BIOLOGICAL OVERVIEW

Molecular insights into the interaction of PYM with the Mago-Y14 core of the exon junction complex

The exon junction complex (EJC) is deposited on mRNAs as a consequence of splicing and influences postsplicing mRNA metabolism. The Mago-Y14 heterodimer is a core component of the EJC. The protein PYM, the product of the fly within bgcn (wibg) gene, has been identified as an interacting partner of Mago-Y14. PYM is a cytoplasmic RNA-binding protein that is excluded from the nucleus by Crm1. PYM interacts directly with Mago-Y14 by means of its N-terminal domain. The crystal structure of the Drosophila ternary complex at 1.9 Å resolution reveals that PYM binds Mago and Y14 simultaneously, capping their heterodimerization interface at conserved surface residues. Formation of this ternary complex is also observed with the human proteins. Mago residues involved in the interaction with PYM have been implicated in nonsense-mediated mRNA decay (NMD). Consistently, human PYM is active in NMD tethering assays. Together, these data suggest a role for PYM in NMD (Bono, 2004; full text of article).

Full-length Drosophila (Dm) PYM (residues 1-207) interacts with Mago-Y14 directly, as detected by pull-down experiments using recombinant proteins. From previous structural studies, Mago is known to be a single structural unit, whereas Y14 folds into three distinct domains (the N-terminal domain, the RNA-binding-like domain (RBD) and a C-terminal low-complexity region). The C-terminal region of Dm Y14 is not required for Mago binding nor for PYM binding. A minimal Mago-Y14 heterodimer containing only the RBD of Y14 (Mago-Y14DeltaNDeltaC) is able to interact with full-length PYM. Guided by sequence alignments, C-terminally truncated fragments of PYM were tested, and it was observed that PYM 1-108 and PYM 1-58 retain Mago-Y14-binding properties. PYM 1-58 contains the most conserved region of the protein (Bono, 2004).

Drosophila full-length Mago, the Y14 RBD (67-154) and the N-terminal 58 residues of PYM were coexpressed and purified. The crystal structure of the ternary complex was determined at 1.9 Å resolution and refined to an R_free of 24.9% and good stereochemistry. It contains residues 3-35 of PYM, residues 67-153 of the Y14 RBD and residues 4-144 of Mago (with the exception of loops 14-19 and 38-45 that were disordered) (Bono, 2004).

The N-terminal domain of Dm PYM binds as a small globular all-β-domain to both Mago and Y14, capping their heterodimerization interface. The structure of the Mago-Y14 heterodimer is very similar to that reported previously in the absence of PYM. Briefly, Mago consists of an antiparallel β-sheet flanked on one side by two long α-helices (α1 and α3) and a short one (α2). The α-helical surface of Mago interacts with the β-sheet surface of the Y14 RBD. More than 85% of the amino-acid residues of Mago-Y14 superpose with an overall root-mean-square deviation of less than 1.2 Å at their Cα atoms whether in the presence or absence of PYM, and whether comparing the Drosophila or human complexes. The largest differences in general are observed in Mago at the 14-19 loop, which is disordered in the present structure, and at the α2-helix. The lack of major changes in the conformation of the Mago and Y14 proteins suggests that the heterodimer acts as a rather rigid scaffold for PYM binding (Bono, 2004).

The N-terminal region of PYM (3-35) folds with a three-stranded β-sheet and a contiguous β-hairpin, and does not resemble other known structures from database searches using the program DALI. Although the crystallized construct contains 25 additional C-terminal residues, these are disordered in the structure and do not contribute to Mago-Y14 binding. Sequence comparison shows the presence of a 65-residue-long insertion at this domain boundary in the Caenorhabditis elegans homologue. Thus, the structure and sequence comparison data define residues 1-35 as the domain of PYM that interacts with Mago-Y14 (Bono, 2004).

PYM binds at the α-helices of Mago with extensive electrostatic interactions and at the β2-β3 loop of Y14 with hydrophobic interactions. Several solvent-mediated contacts appear to strengthen the interaction, as at least 40 water molecules are found at the interface (Bono, 2004).

The structural and biochemical data raise the question whether PYM associates with Mago-Y14 in the nucleus or whether it is a downstream interaction. Mago and Y14 are nucleocytoplasmic shuttling proteins that localize predominantly in the nucleoplasm and in nuclear speckles. In contrast, PYM is detected in the cytoplasm of Drosophila Schneider (S2) cells. The subcellular localization of PYM is conserved, as human PYM is also detected within the cytoplasm of HeLa cells transiently expressing the protein fused to green fluorescent protein (GFP-PYM) (Bono, 2004).

Despite its cytoplasmic localization at equilibrium, Drosophila PYM is a shuttling protein exported from the nucleus by Crm1. Crm1 is a transport receptor of the karyopherin β (importin β-like) family implicated in the nuclear export of a large number of proteins and whose activity is inhibited by leptomycin B. When HeLa cells are treated with leptomycin B, GFP-PYM accumulates within the nucleoplasm and the nucleolus. This indicates that human PYM is also a shuttling protein exported from the nucleus by Crm1. The accumulation of human PYM within the nucleolus following leptomycin B treatment may reflect a specific interaction with ribosomal subunits or mislocalization due to unspecific interactions with nucleolar components such as ribosomal RNA (Bono, 2004).

Recombinant Mago-Y14 heterodimers do not exhibit general RNA-binding activity in gel shift assays. In contrast, recombinant PYM binds RNA directly, despite showing no sequence homology to known RNA-binding proteins. The PYM-RNA complexes can be supershifted when Mago-Y14 dimers are added to the reactions, indicating that PYM can bind simultaneously to RNA and to Mago-Y14 (Bono, 2004).

Analysis of the structure of the trimeric complex reveals that PYM binding involves the direct contribution of amino-acid residues of Mago that were previously shown to have a role in nonsense-mediated mRNA decay. In the structure of the Drosophila complex, Asp67^Mago and Glu69^Mago interact with Lys25^PYM and Arg27^PYM. In human Mago, a double mutation of the corresponding Asp66^MagoHs and Glu68^MagoHs to Arg affects NMD. This double mutation is likely to cause electrostatic repulsion with the positively charged residues of PYM, suggesting a role for PYM in NMD. To test whether PYM might be active in NMD, a transient transfection assay was used in human cells in which degradation of a reporter mRNA is elicited if a protein involved in NMD is tethered downstream of a stop codon. Tethering PYM to the 3'UTR of a reporter mRNA results in its degradation as detected by Northern blot analysis, indicating that PYM interacts with the components of the NMD machinery (Bono, 2004).

The interaction between Mago-Y14 and PYM is direct and conserved across species. It is surprising that whereas Mago-Y14 is predominantly nuclear, PYM localizes in the cytoplasm at equilibrium. Human PYM accumulates in the nucleoplasm and nucleolus on inhibition of the export receptor Crm1, but not in nuclear speckles as is characteristic for Mago-Y14 localization. Therefore, although it cannot be excluded that PYM might interact with Mago-Y14 in the nucleus, a model is favoured where the recognition is a downstream event occurring in the cytoplasm (Bono, 2004).

The molecular recognition described in this study is mediated by an intricate network of interactions between the N-terminal domain of PYM (residues 3-35, Drosophila numbering) and both Mago and Y14, reinforcing the view that Mago-Y14 functions as a single structural unit. Centrally located within the PYM-interaction surface, residues of Mago were found that affect NMD if mutated. The implication from the structural data that the PYM-interacting surface is important for NMD is supported by tethering experiments showing degradation of an NMD reporter when human PYM is tethered downstream of a stop codon. Thus, PYM is a component of the NMD pathway. The precise molecular mechanism by which PYM has a role in NMD is an open question for further studies (Bono, 2004).

