mago nashi

PROTEIN INTERACTIONS

The RNA-binding protein Tsunagi interacts with Mago Nashi to establish polarity and localize oskar mRNA during Drosophila oogenesi

In Drosophila, formation of the axes and the primordial germ cells is regulated by interactions between the germ line-derived oocyte and the surrounding somatic follicle cells. This reciprocal signaling results in the asymmetric localization of mRNAs and proteins critical for these oogenic processes. Mago Nashi protein interprets the posterior follicle cell-to-oocyte signal to establish the major axes and to determine the fate of the primordial germ cells. Using the yeast two-hybrid system, an RNA-binding protein, Tsunagi, has been identified that interacts with Mago Nashi protein. The proteins coimmunoprecipitate and colocalize, indicating that they form a complex in vivo. Immunolocalization reveals that Tsunagi protein is localized within the posterior oocyte cytoplasm during stages 1-5 and 8-9, and this localization is dependent on wild-type mago nashi function. When tsunagi function is removed from the germ line, egg chambers develop in which the oocyte nucleus fails to migrate, Oskar mRNA is not localized within the posterior pole, and dorsal-ventral pattern abnormalities are observed. These results show that a Mago Nashi-Tsunagi protein complex is required for interpreting the posterior follicle cell-to-oocyte signal to define the major body axes and to localize components necessary for determination of the primordial germ cells (Mohr, 2001).

Database searches reveal that Tsunagi is significantly similar to the ribonucleoprotein (RNP) family of RNA-binding proteins with a single RNP domain and has been evolutionarily conserved. Homologs for Tsunagi exist from the fission yeast Schizosaccharomyces pombe to humans. As with Mago and its homologs, a Tsunagi-related protein is not detectable within the genome of the budding yeast Saccharomyces cerevisiae. The primary structure of Tsunagi is reminiscent of the SR family of splicing factors. Classic SR proteins contain at least one RNP domain followed by carboxy-terminal arginine/serine (RS) dipeptide residues. Each of the presumed Tsunagi homologs exhibits a series of basic residues at their carboxyl terminus, and within this region the vertebrate and yeast Tsunagi contain at least one RS-dipeptide repeat. However, the D. melanogaster and Caenorhabditis elegans proteins do not contain any RS sequences among the basic residues at their carboxyl termini (Mohr, 2001).

Drosophila Tsunagi is 60% identical and 72% similar to the human RNA-binding motif protein 8 (RBM8). RBM8 cDNAs have been used to recover clones encoding the Xenopus laevis protein Y14. Results from several laboratories suggest that Y14 is complexed with mRNAs and proteins to form ribonucleoprotein particles (mRNPs) that are preferentially exported from the nucleus to the cytoplasm. Therefore, in addition to having an RNA-binding motif, there is experimental evidence suggesting that Tsunagi-related proteins bind RNA (Mohr, 2001).

tsunagi is Japanese for 'connection' or 'link.' Hybridization of a probe from tsu cDNA to polytene chromosomes was used to ascertain the location of tsu in the genome. A single focus of hybridization on the right arm of chromosome 2, in polytene chromosome interval 45A4, was detected. Two genomic DNA contigs in the region, Dbp45A and hig, were examined by PCR for the presence of tsu sequence. A 30-40-kb P1 phagemid clone, DS02099 (BDGP), that maps to the distal end of Dbp45A was found to contain tsu (Mohr, 2001).

The Mago-Tsunagi complex localizes within the posterior pole of the oocyte during stages 8 and 9 of oogenesis. Posterior pole localization of OSK mRNA is first detected during stage 9 of oogenesis. Previous studies reveal that OSK mRNA accumulates within the oocyte but fails to localize within the posterior pole of egg chambers from mago¹ mutant females, owing specifically to the inability of the Mago¹ protein to localize within the posterior pole plasm. In mago¹ mutant egg chambers Tsunagi protein fails to localize within the posterior end of the oocyte. The results establish that the Mago-Tsunagi complex is detected within the posterior pole prior to and during the time when OSK mRNA is initially sequestered within this discrete cytoplasmic region of the oocyte, and that detection of the complex within the posterior pole is dependent on wild-type mago function (Mohr, 2001).

