CLIP-seq of eIF4AIII reveals transcriptome-wide mapping of the human exon junction complex

Saulière, Jérôme; Murigneux, Valentine; Wang, Zhen; Marquenet, Emélie; Barbosa, Isabelle; Tonquèze, Olivier Le; Audic, Yann; Paillard, Luc; Crollius, Hugues Roest; Hir, Hervé Le

doi:10.1038/nsmb.2420

Cited by 188 publications

(260 citation statements)

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“…S9). These observations provide additional evidence of the existence of noncanonical eIF4AIII (or EJC)-binding sites that were reported previously (37,42). Although we do not know the exact timing of the replacement of the CBC by eIF4E, this event is triggered by importin α/β (6).…”

Section: Discussionmentioning

confidence: 57%

“…S8). Recently, a transcriptome-wide interaction map of eIF4AIII was generated using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation [HITS-CLIP; also named cross-linking immunoprecipitation sequencing (CLIPSeq)], elucidating that eIF4AIII-binding sites are largely located in the canonical EJC deposition region and in the noncanonical region within the exons (37). Given our finding that eIF4AIII is recruited to the 5′-end of mRNA via its direct interaction with CTIF, we expected to see in the eIF4AIII CLIP data that eIF4AIII-mRNA interactions would be enriched in 5′UTRs of mRNAs.…”

Section: Eif4aiii Selectively Increases Ct Efficiency In An In Vitro mentioning

confidence: 99%

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eIF4AIII enhances translation of nuclear cap-binding complex–bound mRNAs by promoting disruption of secondary structures in 5′UTR

Choe

Ryu

Park

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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It has long been considered that intron-containing (spliced) mRNAs are translationally more active than intronless mRNAs (identical mRNA not produced by splicing). The splicing-dependent translational enhancement is mediated, in part, by the exon junction complex (EJC). Nonetheless, the molecular mechanism by which each EJC component contributes to the translational enhancement remains unclear. Here, we demonstrate the previously unappreciated role of eukaryotic translation initiation factor 4AIII (eIF4AIII), a component of EJC, in the translation of mRNAs bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein 80 (CBP80) and CBP20. eIF4AIII is recruited to the 5′-end of mRNAs bound by the CBC by direct interaction with the CBCdependent translation initiation factor (CTIF); this recruitment of eIF4AIII is independent of the presence of introns (deposited EJCs after splicing). Polysome fractionation, tethering experiments, and in vitro reconstitution experiments using recombinant proteins show that eIF4AIII promotes efficient unwinding of secondary structures in 5′UTR, and consequently enhances CBC-dependent translation in vivo and in vitro. Therefore, our data provide evidence that eIF4AIII is a specific translation initiation factor for CBC-dependent translation.eIF4AIII | cap-binding complex | translation | CTIF | nonsense-mediated mRNA decay I n mammalian cells, translation initiation is driven by two major cap-binding proteins (CBPs). One is the nuclear cap-binding complex (CBC), which is a heterodimer of nuclear CBP80 and CBP20 (CBP80/20) (1), and the other is the eukaryotic translation initiation factor (eIF) 4E, which is a major cytoplasmic CBP (2). The CBC binds the 5′-cap structure of a newly synthesized mRNA in the nucleus and then directly interacts with an eIF4G-like scaffold protein called CBP80/20-dependent translation initiation factor (CTIF) (3). Because CTIF is mostly present on the cytoplasmic side of the nuclear envelope and is found in the nucleus (3), the interaction between the CBC and CTIF may occur either in the nucleus or during mRNA export through the nuclear pore complex. In the cytoplasm, at the 5′-end of mRNA, the CBC-CTIF complex recruits the eIF3 complex (4) and then the 40S small subunit of the ribosome, thereby directing the socalled "first" (or pioneer) round of translation (1).Translation of a CBC-bound mRNA precedes that of the eIF4E-bound mRNA, because the CBC-bound mRNA is a precursor form of the eIF4E-bound mRNA (5). The timing of the replacement of the CBC by eIF4E has not yet been elucidated; however, it is known that the replacement is mediated by the action of importin α/β in a translation-independent manner (6). The replaced eIF4E at the 5′-end of mRNA recruits a scaffold protein, eIF4GI or eIF4GII, which recruits eIF3 and eIF4AI/II and, eventually, the 40S ribosomal subunit. Then, the recruited 40S subunit scans the mRNA until it reaches the authentic translation initiation codon AUG. Efficient ribosome scanning requires either eIF4AI or e...