Exon junction complex subunits are required to splice Drosophila MAP kinase, a large heterochromatic gene

The exon junction complex (EJC) is assembled on spliced mRNAs upstream of exon-exon junctions and can regulate their subsequent translation, localization, or degradation. Mutations in Drosophila mago nashi (mago), which encodes a core EJC subunit, based on their unexpectedly specific effects on photoreceptor differentiation. Loss of Mago prevents epidermal growth factor receptor signaling, due to a large reduction in MAPK mRNA levels. MAPK expression also requires the EJC subunits Y14 and eIF4AIII and EJC-associated splicing factors. Mago depletion does not affect the transcription or stability of MAPK mRNA but alters its splicing pattern. MAPK expression from an exogenous promoter requires Mago only when the template includes introns. MAPK is the primary functional target of mago in eye development; in cultured cells, Mago knockdown disproportionately affects other large genes located in heterochromatin. These data support a nuclear role for EJC components in splicing a specific subset of introns (Roignant, 2010).

The exon junction complex (EJC) plays an important role in coupling nuclear and cytoplasmic events in gene expression; its recruitment allows nuclear pre-mRNA splicing to influence the subsequent fate of the spliced mRNAs. The EJC is assembled onto mRNAs during splicing, 20-24 bases upstream of each exon junction. The DEAD box RNA helicase eIF4AIII is the first subunit to associate with pre-mRNA through interactions with the intron-binding protein IBP160. eIF4AIII then recruits Magoh (known as Mago in Drosophila) and Y14. These three subunits constitute the pre-EJC; the fourth core subunit, MLN51 (Barentsz [Btz] in Drosophila). The EJC is best known for its role in nonsense-mediated decay (NMD), a surveillance mechanism that degrades mRNAs containing premature termination codons (PTCs). In mammals, NMD is greatly enhanced by the presence of a spliceable intron downstream of a PTC and is mediated by the EJC and accessory factors that include three up-frameshift (UPF) proteins. In Drosophila, the EJC has a role in mRNA localization; all four core EJC components are required to localize oskar mRNA to the posterior pole of the oocyte (Roignant, 2010).

This study isolated mutant alleles of mago based on their specific defects in epidermal growth factor receptor (EGFR)-dependent processes in eye development. Phosphorylation of mitogen-activated protein kinase (MAPK) is a critical step in signal transduction downstream of the EGFR and other receptor tyrosine kinases. Loss of mago strongly reduces the total level of the mRNA encoding Rolled (Rl), the Drosophila extracellular signal-regulated kinase (ERK)-related MAPK. Y14 and eIF4AIII, the other two subunits of the pre-EJC, also positively regulate MAPK transcript levels, but Btz does not. An intronless MAPK cDNA is independent of mago and can rescue photoreceptor differentiation in mago mutant clones; inclusion of the introns renders it Mago dependent. Mago does not affect MAPK transcription or mRNA stability but alters its splicing pattern. MAPK is a large gene located in heterochromatin; a genome-wide survey of Mago-regulated genes found that genes that shared these features were overrepresented. Based on these observations, it is proposed that the pre-EJC is essential to splice a specific set of transcripts that includes the critical signal transduction component MAPK (Roignant, 2010).

The EJC is thought to bind to all spliced mRNAs independently of their sequence, allowing them to be distinguished from unspliced transcripts in the cytoplasm. Despite these very general binding properties, this study found that loss of core EJC subunits causes surprisingly specific defects. Investigation of the basis for the effect of EJC subunits on one target gene, MAPK, has revealed a function of the pre-EJC during the splicing process (Roignant, 2010).

A genome-wide expression analysis found that loss of Mago reduces the transcript levels of only 7% of the genes expressed in S2R+ cells by 1.5-fold or more. The number of genes directly regulated by the pre-EJC is likely to be much smaller because transcript levels were measured after an extensive period of RNAi treatment that was necessary to eliminate the Mago protein. The ability of MAPK to rescue photoreceptor differentiation in mago mutant clones also suggests that many genes are downregulated as an indirect consequence of loss of MAPK. Similarly, many of the defects of mouse neuroepithelial stem cells heterozygous for Magoh are rescued by restoring the expression of a single gene, Lis1. Cytoplasmic functions of the EJC also show specificity; for instance, the EJC is required to localize oskar mRNA to the posterior of the oocyte but has no effect on the subcellular localization of other spliced mRNAs such as bicoid or gurken. This functional specificity might indicate that EJC components are, in fact, assembled on only a subset of spliced transcripts. Indeed, only the first intron in the oskar transcript contributes to its localization by the EJC. However, experiments in vertebrate and Drosophila cells have found no specific requirement for EJC assembly other than an upstream exon at least 20 bases long. Localization of EJC components to particular cytoplasmic regions in Drosophila oocytes and mammalian neurons may simply represent their selective retention on mRNAs that are translationally repressed (Roignant, 2010).

The importance of MAPK for receptor tyrosine kinase signaling has led to the evolution of multiple mechanisms to regulate its expression as well as its phosphorylation. Other vital targets for the pre-EJC may be found in the ovary. mago and Y14, but not btz, are required early in oogenesis for germline stem cell differentiation and oocyte specification. Because germline inactivation of the Ras pathway has no effect on oogenesis, these functions of Mago and Y14 may reflect a requirement for the pre-EJC to splice transcripts other than MAPK (Roignant, 2010).

The EJC has been shown to act on previously spliced mRNAs in the cytoplasm to increase their translation, direct their subcellular localization, or target them for degradation if they contain premature stop codons. However, none of these mechanisms could explain the strong reduction of MAPK mRNA levels in the absence of pre-EJC subunits. This study has provided several lines of evidence suggesting that the pre-EJC facilitates splicing of a specific subset of introns, including at least one present in the MAPK pre-mRNA. First, MAPK is not an indirect transcriptional target of the pre-EJC because MAPK pre-mRNA is not uniformly reduced in the absence of mago, and Mago is required for the expression of a MAPK genomic construct driven by a heterologous promoter. Second, the EJC-associated splicing factors RnpS1 and SRm160 contribute to maintaining normal MAPK levels, whereas Btz, the only core EJC subunit absent from the spliceosomal complex, is dispensable for MAPK expression. Third, an abnormally spliced MAPK product is detected in Mago-depleted cells. Finally, heterochromatic genes with large introns show an increased propensity for regulation by Mago. Previous experiments did not detect any positive function for the EJC in splicing; however, they were performed in vitro using short introns with strong splice sites and would therefore have missed a function specific to one class of introns (Roignant, 2010).

It will be interesting to determine what features of introns make their splicing dependent on the pre-EJC. The genome-wide analysis points to heterochromatic location and intron size as two characteristics that are likely to be important. Unlike mammalian genomes, the Drosophila genome contains primarily short introns. Large introns are most common in heterochromatic genes such as MAPK, where they are rich in repetitive DNA composed of transposons, retrotransposons, and satellite sequences. Production of endo-siRNAs from such repetitive elements or the presence of splice sites within these elements could interfere with the splicing of the introns they occupy. Chromatin structure might also directly influence splicing, as suggested by recent studies showing differences in nucleosome occupancy and histone modifications between exons and introns and recruitment of splicing regulators by chromatin-binding proteins (Roignant, 2010).

Recognition of splice sites over long distances poses a challenge to the splicing machinery. Splice sites for large introns are initially identified by an exon definition mechanism. The pre-EJC, which is assembled upstream of the 5' splice site during splicing, might interact with other factors across the exon to facilitate recognition of the upstream 3' splice site. Perhaps pre-EJC complexes deposited upstream of introns that can be easily detected due to their small size, strong splice sites, or other features contribute to the subsequent recognition of neighboring introns. Alternatively, because the pre-EJC is assembled prior to exon ligation, it might act during its own recruitment into the spliceosome to promote the second step of splicing. These alternatives cannot be distinguished at present because the measurements of 5' and 3' splice junctions in the MAPK pre-mRNA were made at steady state and thus reflect the balance between transcription, splicing, and degradation. The presence of recursive splice sites that allow large introns to be spliced in multiple steps makes genes less likely to require the EJC. Of interest, recursive splice sites are much less common in vertebrate introns than in Drosophila, suggesting that the EJC-dependent mechanism might be more widely used in higher organisms. The current data challenge the view that the EJC acts only as a marker that affects postsplicing events and suggest that this complex also functions within the nucleus to process a specific set of transcripts (Roignant, 2010).