Mutational analysis of tsu reveals that mothers homozygous or heteroallelic for tsu alleles can produce egg chambers in which OSK mRNA fails to localize within the posterior pole. Consistent with this observation, embryos from tsu mutant females that survive to the time of cellularization lack primordial germ cells. Although posterior pole localization of OSK mRNA is not detected in tsu mutant oocytes, its anterior pole accumulation at stage 8 of oogenesis appears normal. A similar result is observed when germ-line clones lacking tsu⁺ function are examined for the presence and distribution of OSK mRNA. However, when follicle-cell clones are induced, no apparent abnormalities in OSK mRNA localization are detected. The distribution of Mago protein within mutant tsu egg chambers is indistinguishable from wild type, suggesting that posterior pole localization of Mago protein occurs independently of Tsunagi function. These results show that (1) in mutant tsu egg chambers OSK mRNA is transcribed and deposited into the oocyte, (2) anterior localization of OSK mRNA does not require tsu⁺ function, (3) posterior pole localization of Mago protein is independent of tsu⁺ function, and (4) posterior pole accumulation of OSK mRNA is dependent on tsu⁺ germ-line function (Mohr, 2001).

What role might the molecular interaction between Mago and Tsunagi serve in the posterior localization of the Staufen protein/OSK mRNA complex? Several lines of evidence indicate that Mago protein is required to anchor/localize components within the posterior pole that mediate the localization of the Staufen protein/OSK mRNA complex within the pole during stage 9: (1) Mago colocalizes with the Staufen protein/OSK mRNA complex; (2) Mago protein is mislocalized to the same ectopic site as the Staufen protein/OSK mRNA complex in mutants in which oocyte polarity is disrupted; for example, gurken; (3) localization of Mago within the posterior pole (during stage 8) precedes the posterior pole accumulation of the Staufen/OSK mRNA complex and is not dependent on localization of the complex. The evidence indicates that posterior pole localization of Mago occurs independently from the Staufen protein/OSK mRNA complex and Tsunagi protein. Mago's ability to interact with a particular cytoplasmic location independently of the components that it localizes suggests that it may serve as an adaptor that recognizes a specific site within the posterior pole of the oocyte (Mohr, 2001).

Given its interaction with Mago protein, it is likely that Tsunagi is required for anchoring the Staufen protein/OSK mRNA complex within the posterior pole but not for its transport to this cytoplasmic destination. In agreement with this conclusion, the distribution of Tsunagi protein during multiple stages of oogenesis is indistinguishable from that of Mago protein. If Tsunagi protein were involved in transporting the Staufen protein/OSK mRNA complex to the posterior pole, its subcellular distribution would more closely reflect that of Staufen protein and OSK mRNA, which mirror one another during oogenesis. In addition, RNA-binding experiments have failed to reveal interaction between Tsunagi protein and OSK mRNA (Mohr, 2001).

At least two simple models can be presented to explain the results obtained to date. In the first model, Mago protein might act as an adaptor molecule to anchor Tsunagi protein and associated mRNAs within the posterior cytoplasm of the oocyte. Although this model is appealing, it does not take into consideration the colocalization of Mago and Tsunagi proteins within nuclei. Another model is that Mago and Tsunagi proteins are assembled onto mRNP complexes to mark them for transport to and localization within the posterior pole. This alternative model takes into account the nuclear and cytoplasmic colocalization of the proteins but does not exclude the possibility that the Mago protein can act as an adaptor, linking mRNPs to a specific subcellular site. Importantly, this latter model is in agreement with experimental results showing that Y14 (a Xenopus laevis homolog of Tsunagi) shuttles from the nucleus to the cytoplasm in association with mRNAs and is retained on the mRNAs when they are exported to the cytoplasm. Therefore, these studies establish a role for Tsunagi protein in anchoring mRNAs within the posterior pole of the oocyte when associated with Mago protein, suggesting that homologs of Tsunagi in other organisms may also be involved in RNA localization. Regardless of the model, once mRNPs are localized within the posterior pole, translation of the complexed RNAs would result in the production of proteins essential for the assembly of germ-plasm-specific components, such as Staufen protein/OSK mRNA and as yet unidentified mRNAs (Mohr, 2001).

