of four proteins that form a dynamic binding platform for a variety of peripheral factors involved in mRNA metabolism. In the complex, mRNA binding is mediated by the DEAD-box protein eIF4AIII, and inhibition of its ATPase activity forms the mechanistic basis for the long-term stability of the complex. Recent crystal structures of the exon junction complex and eIF4AIII have provided the structural framework for investigating the function of the eIF4AIII ATPase and for localisation of surface patches involved in binding peripheral factors. Additionally, by comparison with the structure of a second DEAD-box protein also bound to RNA and ATP, general principles for the ATPase and unwinding/mRNP remodelling activities for this important group of enzymes can be proposed on the basis of atomic structures.     

The RNA-binding protein Y14 inhibits mRNA decapping and modulates processing body formation

The exon-junction complex (EJC) deposited on a newly spliced mRNA plays an important role in subsequent mRNA metabolic events. Here we show that an EJC core heterodimer, Y14/Magoh, specifically associates with mRNA-degradation factors, including the mRNA-decapping complex and exoribonucleases, whereas another core factor, eIF4AIII/MLN51, does not. We also demonstrate that Y14 interacts directly with the decapping factor Dcp2 and the 5′ cap structure of mRNAs via different but overlapping domains and that Y14 inhibits the mRNA-decapping activity of Dcp2 in vitro. Accordingly, overexpression of Y14 prolongs the half-life of a reporter mRNA. Therefore Y14 may function independently of the EJC in preventing mRNA decapping and decay. Furthermore, we observe that depletion of Y14 disrupts the formation of processing bodies, whereas overexpression of a phosphomimetic Y14 considerably increases the number of processing bodies, perhaps by sequestering the mRNA-degradation factors. In conclusion, this report provides unprecedented evidence for a role of Y14 in regulating mRNA degradation and processing body formation and reinforces the influence of phosphorylation of Y14 on its activity in postsplicing mRNA metabolism.     

MLN51 stimulates the RNA-helicase activity of eIF4AIII

The core of the exon-junction complex consists of Y14, Magoh, MLN51 and eIF4AIII, a DEAD-box RNA helicase. MLN51 stimulates the ATPase activity of eIF4AIII, whilst the Y14-Magoh complex inhibits it. We show that the MLN51-dependent stimulation increases both the affinity of eIF4AIII for ATP and the rate of enzyme turnover; the K(M) is decreased by an order of magnitude and k(cat) increases 30 fold. Y14-Magoh do inhibit the MLN51-stimulated ATPase activity, but not back to background levels. The ATP-bound form of the eIF4AIII-MLN51 complex has a 100-fold higher affinity for RNA than the unbound form and ATP hydrolysis reduces this affinity. MLN51 stimulates the RNA-helicase activity of eIF4AIII, suggesting that this activity may be functionally important.     

Flexibility in the site of exon junction complex deposition revealed by functional group and RNA secondary structure alterations in the splicing substrate

The exon junction complex (EJC) is critical for mammalian nonsense-mediated mRNA decay and translational regulation, but the mechanism of its stable deposition on mRNA is unknown. To examine requirements for EJC deposition, we created splicing substrates containing either DNA nucleotides or RNA secondary structure in the 5′ exon. Using RNase H protection, toeprinting, and coimmunoprecipitation assays, we found that EJC location shifts upstream when a stretch of DNA or RNA secondary structure appears at the canonical deposition site. These upstream shifts occur prior to exon ligation and are often accompanied by decreases in deposition efficiency. Although the EJC core protein eIF4AIII contacts four ribose 2′OH groups in crystal structures, we demonstrate that three 2′OH groups are sufficient for deposition. Thus, the site of EJC deposition is more flexible than previously appreciated and efficient deposition appears spatially limited.     

Biochemical analysis of the EJC reveals two new factors and a stable tetrameric protein core

The multiprotein exon junction complex (EJC) is deposited on mRNAs upstream of exon-exon junctions as a consequence of pre-mRNA splicing. In mammalian cells, this complex serves as a key modulator of spliced mRNA metabolism. To date, neither the complete composition nor the exact assembly pathway of the EJC has been entirely elucidated. Using in vitro splicing and a two-step chromatography procedure, we have purified the EJC and analyzed its components by mass spectrometry. In addition to finding most of the known EJC factors, we identified two novel EJC components, Acinus and SAP18. Heterokaryon analysis revealed that SAP18 is a shuttling protein whereas Acinus is restricted to the nucleus. In MS2 tethering assays Acinus stimulated gene expression at the RNA level, while MLN51, another EJC factor, stimulated mRNA translational efficiency. Using tandem affinity purification (TAP) of proteins overexpressed in HeLa cells, we demonstrated that Acinus binds directly to another EJC component, RNPS1, while stable association of SAP18 to form the trimeric apoptosis and splicing associated protein (ASAP) complex requires both Acinus and RNPS1. Using the same methodology, we further identified what appears to be the minimal stable EJC core, a heterotetrameric complex consisting of eIF4AIII, Magoh, Y14, and MLN51.     

Transcriptome-wide modulation of splicing by the exon junction complex

Background

The exon junction complex (EJC) is a dynamic multi-protein complex deposited onto nuclear spliced mRNAs upstream of exon-exon junctions. The four core proteins, eIF4A3, Magoh, Y14 and MLN51, are stably bound to mRNAs during their lifecycle, serving as a binding platform for other nuclear and cytoplasmic proteins. Recent evidence has shown that the EJC is involved in the splicing regulation of some specific events in both Drosophila and mammalian cells.