The transcriptome-wide landscape and modalities of EJC binding in adult Drosophila

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. This study has 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. The EJC was found 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 not been reported in mammals, specify EJC assembly on a biologically coherent set of transcripts in Drosophila (Obrdlik, 2019).

The exon junction complex (EJC) consists of a heterotetramer core composed of eIF4AIII, Mago, Y14 (Tsunagi), and Barentsz (Btz) and auxiliary factors that form the EJC periphery. The complex assembles on mRNAs during splicing, -20 to -24 nt upstream of exon-exon junctions. EJC assembly is a multi-step process that begins with CWC22-mediated deposition of the DEAD-box helicase eIF4AIII on nascent pre-mRNAs (Alexandrov, 2012; Barbosa, 2012; Steckelberg, 2015) and is followed by recruitment of Mago and Y14, forming a pre-EJC intermediate. The pre-EJC is stably bound to RNA because of the ATPase-inhibiting activity of the (non-RNA-binding) Mago-Y14 heterodimer, which 'locks' eIF4AIII helicase in its RNA-bound state. Once formed, the pre-EJC is completed by recruitment of Barentsz (Btz), forming mature EJCs (Obrdlik, 2019).

The roles of the EJC in post-transcriptional control of gene expression are manifold. In the nucleus, EJC subunits have a role in splicing, mRNA export, and nuclear retention of intron-containing RNAs. In the cytoplasm, the EJC is reported to play a role in translation, nonsense-mediated decay (NMD) , and RNA localization. Although most EJC functions appear conserved, in Drosophila the EJC is not crucial for NMD, but it is essential for oskar mRNA localization within the developing oocyte. To better understand the engagement of the EJC in the fly, a strategy to stabilize mRNA binding proteins (mRBPs) associated with their RNA templates has been developed within multi-protein messenger ribonucleoprotein (mRNP) assemblies, and the EJC mRNA interactome was defined in adult Drosophila melanogaster. Through the use of the crosslinking agent dithio(bis-) succinimidylpropionate (DSP), the method captures stable and transient protein interactions in close proximity and allows definition of the binding sites of specific protein (holo-)complexes associated with their RNA templates (isolation of protein complexes and associated RNA targets [ipaRt]). This analysis of EJC-protected sites defined by ipaRt reveals that in Drosophila, EJC binding occurs at canonical deposition sites, with a median coordinate ~22 nt upstream of exon-exon junctions. Although in mammals EJC-mediated protection outside canonical sides was reported, this study finds that in Drosophila the degree of non-canonical EJC-mediated RNA protection is minimal. In Drosophila RNA polymerase II transcripts protected primarily by the EJC derive from genes involved in differentiation or development, while mRNAs protected primarily by mRBPs derive from genes with homeostatic functions. This analysis suggests that the EJC's bias for transcripts in Drosophila is a consequence of several modalities in the genes' architecture, particularly splice site number and intron length. Moreover, EJC binding is enhanced by adjacent RNA secondary structures and CUG-rich hexamers located 3' to the EJC binding site. These modalities were not identified in previous studies of mammalian EJC binding, reflecting either greater specificity of this method for fully assembled EJCs or differences in EJC binding between flies and human. This study provides a comprehensive transcriptome-wide view of EJC-RNA interactions in a whole organism and unravels RNA modalities that contribute to the unforeseen biological coherence of the bound transcripts (Obrdlik, 2019).

This study has profiled the landscape of EJC binding across the transcriptome of a whole animal, Drosophila melanogaster, and determined the parameters that influence the distribution of the complex on RNAs in the organism. Previous knowledge of EJC-RNA interactions was based on UV-crosslinking experiments in specific cell types grown as homogeneous cultures for the individual studies. Although UV crosslinking remains a method of choice for identification of protein binding sites on nucleic acids, because of the inefficient penetration of UV light into tissues and organisms, the method is most useful when applied to cells in culture. In contrast, this analysis of EJC distribution in the tissues of whole Drosophila flies was made possible by ipaRt, which uses the crosslinking agent DSP to freeze protein-protein interactions within otherwise dynamic RNP complexes, such as the EJC (Obrdlik, 2019).

DSP-mediated covalent bond formation between the RNA helicase eIF4AIII and the Mago-Y14 heterodimer is shown to preserve EJCs in their 'locked' state on mRNAs and that efficient recovery of the bound RNAs does not require their crosslinking to eIF4AIII using UV light. The 'ipaRt' approach, like CLIP and iCLIP, enables highly stringent washing of the samples. In support of the robustness and reliability of the DSP-based assays, this study demonstrated high reproducibility not only among technical but also biological replicates of EJC ipaRt, as well as mRBP footprinting sequencing results (Obrdlik, 2019).

Furthermore, ipaRt allows enables the use of non-RNA-binding subunits of the EJC, such as Mago, as immunoprecipitation baits. This is highly relevant in the context of the EJC, as it has been shown that its RNA-binding subunit, the RNA helicase eIF4AIII, may have other, EJC-independent functions in the cell. ipaRt afford the option of using Mago as a EJC bait, and indeed this is a main reason for the high-quality definition of the EJC binding landscape in the fly cytoplasm that was achieved. The protection site reads obtained from EJC ipaRt map almost exclusively to canonical EJC deposition sites with a median protection ~22 nt of the upstream exon's 3' end. In contrast to mammalian EJC CLIP and RIP studies, in which eIF4AIII served as an immunoprecipitation bait, EJC ipaRt reads mapping to regions distant from canonical deposition sites are of low abundance and sequencing coverage. Although this discrepancy could reflect differences in EJC engagement in humans and Drosophila, it more likely reflects the choice of bait or the cell compartment in which the analysis was executed. Indeed, a recent study in human cells revealed that when the cytoplasmic EJC component Btz was chosen as the bait rather than eIF4AIII, the proportion of non-canonical EJC deposition sites was negligible (Obrdlik, 2019).

Finally, in ipaRt the DSP crosslinker is applied ex vivo during tissue disruption and does not require inhibition of translation in vivo. Therefore ipaRt is considered a method of choice for functional investigations of protein-RNA complexes in fully developed organisms and tissues (Obrdlik, 2019).

Through this analysis, factors were defined that contribute to or inhibit EJC assembly on mRNAs and at individual exon-exon junctions in Drosophila. From this it is deduced that the landscape of EJC binding to RNAs is sculpted through regulation of EJC assembly at two levels in the fly (Obrdlik, 2019).

At the upstream regulatory level, the degree to which EJCs are assembled on an mRNA is dictated by the complexity of the gene's architecture: mRNAs produced from genes of simple architecture are marked by fewer EJCs, while mRNAs from genes of complex architecture, comprising multiple splice sites and long introns, are EJC bound to a higher degree. Given that EJCs assemble on mRNAs concomitantly with splicing, it is not surprising that mRNAs of genes containing a greater number of introns are more likely to be EJC bound. However, the finding that the enhancing effect on EJC binding provoked by large introns is not restricted to flanking junctions but occurs at junctions mRNA-wide is unexpected. Loss-of-function experiments indicate that the EJC participates in exon definition during splicing of long intron-containing genes in Drosophila, particularly in definition of exons proximal to the long introns. The data exclude any significant bias toward EJC assembly in proximity to long-intron splice junctions. Instead they reveal a general enhancement of EJC binding at exon-exon junctions throughout transcripts of long-intron genes. Therefore, it is concluded that stable binding of EJCs within mRNAs of long-intron genes is not the result of EJC engagement in exon definition. Instead, it is proposed that the high degree of EJC binding to long-intron transcripts derives from the increased number and resting time of co-transcriptionally assembled spliceosomes on the nascent transcripts, which would increase the probability of CWC22-dependent eIF4AIII recruitment to pre-mRNAs during splicing (Obrdlik, 2019).