Given the role of the Mago-Tsunagi complex in the localization of OSK mRNA and Staufen protein, it is possible to envision a similar function for the complex during early oogenesis (stages 1-5). That is, during early oogenesis a Mago-Tsunagi complex is likely to be required for localizing mRNAs that encode components necessary to interpret the posterior follicle cell-to-oocyte signal (Mohr, 2001).

Aberrant AP axis formation during oogenesis in tsu mutant egg chambers is revealed by two phenotypes: (1) the migration of the oocyte nucleus from a posterior to an anterior position can be abnormal in tsu mutant egg chambers; (2) markers of microtubule organization such as Kinesin::ß-gal and Nod::ß-gal are found at ectopic sites within tsu mutant oocytes. These are phenotypes that are reminiscent of defects observed in mutants in which Gurken signaling is disrupted. When Gurken signaling is blocked, disassembly of the MTOC at the posterior pole is inhibited and the oocyte nucleus fails to migrate to the anterior pole. Anomalies in nuclear migration and in the distribution of markers used to assess the integrity of the microtubule network are also detected in mago mutant egg chambers. Disassembly of the MTOC has not been examined in tsu mutant egg chambers but is aberrant in mago mutant oocytes. The fact that Mago and Tsunagi proteins cooperate to establish the AP axis of the oocyte suggests that in oocytes from tsu mutant mothers the MTOC at the posterior pole may not disassemble (Mohr, 2001).

Mutations that disrupt Gurken signaling also alter the fates of follicle cells, causing posterior follicle cells to develop as anterior follicle cells. In egg chambers derived from tsu mutant females the accumulation and function of GRK mRNA and protein during stages 1-6 (the time when posterior follicle cell fates are specified) are apparently normal. This is also evident from the fact that follicle cell fates are properly specified in tsu mutant egg chambers, as determined by the formation of the aeropyle (a structure produced by the posterior follicle cells) and by monitoring egg chambers with follicle cell markers that reveal their fates. Importantly, germ-line and follicle-cell clonal analysis indicates that tsu⁺ function is necessary within the germ line but not in follicle cells. Therefore, it appears that the patterning abnormalities detected when egg chambers lack wild-type tsu function arise from a requirement for Tsunagi protein in mediating the response of the oocyte to the posterior follicle cell-to-oocyte signal (Mohr, 2001).

In situ hybridization and immunolocalization reveal that GRK mRNA and protein are altered during stages 9-10 in oocytes from tsu mutant females. The amount of detectable GRK mRNA and protein is often reduced relative to wild type or undetectable above background. In contrast, the amount of GRK mRNA and protein is indistinguishable from wild type in mago mutant egg chambers and they are both properly localized. The differences in GRK mRNA and protein in mago mutant and tsu mutant egg chambers during stages 9-10 suggest that the DV pattern abnormalities in tsu mutant egg chambers are not caused solely by the altered migration of the oocyte nucleus, and although the two proteins cooperate in specific developmental processes, Mago and Tsunagi proteins are likely to function independently in other aspects of oocyte development (Mohr, 2001).

DV patterning of the egg chamber occurs during midoogenesis from restriction of Gurken signaling to the future dorsal side of the egg chamber. The asymmetric localization of Gurken signaling is achieved by (1) migration of the oocyte nucleus to an anterior cortical position; (2) transport of GRK mRNA from the oocyte nucleus to the dorsal-anterior corner of the oocyte; (3) anchoring of GRK mRNA within the dorsal-anterior corner of the oocyte, and (4) translation of the spatially restricted GRK mRNA. Squid protein, a heterogeneous nuclear RNA-binding protein (hnRNP), has been implicated in the export of GRK mRNA from the nucleus and in its delivery to an anchor within the cytoplasm (Mohr, 2001).