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Section: Discussionmentioning

confidence: 57%

Section: Eif4aiii Selectively Increases Ct Efficiency In An In Vitro mentioning

confidence: 99%

eIF4AIII enhances translation of nuclear cap-binding complex–bound mRNAs by promoting disruption of secondary structures in 5′UTR

Choe

Ryu

Park

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…We and others recently have discovered that EJCs are not equally distributed onto spliced mRNAs (35,36). Moreover, no one knows whether the composition of EJCs is identical at every junction onto every mRNA.…”

Section: Discussionmentioning

confidence: 99%

EJC core component MLN51 interacts with eIF3 and activates translation

Chazal

Daguenet

Wendling

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The multiprotein exon junction complex (EJC), deposited by the splicing machinery, is an important constituent of messenger ribonucleoprotein particles because it participates to numerous steps of the mRNA lifecycle from splicing to surveillance via nonsense-mediated mRNA decay pathway. By an unknown mechanism, the EJC also stimulates translation efficiency of newly synthesized mRNAs. Here, we show that among the four EJC core components, the RNA-binding protein metastatic lymph node 51 (MLN51) is a translation enhancer. Overexpression of MLN51 preferentially increased the translation of intron-containing reporters via the EJC, whereas silencing MLN51 decreased translation. In addition, modulation of the MLN51 level in cell-free translational extracts confirmed its direct role in protein synthesis. Immunoprecipitations indicated that MLN51 associates with translation-initiating factors and ribosomal subunits, and in vitro binding assays revealed that MLN51, alone or as part of the EJC, interacts directly with the pivotal eukaryotic translation initiation factor eIF3. Taken together, our data define MLN51 as a translation activator linking the EJC and the translation machinery.

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“…Two very recent publications challenged this strict correlation between introns and EJCs by revealing that only ∼80% of spliced exon-exon junctions carry an EJC at the classical −24 nt position, and on the other hand, 40%-50% of the EJCs detected in these studies located to noncanonical positions (Sauliere et al 2012;Singh et al 2012). Thus, we do not know with absolute certainty whether our mini-μ and β-Globin reporter mRNAs containing the 3 ′ -most intron indeed harbor an EJC downstream from the PTC.…”

Section: Relaxed Correlation Between Intron Removal and Ejc Assembly mentioning

confidence: 99%

“…The EJC is a multimeric protein complex deposited by the splicing machinery 24 nt upstream of the spliced exon-exon boundary in metazoans (Le Hir et al 2000;Sauliere et al 2012;Singh et al 2012). The EJC core consists of the four proteins eIF4AIII, Y14, MAGOH, and MLN51 (also known as Barentz) (Andersen et al 2006;Bono et al 2006), and additional factors associate with the EJC core in a dynamic way, including Pinin, SKAR, SRm160, RNPS1, Acinus, SAP18, UAP56, Aly/REF, and the two NMD factors UPF3b and SMG6 (Le Hir et al 2000Kashima et al 2010;Bono and Gehring 2011;Murachelli et al 2012).…”

Section: Introductionmentioning

confidence: 99%

Comparison of EJC-enhanced and EJC-independent NMD in human cells reveals two partially redundant degradation pathways

Metze

Herzog

Ruepp

et al. 2013

RNA

122

126

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Nonsense-mediated mRNA decay (NMD) is a eukaryotic post-transcriptional gene regulation mechanism that eliminates mRNAs with the termination codon (TC) located in an unfavorable environment for efficient translation termination. The best-studied NMD-targeted mRNAs contain premature termination codons (PTCs); however, NMD regulates even many physiological mRNAs. An exon-junction complex (EJC) located downstream from a TC acts as an NMD-enhancing signal, but is not generally required for NMD. Here, we compared these "EJC-enhanced" and "EJC-independent" modes of NMD with regard to their requirement for seven known NMD factors in human cells using two well-characterized NMD reporter genes (immunoglobulin μ and β-Globin) with or without an intron downstream from the PTC. We show that both NMD modes depend on UPF1 and SMG1, but detected transcript-specific differences with respect to the requirement for UPF2 and UPF3b, consistent with previously reported UPF2-and UPF3-independent branches of NMD. In addition and contrary to expectation, a higher sensitivity of EJC-independent NMD to reduced UPF2 and UPF3b concentrations was observed. Our data further revealed a redundancy of the endo-and exonucleolytic mRNA degradation pathways in both modes of NMD. Moreover, the relative contributions of both decay pathways differed between the reporters, with PTC-containing immunoglobulin μ transcripts being preferentially subjected to SMG6-mediated endonucleolytic cleavage, whereas β-Globin transcripts were predominantly degraded by the SMG5/SMG7-dependent pathway. Overall, the surprising heterogeneity observed with only two NMD reporter pairs suggests the existence of several mechanistically distinct branches of NMD in human cells.

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CLIP-seq of eIF4AIII reveals transcriptome-wide mapping of the human exon junction complex

Cited by 188 publications

References 53 publications

eIF4AIII enhances translation of nuclear cap-binding complex–bound mRNAs by promoting disruption of secondary structures in 5′UTR

eIF4AIII enhances translation of nuclear cap-binding complex–bound mRNAs by promoting disruption of secondary structures in 5′UTR

EJC core component MLN51 interacts with eIF3 and activates translation

Comparison of EJC-enhanced and EJC-independent NMD in human cells reveals two partially redundant degradation pathways

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