At the downstream regulatory level, after EJC assembly rates at transcripts are defined, deposition of EJCs along mRNA exon-exon junctions is modulated by the structural and sequence context of the splice sites. dsRNA stem structures in exon-exon junctions of Drosophila mRNAs either antagonize EJC assembly when present within canonical EJC deposition sites or enhance EJC assembly when located in the vicinity of the EJC deposition site. Absence of dsRNA within the EJC binding moiety is in agreement with reported preference of EJCs for ssRNA. It remains to be elucidated how and why EJC binding is positively affected when RNA stem structures are found in its direct proximity on the bound template (Obrdlik, 2019).

Although it is likely that the structural context of exon-exon junctions in Drosophila directly influences the degree of EJC assembly, sequence composition-derived effects on EJC binding to mRNA are a consequence of the assigned roles of these sequences during pre-mRNA splicing. This study has demonstrated that exon-exon junctions with strong 5' and strong 3' splice sites (SSs) are biased toward junctions with enhanced EJC binding. For the regulation of weak 5' and 3' SSs, which commonly occur at alternatively spliced junctions, cis-acting splicing regulatory elements (SREs) were shown to be of importance. In light of the negative impact of alternative splicing at the level of EJC mRNA binding, it is not surprising that conventional exonic splice enhancers (ESEs) and exonic splicing silencers (ESSs) hardly affect EJC binding at the level of individual exon-exon junctions. Whether the position-dependent bias mediated by the UUU-triplet- and CUG-triplet-containing hexamers toward inhibited or enhanced EJC binding that this study has discovered in the Drosophila dataset is due to a direct or indirect influence of these hexamers on splicing remains to be addressed. UUU-triplet-containing hexamers, which are strongly biased against EJC binding, could potentially function as yet undefined 5'ESS in Drosophila. Interestingly, CUG-triplet-containing hexamers, which are strongly biased toward enhanced EJC binding, share sequence similarity with a previously predicted CUG containing 5'ESE of short intron splice sites. It appears likely that the CUG-triplet and UUU-triplet hexamers exert their effect on EJC binding as a yet undefined class of SREs (Obrdlik, 2019).

In agreement with reports in mammals, the extent of EJC occupancy varies between mRNAs and exon-exon junctions also in Drosophila. The splice site score next to a junction correlates with increased EJC deposition in the fly, and this relationship between splicing efficiency and EJC deposition has also been proposed in mammalian studies. Analysis of published mammalian Btz iCLIP data revealed several modalities that correlate with the increased binding landscape of the EJC on mRNAs in both mammals and Drosophila, including the large number of introns, high transcript abundance, and sequence context of individual exon-exon junctions. Interestingly, the presence of long introns has a slightly negative effect and the amount of alternative splicing a slightly positive effect on EJC occupancy in mammals; the latter agrees with previous observations. Studies in cultured mammalian cells have reported that EJC-enriched junctions contain a relatively high proportion of 'non-canonical' protection sites, which were enriched for RBP consensus sequences of the SR protein family. Analysis of mammalian Btz iCLIP data confirms that presence of ESEs in upstream exons and 5' intronic splicing enhancers (ISE)s in introns correlates with enhanced EJC binding. Moreover, a group of junctions have been identified in mammals containing AGAA hexamers that are biased for enhanced EJC binding, but their effects are not especially strong near the canonical EJC deposition site. These hexamers match the AGAA-encompassing consensus sequence of the mammalian SR protein SRSF10, known to function as splicing enhancers, and have been found previously in EJC bound exon-exon junctions. Not only do the in silico results agree with these reports and support the proposed cooperative binding of EJC with SR proteins, they also partially explain the EJC's preference in mammals for alternatively spliced mRNAs (Obrdlik, 2019).

One observation deriving from this analysis of published mammalian Btz iCLIP datasets is surprising. Although junctions in Drosophila are observed to be enhanced or inhibited in EJC binding by specific base-pairing probability (bpp) profiles, thus by specific RNA folding categories, it was not possible to detect any striking difference between overall bpp profiles of exon-exon junctions with enhanced or inhibited EJC binding in mammals. Indeed, the only aspect of RNA structure shared by mammals and Drosophila is the negative effect of dsRNA when directly overlapping with the canonical EJC deposition site. In Drosophila, however, the presence of dsRNA close to canonical deposition sites enhances EJC binding, an effect that is not observed in mammalian cells (Obrdlik, 2019).

The findings regarding the differences in the RNA modalities enriched at highly occupied mammalian and Drosophila EJC sites provide insight into the expansion of functions of the EJC during eukaryotic evolution. Spliceosome catalyzed splicing reactions are bidirectional, and efficient formation of exon-exon junctions during RNA maturation is achieved by Prp22-induced release of spliceosomes from mRNAs. The EJC is absent in organisms with low rates of RNA splicing, such as Saccharomyces cerevisiae, but present in organisms with high splicing rates, such as Schizosaccharomyces pombe. This suggests that with the increased demand for splicing accuracy in higher eukaryotes, the EJC evolved to function as an exon-exon junction 'lock' hindering spliceosome reassembly at spliced exon-exon junctions. Because EJC binding in the fly is enhanced at strong splices sites, but is not affected by splicing enhancer elements, and is not biased toward alternatively spliced mRNAs, it is proposed that the EJC preserved its primary function as such a lock in Drosophila. Two recent studies provide evidence that also in mammals bound EJCs hinder spliceosome assembly, suppressing recursive splicing (RS) of RS exons. The previously reported importance of EJC for splicing fidelity, and the current observations on the mode of EJC binding to transcripts in the fly revealing its independence from splicing regulatory elements indeed supports that the EJC's most conserved function is to ensure splicing irreversibility (Obrdlik, 2019).

The EJC further evolved to become a central component of the NMD pathway in mammals, in which more than 95% of all genes are alternatively spliced. This may explain why EJCs in mammals are enriched on alternatively spliced transcripts. In Drosophila, in which only 30% of all genes appear to be alternatively spliced, the EJC is not a component of the main NMD pathway. It is proposed that although the EJC-NMD pathway evolved before segregation of the proto- and deuterostome clades, it gained importance by complementing the faux 3'UTR-NMD pathway during the evolution of vertebrates, for which RNA surveillance and spatiotemporal control of gene expression are essential (Obrdlik, 2019).

Similarly, recruitment of the EJC and interacting proteins upon splicing to facilitate mRNA localization so far seems exclusive to Drosophila. Two Drosophila-specific features that modulate EJC binding, namely, the presence of a large intron within a gene and secondary structure near the junction, are also predictive of mRNA localization. Although the precise strength of association between these features and mRNA localization remains to be verified with larger and more quantitative datasets, previous studies with the SOLE in oskar RNA have shown that RNA structure and EJC binding are indeed crucial for oskar mRNA localization (Obrdlik, 2019).

The mago nashi locus encodes an essential product required for germ plasm assembly in Drosophila

The name mago nashi (Japanese for 'without grandchildren') reflects the basis for identification of the gene, a screen for grandchildless-like maternal effect mutations resulting in sterility of the progeny of homozygous females. Examination of embryos collected from such mago mutant females shows no functional posterior determinants in the posterior pole. In viable embryos from mago mutant females, polar granules are reduced or absent and germ cells fail to form (Boswell, 1994).

Clues as to protein function are often obtained from the protein sequence itself. Molecular characterization of the mago nashi locus was not very informative. The mago gene codes for a protein with no identifiable sequence motifs (Newmark, 1994).

What then is the function of Mago protein? Valuable clues to a developmentally complex answer come from a study of egg phenotypes, nuclear migration, microtubule polarity and the localization of Mago protein. Approximately 38% of the eggs produced when mutant mago females are shifted to 17 degrees C. from 25 degrees are ventralized, compared to only 1% for females maintained at the higher temperature. Ventralized mago eggs cannot become fertilized due to a defective micropyle canal. At stage 7 of oogenesis, the oocyte nucleus migrates from the posterior of the oocyte to the anterodorsal cortical postion. This movement is critical for suppression of ventral cell fate and establishment of dorsal follicular cell fates because it allows apical localization of Gurken mRNA, and the spatially restricted synthesis of Grk protein. The number of females with mislocalized oocyte nuclei corresponds to the number of ventralized eggs, suggesting that the ventralized phenotype observed in mago eggs results from the failure of nuclear migration to the anterodorsal cortex during oogenesis (Newmark, 1997).

mago egg chambers also display defects in the organization of the oocyte microtubule cytoskeleton. In mago mutants Bicoid mRNA accumulates at both poles and tagged kinesin accumulates in the center of the oocyte instead of to the normal posterior localization (Newmark, 1997). Using a new in vivo marker for microtubules, it has been shown that mago nashi mutant oocytes develop a symmetric microtubule cytoskeleton that leads to the transient localisation of Bicoid mRNA to both poles (Micklem, 1997). Both these results indicate that polarization of the microtubule cytoskeleton is abnormal in egg chambers from mago females. This defect results in a lack of localization of Staufen protein to the posterior pole plasm (Newmark, 1994).