The Tsunagi homolog Y14 was originally identified through its interaction in a yeast two-hybrid screen with Ran-binding protein 5 (RanBP5). RanBP5 is related to the nuclear transporter receptor proteins importin-ß and transportin, proteins that are part of the nuclear import/export machinery. Like Squid protein, RanBP5 has been shown to interact physically with transportin. Based on the association of Squid protein and Tsunagi homologs with cellular components required in nuclear import and export, biochemical evidence indicating that the two proteins function in RNA export and the genetic evidence provided here, it is reasonable to propose that Tsunagi protein and Squid protein may interact with the nucleocytoplasmic transport machinery to regulate export and/or anchoring of GRK mRNA within the dorsal-anterior corner of the oocyte (Mohr, 2001).

Several roles for Tsunagi protein in the export and/or anchoring of GRK mRNA are consistent with the data presented and the known biochemical roles of Tsunagi homologs. (1)Tsunagi protein may be necessary to maintain the stability of GRK mRNA during midoogenesis. (2) Splicing-dependent export of GRK mRNA from the oocyte nucleus may require Tsunagi⁺ protein. The RNA may not be detectable within the nucleus owing to rapid degradation of unspliced pre-mRNAs, as has been shown to occur in yeast, or it may be exported from the nucleus by a generalized export mechanism. (3)The protein may be required for the export and/or localization of GRK mRNA within the dorsal-anterior cortex. Further experimentation will be necessary to determine the molecular function of Tsunagi protein in the stability, export, and/or anchoring of GRK mRNA during midoogenesis (Mohr, 2001).

The molecular and genetic analysis of tsunagi has revealed that the encoded protein functions in at least three distinct processes during Drosophila oogenesis. In early oogenesis (stages 1-5), Tsunagi protein forms a complex with Mago protein that is critical for interpreting the posterior follicle cell-to-oocyte signal. During stages 8 and 9 of oogenesis, Tsunagi protein cooperates with Mago protein to localize components necessary for anchoring OSK mRNA within the posterior pole. Although other interpretations are possible, a simple model suggests that both during stages 1-5 and 8-9 the Mago-Tsunagi complex localizes RNAs encoding proteins that are essential for mediating axis formation and assembly of the germ plasm. At stages 9-10, Tsunagi protein has a function independent of Mago protein that is crucial for the export and/or localization of GRK mRNA. Tsunagi interacts with a component of the cellular localization machinery, suggesting that homologs of Tsunagi may also be involved in RNA localization (Mohr, 2001).

Drosophila Y14 shuttles to the posterior of the oocyte and is required for oskar mRNA transport

mRNA localization is a powerful and widely employed mechanism for generating cell asymmetry. In Drosophila, localization of mRNAs in the oocyte determines the axes of the future embryo. Oskar mRNA localization at the posterior pole is essential and sufficient for the specification of the germline and the abdomen. Its posterior transport along the microtubules is mediated by Kinesin I and several proteins, such as Mago-nashi, which, together with ^Oskar mRNA, form a posterior localization complex. It was recently shown that human Y14, a nuclear protein that associates with mRNAs upon splicing and shuttles to the cytoplasm, interacts with MAGOH, the human homolog of Mago-nashi. Drosophila Y14 interacts with Mago-nashi in vivo. Immunohistochemistry reveals that Y14 is predominantly nuclear and colocalizes with Oskar mRNA at the posterior pole. In y14 mutant oocytes, Oskar mRNA localization to the posterior pole is specifically affected, while the cytoskeleton appears to be intact. These findings indicate that Y14 is part of the Oskar mRNA localization complex and that the nuclear shuttling protein Y14 has a specific and direct role in Oskar mRNA cytoplasmic localization (Hachet, 2001).

