mago nashi
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).
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/Tsunagi, 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 y14EP(2)0567 allele on the distribution of three localized mRNAs, Oskar, Gurken, and Bicoid, was investigated by in situ hybridization. For this purpose, homozygous y14EP(2)0567 germline clones were generated by using the FLP/FRT ovoD 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 y14EP(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 y14EP(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 y14EP(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 y14EP(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).
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).
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 Rfree 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, Asp67Mago and Glu69Mago interact with Lys25PYM and Arg27PYM. In human Mago, a double mutation of the corresponding Asp66MagoHs and Glu68MagoHs 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).
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).
oskar messenger RNA localization at the posterior pole of the Drosophila oocyte is essential for germline and abdomen formation in the future embryo. The nuclear shuttling proteins Y14/Tsunagi and Mago nashi are required for oskar mRNA localization, and they co-localize with oskar mRNA at the posterior pole of the oocyte. Their human homologues, Y14/RBM8 and Magoh, are core components of the exon-exon junction complex (EJC). The EJC is deposited on mRNAs in a splicing-dependent manner, 20-24 nucleotides upstream of exon-exon junctions, independently of the RNA sequence. This indicates a possible role of splicing in oskar mRNA localization, challenging the established notion that the oskar 3' untranslated region (3'UTR) is sufficient for this process. This study shows that splicing at the first exon-exon junction of oskar RNA is essential for oskar mRNA localization at the posterior pole. The issue of sufficiency of the oskar 3'UTR for posterior localization was revisited and it was shown that the localization of unrelated transcripts bearing the oskar 3'UTR is mediated by endogenous oskar mRNA. The results reveal an important new function for splicing: regulation of messenger ribonucleoprotein complex assembly and organization for mRNA cytoplasmic localization (Hachet, 2004).
To address the requirement of splicing for oskar mRNA localization, the effect of deleting oskar introns on mRNA localization was tested by a transgenic approach. oskar mRNA localization was evaluated in the oskA87/Df(3R)pXT103 background, in which no endogenous oskar RNA is produced. oskA87/Df(3R)pXT103 ovaries undergo an early arrest of oogenesis that can be rescued by transgenic oskar mRNA, providing a phenotypic confirmation of efficient transgene expression. Because the only source of oskar mRNA in these experiments is transgenic, each genotype is referred to by the name of the oskar transgene (Hachet, 2004).
The localization was tested of oskar mRNA produced from the intronless transgene oskδi(1,2,3), in which the three oskar introns i1, i2 and i3 were deleted. During early oogenesis, oskδi(1,2,3) mRNA is correctly transported from the nurse cells to the oocyte indicating that oskar introns are dispensable both for nuclear export and the early phase of oskar mRNA transport. A defect in oskδi(1,2,3) mRNA localization becomes evident during mid-oogenesis. At stage 8, oskδi(1,2,3) mRNA distribution seems diffuse compared with oskWT mRNA. At stage 9, whereas oskWT mRNA accumulates at the posterior with a transient accumulation at the anterior corners, oskδi(1,2,3) mRNA is distributed throughout the entire ooplasm. During late oogenesis, only a small amount of oskδi(1,2,3) mRNA is detected in an extended posterior crescent, most probably reflecting local anchoring of mRNA randomly distributed in the posterior area during mid-oogenesis. Consistent with the oskδi(1,2,3) mRNA localization defect was the finding that Staufen protein, a marker for oskar mRNA, is similarly mislocalized. Although oskar mRNA levels are similar in oskWT and oskδi(1,2,3) ovaries, oskδi(1,2,3) mRNA is poorly translated, presumably reflecting the requirement of localization for oskar mRNA translation. Finally, more than two-thirds of the embryos from eggs laid by oskδi(1,2,3) females fail to hatch and show a severe posterior group phenotype (Hachet, 2004).