Marker studies show that regardless of the temperature at which mago females are reared, posterior follicle cells are specified properly. This suggests that the earlier gurken signaling from the posterior of the oocyte occurs normally and indicates that mago functions within the oocyte to mediate the return signal(s) sent from the posterior follicle cells to the oocyte. In the absence of wild-type mago function, the reorganization of the microtubule network, essential for relocation of the nucleus from the posterior to the anterior/dorsal part of the oocyte, fails to occur, and axis formation and subsequent germ-plasm assembly is defective (Newmark, 1997).

In the germarium, Mago protein is associated with all germ-line nuclei. During stages 1 and 2 of oogenesis, after the cyst leaves the germarium, Mago is detected within the nurse cell nuclei. By oocytic stage 3-4, Mago is detected in the oocyte, both within the nucleoplasm and in the cytoplasm. Mago protein is also associated with the posterior-most region of the oocyte. This posterior localization is observed during two distinct periods of oogenesis, the first during stages 3 to 5, and the second during stages 8 and 9. During stages 6 and 7, Mago is not detected at the posterior pole. By stage 10 Mago is once again no longer detectable at the posterior pole. Mago protein is also detected in the somatic follicle cell nuclei. Mutant Mago protein is not detected within the posterior pole during oogenesis, demonstrating that posterior pole localization (but not nuclear localization) is required for correct microtubule polarity and assembly of a functional posterior pole plasm. Thus mago is considered a posterior group gene (Newmark, 1997).

Drosophila axis formation requires a series of inductive interactions between the oocyte and the somatic follicle cells. Early in oogenesis, Gurken protein is produced in the posterior part of the oocyte to induce the adjacent posterior follicle cells to adopt a posterior cell fate. These cells subsequently send an unidentified signal back to the oocyte to induce the formation of a polarised microtubule array that defines the anterior-posterior axis. The polarised microtubules also direct the movement of the nucleus and Gurken mRNA from the posterior to the anterior/dorsal part of the oocyte, where Gurken signals a second time to induce the dorsal follicle cells, thereby polarizing the dorsal-ventral axis. Mago nashi protein is required in the germ line for the transduction of the polarizing signal from the posterior follicle cells (Micklem, 1997 and Newmark, 1997). The apparent absence of Mago from the posterior pole during stages 6 and 7 may reflect a reorganization of the oocyte cytoskeleton (See betaTubulin56D) that takes place at these stages (Theurkauf, 1992).

The protein Mago provides a link between splicing and mRNA localization

The proteins Mago and Y14 (FlyBase name: Tsunagi) are evolutionarily conserved binding partners. Y14 is a component of the exon-exon junction complex (EJC), deposited by the spliceosome upstream of messenger RNA (mRNA) exon-exon junctions. The EJC is implicated in post-splicing events such as mRNA nuclear export and nonsense-mediated mRNA decay. Drosophila Mago is essential for the localization of oskar mRNA to the posterior pole of the oocyte, but the functional role of Mago in other species is unknown. Mago is shown to be a bona fide component of the EJC. Like Y14, Mago escorts spliced mRNAs to the cytoplasm, providing a direct functional link between splicing and the downstream process of mRNA localization. Mago/Y14 heterodimers are essential in cultured Drosophila cells. Taken together, these results suggest that, in addition to its specialized function in mRNA localization, Mago plays an essential role in other steps of mRNA metabolism (Le Hir, 2001b).

Messenger RNAs (mRNAs) exist in the cell in dynamic association with multiple proteins, of which many bind cotranscriptionally and accompany the mRNA to the cytoplasm. Components of the splicing machinery (including the spliceosomal U snRNPs) are also loaded onto nascent transcripts, but while U snRNPs and most splicing factors dissociate from the spliced mRNA after completion of the splicing reaction, specific proteins bind to mRNAs as a consequence of splicing. Indeed, it has been shown that the spliceosome imprints the mRNA product by depositing several proteins 20-24 nucleotides (nt) upstream of mRNA exon-exon junctions (Le Hir, 2000a, b, 2001a; Kim, 2001a; Le Hir, 2001a: Le Hir, 2001b and references therein).

When assembled in vitro, the so-called exon-exon junction complex (EJC) contains at least five proteins: SRm160, DEK, RNPS1, Y14 and REF/Aly (Le Hir, 2000a, b). SRm160, DEK and RNPS1 are related to the splicing process. REF/Aly have been implicated in mRNA nuclear export by interacting with members of the TAP/NXF family of mRNA export receptors. Consistently, in vivo the EJC facilitates the recruitment of the heterodimeric nuclear export receptor TAP/p15 (NXF1/p15) to spliced mRNAs (Le Hir, 2001b and references therein).

In contrast to the other EJC components, the RNA-binding protein Y14 (also known as RBM8) remains bound to the mRNP after translocation through nuclear pore complexes. In this context, it has been proposed that Y14 communicates the position of introns to the cytoplasm and may play a role in nonsense-mediated mRNA decay (NMD). NMD is the process by which, if an in-frame stop codon is located some distance upstream of at least one exon-exon junction, it is generally recognized as premature and the mRNA is targeted for degradation. The proteins UPF1, UPF2 and UPF3 are required for NMD. UPF3 and UPF2 are recruited to the mRNP via interactions with components of the EJC, such as Y14 and RNPS1 (Le Hir, 2001b and references therein).

Recently, Y14 was found in yeast two-hybrid screens when the human protein Mago (Hs MGN) was used as a bait (Zhao, 2000). The interaction between Mago and Y14 was independently confirmed by Mingot (2001), who identified Mago/Y14 heterodimers as the import substrate of importin-13. Drosophila Mago (also known as Dm MGN) is required for the definition of the anteroposterior and dorsoventral axis in Drosophila and for the localization of oskar mRNA to the posterior pole of the oocyte (Le Hir, 2001b and references therein).

The conserved Mago/Y14 heterodimer specifically associates with EJC containing spliced mRNAs both in vitro and in vivo and accompanies these mRNAs to the cytoplasm. Moreover, both Mago and Y14 are essential in Drosophila cells and colocalize in the nucleus, both in Drosophila and HeLa cells. This raises the possibility that factors involved in mRNA localization in the cytoplasm are loaded onto the nuclear mRNA during splicing and escort it to its final cytoplasmic destination (Le Hir, 2001b).

Y14 and Mago are 63% and 88% identical to their human counterparts, suggesting that their interaction is conserved. Indeed, untagged Hs or Dm Y14 copurify with glutathione S-transferase (GST) fusions of Hs MGN or Mago when the proteins are coexpressed in Escherichia coli. Conversely, GST-Hs Y14 pulls down untagged Hs MGN from total lysates of E. coli expressing both proteins. The Mago/Y14 interaction occurs in the presence of RNase A, indicating that it is not RNA-mediated. When the GST tag is removed by cleavage with TEV protease and Mago/Y14 complexes are further purified by gel filtration, the two subunits are recovered in a stoichiometric ratio and the apparent molecular weight of the complex is consistent with that of the heterodimer (Le Hir, 2001b).