Y14 and Mago-nashi are highly conserved proteins, from unicellular eukaryotes to vertebrates. MAGOH and Mago-nashi share 89% identity, and human and Drosophila Y14 share 59% identity. However, the Y14 homologs differ noticeably in one respect: Drosophila Y14 lacks most of the SR domain present in the human form. To test whether the Y14:MAGOH interaction is conserved in Drosophila, the Drosophila y14 gene was cloned and an interaction test was designed, making use of the LexA two-hybrid system. An interaction between Mago-nashi and Drosophila Y14 was detected in both assay orientations, using two independent Mago-nashi baits that differ with regard to inclusion of a nuclear localization signal (NLS) (Hachet, 2001).

To address the physiological relevance of the Y14:Mago-nashi interaction, an antibody was raised against Y14 and coimmunoprecipitations were performed from ovarian extracts of myc-mago transgenic flies, which produce a Myc-mago protein in a wild-type background. Y14 can be coimmunoprecipitated with the Myc-mago protein when using the anti-Myc antibody. Mago-nashi coimmunoprecipitates with Y14 when wild-type ovarian extracts are treated with anti-Y14 antibody, showing that the in vivo interaction is detected in both orientations. More than 50% of Mago-nashi coimmunoprecipitates with Y14. This indicates that the Y14:Mago-nashi interaction is remarkably robust and that a large proportion of Y14 is associated with Mago-nashi in the ovary (Hachet, 2001).

To investigate the subcellular localization of Y14 in Drosophila egg chambers, in situ immunostaining was performed using affinity-purified anti-Y14 antibody. The in situ localization study highlights the predominantly nuclear localization of Drosophila Y14, as is the case for the human protein. This distribution correlates with that of Mago-nashi and is consistent with the detected protein interaction. Interestingly, Y14 also shows a cytoplasmic distribution. During the early stages of oogenesis, Y14 is enriched in the posterior half of the oocyte, like Mago-nashi. After stage 7, Y14 localizes to the posterior of the oocyte. Thus, Y14 is asymmetrically distributed in the oocyte, where it colocalizes with Mago-nashi at the posterior pole. This suggests that Y14 might be part of the oskar mRNA localization complex (Hachet, 2001).

To test whether Y14 shuttles with the Oskar mRNA posterior transport machinery, the localization of Y14 was examined in a genetic context in which Oskar mRNA and its partner, the double-stranded RNA binding protein, Staufen, were mislocalized. To do so, a par-1 mutant combination (9A/w3) was employed in which Oskar mRNA is targeted to an ectopic site, due to misorientation of the microtubule network. In this genetic background, as in the wild-type, Y14 colocalizes with Staufen, indicating that Y14 travels with the Oskar mRNA localization complex. This supports the idea that Y14 may be part of this complex (Hachet, 2001).

To analyze the function of Y14 in vivo, a genetic analysis of the y14 gene was performed. Through database searches, a transposable element (EP element) inserted 70 base pairs upstream of the y14 start codon was identified in the EP(2)0567 line. This line is homozygous lethal, and lethality is observed when the EP element is placed over three independent chromosomal deletions covering the y14 locus. Hence, the lethality is closely associated with the EP element insertion. In addition, the lethality can be rescued by mobilizing the transposable element. This indicates that the EP(2)0567 insertion affects the expression of an essential gene. Transposable element insertions are known to interfere with the expression of downstream coding sequences. Since this mutation is lethal, in order to test whether y14 expression is affected by the EP insertion, homozygous clones were generated in mosaic tissue, making use of the FLP/FRT system. Homozygous mutant clones were marked by the absence of a GFP signal, whereas heterozygous and wild-type cells were marked by a GFP signal of proportional intensity. This clonal analysis revealed that the level of Y14 expression is reduced in heterozygous compared to wild-type cells. Importantly, no Y14 protein was detected in EP(2)0567 homozygous cells, either in somatic or germline clones. This shows that EP(2)0567 strongly reduces or even completely abolishes the expression of y14 and thus constitutes a strong y14 allele. This is confirmed by the fact that the excision of the EP element restores Y14 expression (Hachet, 2001).