Results

Here, we show that knockdown of EJC core proteins causes widespread alternative splicing changes in mammalian cells. These splicing changes are specific to EJC core proteins, as knockdown of eIF4A3, Y14 and MLN51 shows similar splicing changes, and are different from knockdown of other splicing factors. The splicing changes can be rescued by a siRNA-resistant form of eIF4A3, indicating an involvement of EJC core proteins in regulating alternative splicing. Finally, we find that the splicing changes are linked with RNA polymerase II elongation rates.

Conclusion

Taken together, this study reveals that the coupling between EJC proteins and splicing is broader than previously suspected, and that a possible link exists between mRNP assembly and splice site recognition.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0551-7) contains supplementary material, which is available to authorized users.     

A quantitative high-throughput in vitro splicing assay identifies inhibitors of spliceosome catalysis

Despite intensive research, there are very few reagents with which to modulate and dissect the mRNA splicing pathway. Here, we describe a novel approach to identify such tools, based on detection of the exon junction complex (EJC), a unique molecular signature that splicing leaves on mRNAs. We developed a high-throughput, splicing-dependent EJC immunoprecipitation (EJIPT) assay to quantitate mRNAs spliced from biotin-tagged pre-mRNAs in cell extracts, using antibodies to EJC components Y14 and eukaryotic translation initiation factor 4aIII (eIF4AIII). Deploying EJIPT we performed high-throughput screening (HTS) in conjunction with secondary assays to identify splicing inhibitors. We describe the identification of 1,4-naphthoquinones and 1,4-heterocyclic quinones with known anticancer activity as potent and selective splicing inhibitors. Interestingly, and unlike previously described small molecules, most of which target early steps, our inhibitors represented by the benzothiazole-4,7-dione, BN82685, block the second of two trans-esterification reactions in splicing, preventing the release of intron lariat and ligation of exons. We show that BN82685 inhibits activated spliceosomes' elaborate structural rearrangements that are required for second-step catalysis, allowing definition of spliceosomes stalled in midcatalysis. EJIPT provides a platform for characterization and discovery of splicing and EJC modulators.     

mRNA biogenesis-related helicase eIF4AIII from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is an important factor for abiotic stress adaptation

Similar to other plant species, Arabidopsis has a huge repertoire of predicted helicases, including the eIF4AIII factor, a putative component of the exon junction complex related to mRNA biogenesis. In this article, we integrated evolutionary and functional approaches to have a better understanding of eIF4AIII function in plants. Phylogenetic analysis showed that the mRNA biogenesis-related helicase eIF4AIII is the ortholog of the stress-related helicases PDH45 from Pisum sativum and MH1 from Medicago sativa, suggesting evolutionary and probably functional equivalences between mRNA biogenesis and stress-related plant helicases. Molecular and genetic analyses confirmed the relevance of eIF4AIII during abiotic stress adaptation in Arabidopsis. Therefore, in addition to its function in mRNA biogenesis, eIF4AIII can play a role in abiotic stress adaptation.     

The Hierarchy of Exon-Junction Complex Assembly by the Spliceosome Explains Key Features of Mammalian Nonsense-Mediated mRNA Decay

Exon junction complexes (EJCs) link nuclear splicing to key features of mRNA function including mRNA stability, translation, and localization. We analyzed the formation of EJCs by the spliceosome, the physiological EJC assembly machinery. We studied a comprehensive set of eIF4A3, MAGOH, and BTZ mutants in complete or C-complex–arrested splicing reactions and identified essential interactions of EJC proteins during and after EJC assembly. These data establish that EJC deposition proceeds through a defined intermediate, the pre-EJC, as an ordered, sequential process that is coordinated by splicing. The pre-EJC consists of eIF4A3 and MAGOH-Y14, is formed before exon ligation, and provides a binding platform for peripheral EJC components that join after release from the spliceosome and connect the core structure with function. Specifically, we identified BTZ to bridge the EJC to the nonsense-mediated messenger RNA (mRNA) decay protein UPF1, uncovering a critical link between mRNP architecture and mRNA stability. Based on this systematic analysis of EJC assembly by the spliceosome, we propose a model of how a functional EJC is assembled in a strictly sequential and hierarchical fashion, including nuclear splicing-dependent and cytoplasmic steps.     

Eukaryotic Translation Initiation Factor 4AIII (eIF4AIII) Is Functionally Distinct from eIF4AI and eIF4AII

Eukaryotic initiation factor 4A (eIF4A) is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5' proximal secondary structure of eukaryotic mRNAs to facilitate attachment of the 40S ribosomal subunit. eIF4A functions in a complex termed eIF4F with two other initiation factors (eIF4E and eIF4G). Two isoforms of eIF4A, eIF4AI and eIF4AII, which are encoded by two different genes, are functionally indistinguishable. A third member of the eIF4A family, eIF4AIII, whose human homolog exhibits 65% amino acid identity to human eIF4AI, has also been cloned from Xenopus and tobacco, but its function in translation has not been characterized. In this study, human eIF4AIII was characterized biochemically. While eIF4AIII, like eIF4AI, exhibits RNA-dependent ATPase activity and ATP-dependent RNA helicase activity, it fails to substitute for eIF4AI in an in vitro-reconstituted 40S ribosome binding assay. Instead, eIF4AIII inhibits translation in a reticulocyte lysate system. In addition, whereas eIF4AI binds independently to the middle and carboxy-terminal fragments of eIF4G, eIF4AIII binds to the middle fragment only. These functional differences between eIF4AI and eIF4AIII suggest that eIF4AIII might play an inhibitory role in translation under physiological conditions.