In Drosophila oocytes Mago is predominantly nuclear, although a fraction of the protein accumulates within the posterior pole plasm. Consistently, in transiently transfected SL2 cells zz-tagged Mago and Dm Y14 localize within the nucleus and are excluded from the nucleolus. Similarly, when hs MGN is transiently expressed in HeLa cells as a fusion with green fluorescent protein (GFP), it localizes in the nucleoplasm and is excluded from the nucleolus. Although the GFP signal is widespread in the nucleoplasm, the staining is not homogenous and sites of higher concentration in speckled domains are observed. These domains colocalize with the structures labeled by the antibody NM4 directed to SR proteins. The localization of Mago in the nucleoplasm and in speckled domains is similar to that reported for Y14 (Le Hir, 2001b).

Based on the heterodimerization of Mago with Y14 and its localization into nuclear speckles, attempts were made to determine whether Mago is a component of the EJC. To this end, ß-globin pre-mRNAs containing either a 38- or a 17-nt exon 1 (named ß/38 and ß/17, respectively) were coincubated in HeLa cell nuclear extracts to generate spliced mRNAs. Because the EJC is deposited on spliced mRNAs more than 20 nt upstream of the exon-exon junction (Le Hir, 2000b, 2001a), the spliced ß/17 mRNA with a 5' exon shorter than 20 nt does not carry the EJC (Le Hir, 2001a). Splicing reactions were supplemented with recombinant Mago or Mago/Y14 complexes having a GST tag fused N-terminally to Mago. Recombinant GST served as negative control. After splicing, these reactions were subjected to immunoprecipitation (IP) with anti-GST antibodies and the coimmunoprecipitated (coIP) RNA fragments monitored by denaturing PAGE. In extracts supplemented with Mago/Y14 complexes, the spliced ß/38 mRNA was precipitated above the background levels observed in extracts supplemented with GST. In contrast, RNAs without the EJC such as the pre-mRNAs or the spliced ß/17 mRNA were not precipitated above background levels. GST-Mago alone did not significantly associate with spliced ß/38 mRNA, suggesting that Mago is recruited to spliced mRNPs only as a heterodimer with Y14. These results also indicate that the association of recombinant Mago/Y14 complexes with ß/38 mRNA is specific and not due to an artificial tethering of recombinant GST-Mago to the EJC through endogenous Y14. Next it was determined that Mago binds to the mRNA at the position where the EJC is deposited. Taken together, these results show that recombinant Mago/Y14 heterodimers specifically associate with spliced mRNAs and that deposition of Mago/Y14 complexes is spatially restricted to the mRNA fragment carrying the EJC (Le Hir, 2001b).

To investigate whether Mago remains associated with spliced mRNAs after export to the cytoplasm, full-length Ftz or ß-globin pre-mRNAs were coinjected into oocyte nuclei along with recombinant GST-Mago/Y14 or GST and the mixture of control RNAs. Like Y14, Mago remains bound to the cytoplasmic mRNA after export. Mago alone does not significantly associate with mRNA suggesting that there is not a large pool of free endogenous Y14 in the oocyte (Le Hir, 2001b).

In order to gain additional insight into the role of Mago and Y14 in vivo, the endogenous proteins were depleted from SL2 cells by means of double-stranded (ds) RNAi. The effect of these depletions was compared with the phenotype observed when the essential mRNA export receptor NXF1 or the NMD factor UPF1 are depleted. Depletion of Mago or Y14 inhibits cell growth. This inhibition is detected 4 days after transfecting the corresponding dsRNAs and parallels the inhibitory effect observed when UPF1 is depleted. However, it is not as dramatic as the inhibition observed when cells are depleted of NXF1 (Le Hir, 2001b).

The efficiency and specificity of the depletion was investigated by Western blot with antibodies raised against the recombinant proteins. The steady-state expression level of Mago was reduced to ~5%-10% of the level detected in untreated cells, 4-6 days after transfecting Mago dsRNA. Surprisingly, a similar reduction of Mago protein level was observed when cells were transfected with Y14 dsRNA. Conversely, Y14 protein levels were reduced in cells transfected with Mago dsRNA, although not as efficiently as when Y14 dsRNA was transfected. These results are consistent with the observation that Mago and Y14 form heterodimers and suggest that depletion of one subunit affects the expression level of the second component of the heterodimer (Le Hir, 2001b).

Mago and Y14 are highly conserved and ubiquitously expressed proteins in metazoans. The genome of Schizosaccharomyces pombe also encodes Mago and Y14 homologs, but no obvious homologs are encoded by the Saccharomyces cerevisiae genome. The conservation of Mago/Y14 in metazoans and in S. pombe and their ubiquitous pattern of expression suggest an essential role for these proteins, in addition to the specialized role of Mago in the localization of oskar mRNA in Drosophila oocytes. Indeed, as components of the EJC, Mago/Y14 are likely to associate with most, if not all, spliced mRNAs and may have a more general function in post-splicing mRNA metabolism (Le Hir, 2001b).

Depletion of Mago or Y14 from SL2 cells inhibits growth. The data suggest that this inhibition cannot be attributed to a general block of splicing or mRNA export. However, it cannot be ruled out that Mago/Y14 are required for splicing and/or export of a subset of essential mRNAs. Y14 has been implicated in NMD; remarkably, depletion of UPF1 from SL2 cells inhibits growth with similar kinetics as those observed when Mago or Y14 are depleted. The growth arrest upon Mago or Y14 depletion may then arise from a deficiency in NMD. Certainly, this possibility requires further investigation, in particular the analysis of mRNAs carrying premature stop codons (Le Hir, 2001b).

Although Mago is not an RNA-binding protein, mutations in the mago nashi gene affect the localization of oskar mRNA to the posterior pole of Drosophila oocytes. mRNA localization depends on the presence of different cis-acting RNA sequences, many of which fall within the 3' untranslated region (3' UTR) of the mRNA. These sequence elements recruit specific trans-acting factors. Thus, a possible role of Mago in oskar mRNA localization may be to facilitate the recruitment and/or to stabilize proteins bound to the localization signals present at the 3' UTR of this transcript. In this context, it is interesting to note that the protein Barentsz, which is also required for the posterior localization of oskar mRNA, fails to associate with oskar mRNA in mago nashi mutants. Thus, despite their association with bulk mRNA, Mago/Y14 heterodimers may play specific roles in multiple steps of RNA metabolism in both the nucleus and the cytoplasm (Le Hir, 2001b).

GENE STRUCTURE

The untranslated region of the 1.1 kb transcript contains three AUUA sites not included in the 0.7 kb transcript. These sites have been implicated in mRNA instability (Newmark, 1994 and references).

Bases in 5' UTR - 109

Exons - 2

Bases in 3' UTR - 531 and 151

PROTEIN STRUCTURE

Amino Acids - 147

Structural Domains

The Mago protein is slightly acidic and contains a high percentage of charged residues. The C-terminus is hydrophobic. A striking similarity is observed with a C. elegans protein, Ce-mago. The proteins share 78% sequence identity and 86% conservation over a region of 101 amino acids (Newmark, 1994).

EVOLUTIONARY HOMOLOGS

Homologs of Drosophila mago nashi have been detected in C. elegans (ce-mago), Xenopus laevis (xl-mago), and M. musculus (mm-mago). xl-mago and mm-mago are 88% identical to Drosophila mago indicating that Mago is highly conserved. A single copy of ce-mago is sufficient to complement the axis defects and sterility of a Drosophila mago mutant. Thus ce-mago is able to provide mago+ function required for both germ-plasm assembly and zygotic viability (Newmark, 1997).