Because Y14 is likely to belong to the Oskar mRNA localization complex, whether Y14 has a role in Oskar mRNA localization was investigated. The effect of the y14^EP(2)0567 allele on the distribution of three localized mRNAs, Oskar, Gurken, and Bicoid, was investigated by in situ hybridization. For this purpose, homozygous y14^EP(2)0567 germline clones were generated by using the FLP/FRT ovo^D system, which allows only homozygous mutant germline clones to proceed through oogenesis. Interestingly, the analysis of Oskar mRNA distribution revealed that the early transport of Oskar mRNA is not affected by the y14^EP(2)0567 mutation. From stages 3 to 5, Oskar mRNA accumulates in the oocyte, showing that its transport from the nurse cells to the oocyte does not depend on Y14. At stage 7, when the microtubule network is reoriented, while Oskar mRNA is transported toward the posterior in the wild-type, in y14^EP(2)0567 egg chambers, Oskar mRNA fails to localize at the posterior. This defect in Oskar mRNA posterior localization is persistent, since Oskar mRNA never accumulates at the posterior pole and eventually diffuses throughout the oocyte during the ooplasmic streaming that occurs at stage 10b. In contrast, both Gurken and Bicoid mRNAs reach their final destination in y14 mutant egg chambers, even if the amount of localized mRNA seems reduced. This shows that their localization competence per se is not altered in the absence of Y14. As Staufen colocalizes with Oskar mRNA at the oocyte posterior, the effect of y14^EP(2)0567 on Staufen protein distribution was examined. As expected, Staufen accumulates properly in the oocyte during the early stages, but is not observed at the posterior pole after stage 9, in contrast to the wild-type. Instead, Staufen is detected throughout the oocyte and at the anterior pole, confirming the Oskar mRNA mislocalization phenotype. Staufen localization is rescued by excision of the P element. In addition, no Oskar protein was detected at the posterior of y14^EP(2)0567 egg chambers, consistent with the defect in Oskar mRNA localization, because Oskar mRNA translation is only activated at the posterior pole. The in situ localization study reveals that Y14 is specifically required for Oskar mRNA posterior localization, suggesting that Y14, like Mago-nashi, may have a direct function in the posterior transport of Oskar mRNA (Hachet, 2001).

During later stages of oogenesis, the nurse cells degenerate, expelling their entire cytoplasm into the oocyte in a process called 'dumping'. The y14 mutation leads to a dumpless phenotype; laid eggs are smaller than wild-type and are unfertilized. This prevented an analysis of the effect of the mutation on embryonic patterning (Hachet, 2001).

Since human Y14 has been described as imprinting mRNAs upon splicing, it is tempting to imagine that recruitment of the Y14:Mago-nashi complex upon splicing of Oskar mRNA constitutes a critical step in the assembly of the posterior transport machinery. If this were the case, this mechanism would constitute a new function for the splicing event, the deposit of a landmark addressing mRNAs to their cytoplasmic destinations (Hachet, 2001).

A novel mode of RBD-protein recognition in the Y14-Mago complex

Y14 and Mago are conserved eukaryotic proteins that associate with spliced mRNAs in the nucleus and remain associated at exon junctions during and after nuclear export. In the cytoplasm, Y14 is involved in mRNA quality control via the nonsense-mediated mRNA decay (NMD) pathway and, together with Mago, is involved in localization of osk mRNA. The crystal structure of the complex between Drosophila Y14 and Mago was determined at a resolution of 2.5 Å. The structure reveals an atypical mode of protein-protein recognition mediated by an RNA-binding domain (RBD). Instead of binding RNA, the RBD of Y14 engages its RNP1 and RNP2 motifs to bind Mago. Using structure-guided mutagenesis, it has been shown that Mago is also a component of the NMD pathway, and that its association with Y14 is essential for function. Heterodimerization creates a single structural platform that interacts with the NMD machinery via phylogenetically conserved residues (Fribourg, 2003).