oskar introns were systematically deleted to assess their relative contribution in oskar mRNA localization. The results show that, whereas the mRNAs produced from all i1-containing transgenes are localized (oskWT, oskδi(2,3), oskδi2 and oskδi3), those produced from all i1-deleted transgenes are mislocalized, although they accumulate correctly in the oocyte (oskδi(1,2,3), oskδi(1,3), oskδi(1,2) and oskδi1). Although the absence of i2 and i3 does not affect oskδi(2,3) mRNA localization, oskδi1 mRNA fails to localize at the posterior pole of stage 9 and 10 oocytes, although i2 and i3 are correctly spliced. Confirming the previously reported oskar mRNA-dependent localization of Y14, Y14 fails to localize in oskδi1 oocytes. The fact that oskδi(2,3) mRNA supports Y14 localization to the posterior shows that splicing of i1 is sufficient for the association of Y14 with oskar mRNA. Thus, these results reveal an unexpected and distinct role of i1, compared with i2 and i3, in oskar mRNA localization. They indicate either that i1 contains sequence-specific information or that splicing at the i1 position is essential for oskar mRNA localization (Hachet, 2004).
To discriminate between these two possibilities, the localization was tested of mRNAs produced by a transgene in which the i1 sequence was replaced by i3, called osk(i3 in i1). osk(i3 in i1) mRNA localizes at the posterior, showing that, although i3 is unable to promote oskar mRNA localization when located at its normal position, i3 can functionally substitute for i1 when placed in the i1 context, between exons I and II. Consistent with this was the observation that the EJC component Y14 is recruited by osk(i3 in i1) mRNA, as revealed by the localization of Y14 at the posterior pole, indicating that Y14 recruitment is independent of intron sequence. This demonstrates the importance of splicing at the first exon-exon junction of oskar mRNA, rather than a specific requirement for i1, for oskar mRNA localization. These results show that oskar RNA splicing and localization are mechanistically coupled (Hachet, 2004).
The demonstration that splicing is required for oskar mRNA localization seemingly contradicts previous work reporting that the oskar 3'UTR is sufficient for mRNA targeting to the posterior pole of the oocyte. This conclusion was based on several studies of lacZ-osk3'UTR hybrid RNAs, in which the intronless lacZ gene was fused to the oskar 3'UTR and the chimaeric mRNAs were observed to localize at the oocyte posterior. This apparent contradiction prompted the role of the 3'UTR in oskar mRNA localization to be revisited. The possibility was considered that the localization of lacZ-osk3'UTR mRNA might be influenced by endogenous oskar mRNA, which was present in all previous studies. A direct analysis of lacZ-osk3'UTR mRNA localization in oskar RNA-null oocytes is prevented by the early oogenesis arrest of the oskA87/Df(3R)pXT103 mutant. Therefore the localization of lacZ-osk3'UTR mRNAs was examined in oskWT and oskδi(1,2,3) oocytes, in which transgenic oskar mRNA supports oocyte development beyond the early stages. It was found that although lacZ-osk3'UTR mRNA localizes correctly in oskWT oocytes, it fails to accumulate at the posterior pole of oskδi(1,2,3) oocytes, as revealed by lacZ in situ hybridization. In addition, when placed in the Oskar protein-null background osk84/Df(3R)pXT103, in which the osk84 nonsense mRNA localizes correctly until stage 10 of oogenesis, lacZ-osk3'UTR mRNA also localizes at the posterior pole. Taken together, these results demonstrate that an endogenous source of oskar mRNA is required for lacZ-osk3'UTR localization at the posterior pole, and that this effect is independent of Oskar protein. Localization of the lacZ-osk3'UTR hybrid RNA to the posterior pole is therefore most probably due to its hitchhiking on endogenous oskar mRNA localization complexes, whose assembly involves splicing. These results indicate that the oskar 3'UTR promotes association of the RNA into higher-order oskar messenger ribonucleoprotein (mRNP) complexes. This idea is consistent with estimates indicating that oskar mRNA particles contain about 100 oskar mRNA molecules. The assembly of such multi-mRNP particles might be mediated by protein-protein interactions involving factors bound to the 3'UTR or by direct RNA-RNA interaction as occurs with bicoid mRNA (Hachet, 2004).