Mago nashi is essential for spermatogenesis in the fern Marsilea

Spermatogenesis in fern Marsilea vestita is a rapid process that is activated by placing dry microspores into water. Nine division cycles produce seven somatic cells and 32 spermatids, where size and position define identity. Spermatids undergo de novo formation of basal bodies in a particle known as a blepharoplast. This study investigated mechanisms responsible for spermatogenous initial formation. Mago nashi (Mv-mago) is a highly conserved gene present as stored mRNA and stored protein in the microspore. Mv-mago protein increases in abundance during development and it localizes at discrete cytoplasmic foci (Mago-dots). RNA interference experiments show that new Mv-mago protein is required for development. With Mv-mago silenced, asymmetric divisions become symmetric, cell fate is disrupted, and development stops. The alpha-tubulin protein distribution, centrin translation, and Mv-PRP19 mRNA distribution are no longer restricted to the spermatogenous cells. Centrin aggregations, resembling blepharoplasts, occur in jacket cells. Mago-dots are undetectable after the silencing of Mv-mago, Mv-Y14, or Mv-eIF4AIII, three core components of the exon junction complex (EJC), suggesting that Mago-dots are either EJCs in the cytoplasm, or Mv-mago protein aggregations dependent on EJCs. Mv-mago protein and other EJC components apparently function in cell fate determination in developing male gametophytes of M. vestita (van der Weele, 2007).

mag-1, a nomolog of Drosophila mago nashi, regulates hermaphrodite germ-line sex determination in Caenorhabditis elegans

The Caenorhabditis elegans gene mag-1 can substitute functionally for its homolog mago nashi in Drosophila and is predicted to encode a protein that exhibits 80% identity and 88% similarity to Mago nashi. RNA-mediated interference (RNAi) has been used to analyze the phenotypic consequences of impairing mag-1 function in C. elegans. mag-1(RNAi) causes masculinization of the germ line (Mog phenotype) in RNA-injected hermaphrodites, suggesting that mag-1 is involved in hermaphrodite germ-line sex determination. Epistasis analysis shows that ectopic sperm production caused by mag-1(RNAi) is prevented by loss-of-function (lf) mutations in fog-2, gld-1, fem-1, fem-2, fem-3, and fog-1, all of which cause germ-line feminization in XX hermaphrodites, but not by a her-1(lf) mutation that causes germ-line feminization only in XO males (HER-1 is an inhibitory ligand that binds to TRA-2, which functions as an inhibitor of spermatogenesis). These results suggest that mag-1 interacts with the fog, fem, and gld genes (these act downstream of TRA-2) and acts independently of her-1. It is proposed that mag-1 normally allows oogenesis by inhibiting function of one or more of these masculinizing genes, which act during the fourth larval stage to promote transient sperm production in the hermaphrodite germ line. When the Mog phenotype is suppressed by a fog-2(lf) mutation, mag-1(RNAi) also causes lethality in the progeny embryos of RNA-injected, mated hermaphrodites, suggesting an essential role for mag-1 during embryogenesis. The defective embryos arrest during morphogenesis with an apparent elongation defect. The distribution pattern of a JAM-1::GFP reporter, which is localized to boundaries of hypodermal cells, shows that hypodermis is disorganized in these embryos. The temporal expression pattern of the mag-1 gene prior to and during morphogenesis appears to be consistent with an essential role for mag-1 in embryonic hypodermal organization and elongation (Lim, 2000).

Since mago nashi functions in Drosophila oogenesis, it was thought possible that mag-1 could be a positive regulator of C. elegans oogenesis, and be required either for switching from spermatogenesis to oogenesis or for maintaining oocyte production after the switch occurs. The demonstration that mag-1 is not required for oogenesis appears to rule out these possibilities. The oogenesis defect resulting from mag-1(RNAi) is completely suppressed by mutations in genes required for spermatogenesis that cause feminization of the germ line, supporting the view that instead, mag-1 normally acts to promote oogenesis by preventing spermatogenesis, through interaction with these genes. To allow oogenesis after the switch, the hermaphrodite germ line must repress the function of fem-3, which acts with or through the other fem genes to promote spermatogenesis. Two different mechanisms appear to be involved in negative regulation of fem-3: (1) its function is repressed at the protein level by interaction of FEM-3 with TRA-2 (TRA-2 is the putative receptor for HER-1); (2) its expression is repressed posttranscriptionally by interaction of FBF-1 and FBF-2 with the 3'UTR of fem-3 mRNA (Lim, 2000).

This dual regulation suggests two possible models for the function of mag-1 in preventing spermatogenesis: (1) mag-1 could trigger inactivation of fem-3 function by inhibiting fog-2/gld-1 activity, thereby activating tra-2 expression; (2) mag-1 could trigger posttranscriptional repression of fem-3 expression through or with the repressor functions of fbf-1 and fbf-2. In support of model (1) is the finding that the masculinization of the germ line phenotype resulting from mag-1(RNAi) is suppressed by tra-2(gf) mutations that prevent tra-2 inactivation by fog-2/gld-1, as well as by feminizing lf mutations in fog-2 or gld-1. These results would seem to suggest that mag-1 acts upstream of tra-2 and fog-2/gld-1 (Lim, 2000).

However, model (2) would seem more likely if there were evolutionary conservation of functional relationships between mag-1 and the pumilio homologs fbf-1 and fbf-2. In Drosophila, mago nashi appears to act upstream of nanos, which functions with pumilio in the pathway of posterior group genes required for embryonic abdominal patterning. Members of this pathway also act to control early events in germ-line development as well as oogenesis. Apparently arguing against model (2) is the finding that fog-2(lf) mutations are epistatic to mag-1(RNAi) rather than vice versa, as the model would predict. It is known that fbf(RNAi), in contrast to mag-1(RNAi), is epistatic to fog-2(lf). This result indicates that derepression of fem-3 by fbf(RNAi) can override the ability of TRA-2 (A or B) to functionally repress the resulting FEM-3 product, so that the germ line is masculinized even though tra-2 is fully active. If fem-3 were fully derepressed in the absence of any mag-1 function, then the mag-1 null condition should be epistatic to fog-2(lf) (Lim, 2000).

The findings could be consistent with model (2) if mag-1(RNAi) did not result in a null phenotype or otherwise allow some fbf function so that fem-3 is not completely derepressed. This possibility seems plausible because the mag-1(RNAi) phenotype of germ-line masculinization had to be scored in the injected animals, which may have had enough previously synthesized MAG-1 protein in the germ line to result in partial fem-3 repression. Therefore, the genetic evidence so far is still consistent with mag-1 acting either to repress fog-2/gld-1 function or to repress fem-3 function, and whether one model is preferable to the other cannot yet be determined. It is also possible that mag-1 could act in both these ways (Lim, 2000).

Ce-Y14 and MAG-1, components of the exon-exon junction complex, are required for embryogenesis and germline sexual switching in Caenorhabditis elegans

Y14 is a component of the splicing-dependent exon-exon junction complex (EJC) and is involved in the mRNA quality control system called nonsense-mediated mRNA decay. Together with another EJC component, Mago, the Drosophila homologue DmY14/Tsunagi is required for proper localization of oskar mRNA during oogenesis, a process critical for posterior formation in Drosophila development. The nematode C. elegans Ce-Y14 and MAG-1 (Mago homologue) are required for late embryogenesis and proper germline sexual differentiation. Like in other organisms, Ce-Y14 preferentially binds to spliced mRNA and specifically interacts with MAG-1. Consistent with the evolutionarily conserved interaction between Y14 and Mago homologues, suppression of Ce-Y14 by RNAi resulted in the same phenotypes as those caused by RNAi of mag-1, lethality during late embryogenesis and masculinization of the adult hermaphrodite germline. These results demonstrate that the evolutionarily conserved interaction between two EJC components, Ce-Y14 and MAG-1, has critical developmental roles in C. elegans (Kawano, 2004)

The mammalian homologue of mago nashi encodes a serum-inducible protein

The products of at least 11 maternal effect genes have been shown to be essential for proper germ plasm assembly in Drosophila melanogaster embryos. The mammalian counterpart for one of these genes (named MAGOH for mago nashi homolog) has been isolated and characterized. The predicted amino acid sequence of mouse and human MAGOH are completely identical; MAGOH homologs from the nematode C. elegans and rice grain Oryza sativa also show a remarkable degree of amino acid conservation. MAGOH was mapped to chromosome 1p33-p34 in the human and a syntenic region of chromosome 4 in the mouse. Of note, MAGOH mRNA expression is not limited to germ plasm, but is expressed ubiquitously in adult tissues and can be induced by serum stimulation of quiescent fibroblasts (Zhao, 1998).