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).

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

Pre-mRNA splicing is essential for generating mature mRNA and is also important for subsequent mRNA export and quality control. The splicing history is imprinted on spliced mRNA through the deposition of a splicing-dependent multiprotein complex, the exon junction complex (EJC), at approximately 20 nucleotides upstream of exon-exon junctions. The EJC is a dynamic structure containing proteins functioning in the nuclear export and nonsense-mediated decay of spliced mRNAs. Mago nashi (Mago) and Y14 are core components of the EJC, and they form a stable heterodimer that strongly associates with spliced mRNA. This study reports a 1.85 Å-resolution structure of the Drosophila Mago-Y14 complex. Surprisingly, the structure shows that the canonical RNA-binding surface of the Y14 RNA recognition motif (RRM) is involved in extensive protein-protein interactions with Mago. This unexpected finding provides important insights for understanding the molecular mechanisms of EJC assembly and RRM-mediated protein-protein interactions (Shi, 2003; full text of article).

The specification of both the germ line and abdomen in Drosophila depends on the localization of oskar messenger RNA to the posterior of the oocyte. This localization requires several trans-acting factors, including Barentsz and the Mago-Y14 heterodimer, which assemble with oskar mRNA into ribonucleoprotein particles (RNPs) and localize with it at the posterior pole. Although Barentsz localization in the germ line depends on Mago-Y14, no direct interaction between these proteins has been detected. This study demonstrates that the translation initiation factor eIF4AIII interacts with Barentsz and is a component of the oskar messenger RNP localization complex. Moreover, eIF4AIII interacts with Mago-Y14 and thus provides a molecular link between Barentsz and the heterodimer. The mammalian Mago (also known as Magoh)-Y14 heterodimer is a component of the exon junction complex. The exon junction complex is deposited on spliced mRNAs and functions in nonsense-mediated mRNA decay (NMD), a surveillance mechanism that degrades mRNAs with premature translation-termination codons. Both Barentsz and eIF4AIII are essential for NMD in human cells. Thus, eIF4AIII and Barentsz have been identified as components of a conserved protein complex that is essential for mRNA localization in flies and NMD in mammals (Palacios, 2004).

The exon junction complex (EJC) plays a major role in posttranscriptional regulation of mRNA in metazoa. The EJC is deposited onto mRNA during splicing and is transported to the cytoplasm where it influences translation, surveillance, and localization of the spliced mRNA. The complex is formed by the association of four proteins (eIF4AIII, Barentsz [Btz], Mago, and Y14), mRNA, and ATP. The 2.2 Å resolution structure of the EJC reveals how it stably locks onto mRNA. The DEAD-box protein eIF4AIII encloses an ATP molecule and provides the binding sites for six ribonucleotides. Btz wraps around eIF4AIII and stacks against the 5' nucleotide. An intertwined network of interactions anchors Mago-Y14 and Btz at the interface between the two domains of eIF4AIII, effectively stabilizing the ATP bound state. Comparison with the structure of the eIF4AIII-Btz subcomplex that has also been determined reveals that large conformational changes are required upon EJC assembly and disassembly (Bono, 2006).

DEVELOPMENTAL BIOLOGY

Embryonic

The two messenger RNAs are detected throughout the life cycle. During the first four hours of embryonic development, the 0.7 kb transcript appears to be more abundant than the 1.1 kb transcript. Both mRNAs are detected at similar abundance in larvae, adult males and females, and are in low abundance in late embryos. The expression of MAGO mRNAs in developmental stages beyond oogenesis and early embryogenesis is consistent with a function for the mago product in later developmental events as suggested by the zygotic lethality of several lethal mago alleles (Newmark, 1994).