Although the oskar 3'UTR and associated factors are clearly important for oskar mRNA localization, because oskar transcripts lacking the 3'UTR fail to localize, the data show that oskar mRNA localization requires additional factors recruited to the mRNA upon splicing. Thus, information imparted to oskar RNA in the nucleus during pre-mRNA processing is crucial for the localization of oskar mRNA at the posterior pole of the oocyte cytoplasm. The fact that both splicing and the EJC components Y14 and Mago nashi are essential for oskar mRNA localization indicates that oskar RNA splicing and cytoplasmic localization are mechanistically coupled by the splicing-dependent deposition of the EJC. Unexpectedly, of the three oskar intron positions, only the first is strictly required and functional for oskar mRNA localization, although an EJC is presumably assembled at each oskar mRNA exon-exon junction. This indicates not only that an EJC landmark is required but also that its position is essential for oskar mRNA localization at the oocyte posterior. The importance of the splicing position, and thus of the EJC on oskar mRNA, suggests a structural role of the Y14-Mago nashi heterodimer and Barentsz, in assembly of the oskar mRNA localization complex. The first EJC landmark on oskar mRNA might have a pivotal function in mediating interactions between factors bound to different regions of oskar mRNA, including the 5' cap, the 3'UTR and potentially the coding region. It is proposed that the position of the first oskar EJC landmark is crucial in specifying the architecture of the oskar mRNA localization complex. This structural model could explain why the EJC is not always involved in cytoplasmic mRNA localization and why the transport of gurken and bicoid mRNAs, both of which are produced from intron-containing genes and are thus presumably imprinted with the EJC, seems to be independent of the EJC. The model also suggests that alternatively spliced mRNAs might be directed to different cytoplasmic locations, depending on the formation of alternative mRNP complex architectures (Hachet, 2004).
In humans, EJC imprinting allows the recognition of premature termination codons, triggering mRNA degradation by activation of the nonsense-mediated decay (NMD) pathway. In Drosophila, however, although NMD factors and EJC components are conserved, the recognition of premature termination codons depends neither on the EJC nor on intron position. The involvement of Y14, Magoh, eIF4AIII and Barentsz in NMD in humans and in oskar mRNA localization in Drosophila is striking and suggests the maintenance of an evolutionarily conserved complex with divergent functions. However, this does not exclude a possible involvement of splicing and the EJC in the cytoplasmic localization of some mRNAs in vertebrates. In particular, the localization of Barentsz in hippocampal neurons suggests that the use of these factors in cytoplasmic mRNA localization has been conserved in vertebrates. It will be of particular interest to determine whether the transport of other localized mRNAs is dependent on the EJC and to evaluate the relevance of the conservation of the EJC regarding mRNA cytoplasmic localization (Hachet, 2004).
oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, it was showm that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. Each component of the oskar mRNA complex is shown to play a distinct role in particle formation and transport (Zimyanin, 2008).
The mechanism of osk mRNA localization has been controversial, and a number of competing models have been proposed to explain its targeting to the posterior. This study observed directly how the RNA travels to the posterior by tracking the movements of osk mRNA particles at high temporal resolution in living oocytes. Surprisingly, the results are incompatible with the existing models, leading to proposal of a new mechanism for the localization of the mRNA (Zimyanin, 2008).
(1) It is clear that the mRNA is not transported in a highly directed fashion toward the posterior since the particles move in all directions with only a slight posterior bias. (2) The rapid transport of the osk mRNPs argues against a role for passive diffusion. (3) The results are inconsistent with the two-step model for osk mRNA localization, in which kinesin first transports the RNA away from the anterior and lateral cortex to the oocyte center before it is translocated to the posterior in a second step. osk mRNP particles show a similar behavior in all regions of the oocyte at stage 9, with a consistent small excess of particles moving posteriorly, and this is incompatible with the idea that particles are first transported to the center. Moreover, slow kinesin mutants have an identical effect on the speeds of particle movements in all regions of the oocyte, strongly arguing that kinesin transports the mRNA in a one-step pathway all of the way to the posterior pole (Zimyanin, 2008).
Instead, the data suggest that osk mRNA is localized by a biased random walk, in which each particle undergoes a large number of active movements in many different directions, with a small excess of movements toward the posterior. After hundreds of movements, the 14% excess of posterior movements results in a large net posterior displacement that delivers the mRNA to its destination. Given that 13% of particles are moving at any one time, the average osk mRNP will undergo a net posterior displacement during the 6-10 hr of stage 9 of 112-187 microm (6-10 × 3600 s × 0.13 × 0.04 microm/s). Since this is more than 1.5 times the length of the oocyte (80 microm), this is more than sufficient to produce a robust posterior localization of osk mRNA by the end of stage 9 (Zimyanin, 2008).