MAGOH interacts with a novel RNA-binding protein

MAGOH is the human homolog of Drosophila Mago nashi, a protein that is required for normal germ plasm development in the Drosophila embryo. Using human MAGOH as a bait protein in a yeast two-hybrid screen, four independent cDNA clones were recovered that encode different lengths of a novel protein containing a conserved RNA-binding region. This gene, designated RBM8, encodes a 173-aa protein that has an apparent molecular mass of 26 kDa, as demonstrated by in vitro translation assay. The interaction between MAGOH and RBM8 was demonstrated by both yeast two-hybrid and GST fusion protein pull-down assays. Like MAGOH, RBM8 gene is expressed ubiquitously in human tissues; three species of RBM8 mRNA have been detected. Also similar to MAGOH, RBM8 expression is serum inducible in quiescent NIH3T3 fibroblast cells (Zhao, 2000).

Magoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing-dependent exon-exon junction complex

The RNA-binding protein Y14 binds preferentially to mRNAs produced by splicing and is a component of a multiprotein complex that assembles ~20 nucleotides upstream of exon-exon junctions. This complex probably has important functions in post-splicing events including nuclear export and nonsense-mediated decay of mRNA. Y14 binds to two components, Aly/REF and RNPS1, and to the mRNA export factor TAP. Moreover, magoh, a human homolog of the Drosophila mago nashi gene product, has been identified as a novel component of the complex. Magoh binds avidly and directly to Y14 and TAP, but not to other known components of the complex, and is found in Y14-containing mRNPs in vivo. Importantly, magoh also binds to mRNAs produced by splicing upstream (~20 nucleotides) of exon-exon junctions and its binding to mRNA persists after export. These experiments thus reveal specific protein-protein interactions among the proteins of the splicing-dependent mRNP complex and suggest an important role for the highly evolutionarily conserved magoh protein in this complex (Kataoka, 2001).

It has been suggested that magoh plays important roles in mRNA functions in the cytoplasm, since point mutations in Drosophila mago cause several deficiencies in cytoplasmic events in oocytes. For example, mago mutations cause disruption of the anterior-posterior transport of Oskar and Gurken mRNAs. These mutations also cause mislocalization of the oocyte nucleus to the posterior region. These findings strongly suggest that magoh is involved in mRNA localization in the cytoplasm through interaction with the cytoskeleton. It is likely that the cytoskeleton has an important role in the cytoplasmic sorting and localization of mRNAs. The binding of Y14 persists on newly exported mRNAs in the cytoplasm and Y14 remains bound to the same position after export. If magoh is also present in the cytoplasmic Y14 complex, magoh and Y14 may serve as adaptors for the interaction of mRNAs with microtubule-based cytoskeletons (Kataoka, 2001).

Another possible function for magoh present in the Y14 complex is in nonsense-mediated mRNA decay (NMD). Mutations that cause premature termination can be deleterious, since they may produce C-terminal truncated proteins, often resulting in severe human diseases. NMD is a process that helps avoid accumulation of mutant proteins by degrading the mRNAs that bear such mutations. Although degradation of mRNA through the NMD pathway is thought to occur in the cytoplasm, pre-mRNA splicing has an important role in NMD. Therefore, the exon-exon junction complex is an excellent candidate to mediate transfer of molecular memory of splicing and the position of introns on mRNAs to the cytoplasm. By tethering various components of the exon-exon junction complex downstream of stop codon, RNPS1 was found to recruit activity that mediates NMD. Y14 has a moderate activity in that system. The human proteins involved in NMD were identified and designated as hUpf proteins. hUpf3 interacts with Y14 and is a component of the exon-exon junction complex. Since both Y14 and magoh binding persist on the mRNA in the cytoplasm, it appears likely that Y14 and magoh together communicate the position of exon-exon junctions on mRNAs to the cytoplasm and play a role in NMD. The specific roles of these proteins in NMD remain to be investigated (Kataoka, 2001).

Protein-protein interaction between Y14 and other components of the splicing-dependent exon-exon junction complex has been studied. Y14 interacts with Aly/REF, which has a role in nuclear export of mRNAs. Since RNase A treatment did not affect its binding and the domain on Y14 which binds to Aly/REF is outside of the RBD, the interaction between these two proteins is a protein-protein interaction. These results are consistent with previously reported co-immunoprecipitation of Aly/REF with Y14 from the nucleoplasm even in the presence of RNase A. It is also likely that Y14 and Aly/REF interact with each other in the exon-exon junction complex. Interestingly, Y14 interacts with another component of this complex, RNPS1, and this is only detected after treatment with RNase A. This suggests that this is a protein-protein interaction, but that these proteins may not interact with each other when they are bound to mRNAs. No binding of Y14 to DEK is detected, nor has other evidence been found for conclusions that DEK is a component of the complex. It will be important to determine how and when the proteins of the exon-exon junction complex are recruited to mRNAs (Kataoka, 2001).

An eIF4AIII-containing complex containing Mago is required for mRNA localization and nonsense-mediated mRNA decay

Splicing of pre-mRNA in eukaryotes imprints the resulting mRNA with a specific multiprotein complex, the exon-exon junction complex (EJC), at the sites of intron removal. The proteins of the EJC, Y14, Magoh, Aly/REF, RNPS1, Srm160, and Upf3, play critical roles in postsplicing processing, including nuclear export and cytoplasmic localization of the mRNA, and the nonsense-mediated mRNA decay (NMD) surveillance process. Y14 and Magoh are of particular interest because they remain associated with the mRNA in the same position after its export to the cytoplasm and require translation of the mRNA for removal. This tenacious, persistent, splicing-dependent, yet RNA sequence-independent, association suggests an important signaling function and must require distinct structural features for these proteins. This study describes the high-resolution structure and biochemical properties of the highly conserved human Y14 and Magoh proteins. Magoh has an unusual structure comprised of an extremely flat, six-stranded anti-parallel beta sheet packed against two helices. Surprisingly, Magoh binds with high affinity to the RNP motif RNA binding domain (RBD) of Y14 and completely masks its RNA binding surface.The structure and properties of the Y14-Magoh complex suggest how the pre-mRNA splicing machinery might control the formation of a stable EJC-mRNA complex at splice junctions (Lau, 2003).

Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA

In higher eukaryotes, a multiprotein exon junction complex is deposited on spliced messenger RNAs. The complex is organized around a stable core, which serves as a binding platform for numerous factors that influence messenger RNA function. This study presents the crystal structure of a tetrameric exon junction core complex containing the DEAD-box adenosine triphosphatase (ATPase) eukaryotic initiation factor 4AIII (eIF4AIII) bound to an ATP analog, MAGOH, Y14, a fragment of MLN51, and a polyuracil mRNA mimic. eIF4AIII interacts with the phosphate-ribose backbone of six consecutive nucleotides and prevents part of the bound RNA from being double stranded. The MAGOH and Y14 subunits lock eIF4AIII in a prehydrolysis state, and activation of the ATPase probably requires only modest conformational changes in eIF4AIII motif I (Andersen, 2006).

Dosage-dependent requirements of Magoh for cortical interneuron generation and survival

Embryonic interneuron development underlies cortical function and its disruption contributes to neurological disease. Yet the mechanisms by which viable interneurons are produced from progenitors remain poorly understood. This study demonstrates dosage-dependent requirements of the exon junction complex component Magoh for interneuron genesis in mouse. Conditional Magoh ablation from interneuron progenitors, but not post-mitotic neurons, depletes cortical interneuron number through adulthood, with increased severity in homozygotes. Using live imaging, Magoh deficiency was found to delay progenitor mitotic progression in a dosage-sensitive fashion, with 40% of homozygous progenitors failing to divide. This shows that Magoh is required in progenitors for both generation and survival of newborn progeny. Transcriptome analysis implicates p53 signaling; moreover, p53 ablation in Magoh haploinsufficient progenitors rescues apoptosis, completely recovering interneuron number. In striking contrast, in Magoh homozygotes, p53 loss fails to rescue interneuron number and mitotic delay, further implicating mitotic defects in interneuron loss. These results demonstrate that interneuron development is intimately dependent upon progenitor mitosis duration and uncover a crucial post-transcriptional regulator of interneuron fate relevant for neurodevelopmental pathologies (Sheehan, 2020).

date revised: 3 January 2020

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