Adult

MAGO mRNAs appear to be uniformly expressed throughout the nurse cell-oocyte complex during early oogenesis, and are abundant in nurse cells at stage 10: they appear to be uniformly distributed throughout the embryo by the time of egg deposition (Newmark, 1994).

Effects of mutation or deletion

The mago nashi (mago) locus is a newly identified strictly maternal effect, grandchildless-like gene in Drosophila. In homozygous mutant mago females reared at 17 degrees C, mago+ function is reduced, the inviable embryos lack abdominal segments and 84-98% of the embryos die. In contrast, at 25 degrees C, some mago alleles produce a novel gene product capable of inducing the formation of symmetrical double abdomen embryos. Reciprocal temperature-shift experiments indicate that the temperature-sensitive period is during oogenetic stages 7-14. Embryos collected from mago1 homozygous females contain no apparent functional posterior determinants in the posterior pole. In viable F1 progeny from mago mutant females, regardless of genotype and temperature, polar granules are reduced or absent and germ cells fail to form (the grandchildless-like phenotype). Thus, it is proposed that the mago+ product is a component of the posterior determinative system, required during oogenesis, both for germ cell determination and delineation of the longitudinal axis of the embryo (Boswell, 1991).

In addition to its previously described role in the localisation of Oskar mRNA, the mago nashi gene is required in the germ line for the transduction of the polarising signal from the posterior follicle cells. Using a new in vivo marker for microtubules, it is shown that mago nashi mutant oocytes develop a symmetric microtubule cytoskeleton that leads to the transient localisation of Bicoid mRNA to both poles. The oocyte nucleus often fails to migrate to the anterior, causing the second Gurken signal to be sent in the same direction as the first. This results in a novel phenotype in which the anterior of the egg is ventralized and the posterior dorsalized, demonstrating that the migration of the oocyte nucleus determines the relative orientation of the two principal axes of Drosophila. It is concluded that the mago nashi gene plays two essential roles in Drosophila axis formation: it is required downstream of the signal from the posterior follicle cells for the polarization of the oocyte microtubule cytoskeleton, and has a second, independent role in the localization of Oskar mRNA to the posterior of the oocyte (Micklem, 1997).

Nonsense mutations in mago nashi, as well as a deletion of the 5' coding sequences, result in zygotic lethality. The original mago nashi allele disrupts the localization of Oskar mRNA and Staufen protein to the posterior pole of the oocyte during oogenesis; anterior localization of Bicoid mRNA is unaffected by the mutation. Oscar mRNA remains detectable in the anterior of the oocyte through stage 10 oocytes. Expression of Oscar mRNA in the germarium and through the first seven stages of oogenesis appears normal. Staufen protein appears to be uniformly distributed throughout the oocyte in stages 8 to 10. Normally by stage 10 it is tightly localized to the posterior pole. These results demonstrate that mago nashi encodes an essential product necessary for the localization of germ plasm components to the posterior pole of the oocyte (Newmark, 1994).

Shortly after fertilization in Drosophila embryos, the G-protein alpha subunit, Gi alpha, undergoes a dramatic redistribution. Initially granules containing Gi alpha are present throughout the embryonic cortex but during nuclear cleavage they become concentrated at the posterior pole and are lost by the blastoderm stage. Mutations that eliminate anterior structures (bicoid, swallow, and exuperantia) do not prevent the posterior accumulation of Gi alpha. Likewise, embryos from mothers with dominant gain of function mutations in the Bicaudal D gene show normal polarization of Gi alpha granules. By contrast, a subset of mutations wthat eliminate posterior structures (cappuccino, spire, staufen, mago nashi, valois, and oskar,) prevent the posterior accumulation of Gi alpha. It is important to note that mutations in posterior genes lower in the putative hierarchy (vasa, tudor nanos, and pumilio) do not affect Gi alpha redistribution. From these results it is concluded that Gi alpha redistribution to the posterior pole depends on maternal factors involved in the localization of the posterior morphogen Nanos (Wolfgang, 1995).

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date revised: 15 April 2007  

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