BIOLOGICAL OVERVIEW

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? Valuble 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 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).

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).

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).

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).

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).

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)

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).

date revised: 10 March 2004

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