This model is supported by the observation that the direction of the bias correlates perfectly with the site of osk mRNA accumulation: wild-type oocytes show a posterior bias and posterior localization of the mRNA, whereas the bias is reversed in mago, TmII, and btz mutants, and osk mRNA accumulates at the anterior. The effectiveness of the biased random walk in localizing the RNA is even more clearly demonstrated by stau mutants: most of the mRNA is trapped at the anterior of stau mutant oocytes, and the mRNA that is released into the oocyte cytoplasm moves four times less frequently than in wild-type. Nevertheless, the small number of movements that occur show a normal posterior bias, which leads to a transient posterior enrichment of the mRNA that is lost at later stages because the RNA is not anchored (Zimyanin, 2008).
Similar biased bidirectional transport has been described for lipid droplets in the Drosophila embryo and for many other particles and organelles in other systems. In most cases, the bias depends on the competing activities of motors that move in opposite directions. By contrast, the results indicate that the vast majority of osk mRNA movements are directed toward microtubule plus ends and are mediated by kinesin. (1) When the microtubules are aligned around the cortex by premature cytoplasmic streaming, over 80% of fast-moving particles move in the same direction as the cytoplasmic flows, i.e., toward the plus ends. (2) More than 80% of movements are abolished by null mutations in the Khc. (3) Point mutations in kinesin reduce the speed of particle movements in all directions. Fourth, particles have never been observed that show a clear 180° reversal in the direction of their movement out of more than 3000 particle tracks analyzed, indicating that particles rarely switch between plus and minus end-directed motion (Zimyanin, 2008).
The traditional view of the oocyte microtubule cytoskeleton is that it is polarized along the anterior-posterior axis with minus ends at the anterior and plus ends at the posterior. This view is based on the localization of fusion proteins containing the motor domains of Nod and kinesin to the anterior and posterior of the oocyte, respectively, and the assumption that these act as minus and plus end markers. The microtubule organization appears much more complex, however, when visualized directly: the microtubules appear to be nucleated from both the anterior and lateral cortex and extend in all directions to form an anterior-posterior gradient. The data are consistent with this latter view because osk mRNA particles move in all directions in every region of the oocyte. More importantly, because almost all osk mRNA movements are plus end directed, each RNA track provides a snapshot of the polarity of a microtubule segment. The observation that 57% of tracks have a net posterior vector therefore indicates that the microtubules have only a weak orientation bias toward the posterior. Even if all 10%-20% of kinesin-independent osk mRNA movements are minus end directed, this would still give a posterior bias in microtubule polarity of only 62%. Thus, the data suggest a revised view of the organization of the cytoskeleton, in which the microtubules extend in all directions from the anterior and lateral cortex, with about a 20% excess of microtubules with their plus ends pointing posteriorly. One appealing aspect of this model is that it can reconcile the two opposing views of the microtubule organization. Kinβgal is an unregulated motor that constitutively moves toward the plus ends of microtubules, and it is proposed that it accumulates at the posterior by following a biased random walk similar to osk mRNA. According to this view, it is not a marker for microtubule plus ends but for regions where plus ends are most enriched (Zimyanin, 2008).
Mutants in different components of the osk mRNA localization complex produce very similar phenotypes when analyzed by in situ hybridizations to fixed samples. However, they have different effects on the dynamics of osk mRNA particles. First, hrp48 mutants abolish the formation of visible osk mRNA particles, indicating a requirement for this HnRNPA/B-like protein in the assembly of functional transport particles (Zimyanin, 2008).
Mutants in the EJC components, Mago nashi and Btz, do not affect osk mRNP particle formation but reduce the frequency of particle movement and reverse the bias, so that the particles accumulate at the oocyte anterior. This behavior is what one would expect if the movement is primarily mediated by a minus end-directed motor. Furthermore, the anterior accumulation of osk mRNA in these mutants resembles that of many other mRNAs that are transported into the oocyte by the dynein/Bic-D/Egl pathway, and which are thought to localize to the anterior by default, because this pathway remains active in the oocyte. Thus, the EJC may be required to turn off the dynein/Bic-D/Egl pathway when osk mRNA enters the oocyte so that it can then associate with the kinesin pathway (Zimyanin, 2008).
TmII mutants have the same effects on osk mRNP dynamics as EJC mutants, suggesting that Tropomyosin is required for the same step in localization. This raises the possibility that Tropomyosin plays a role in either the recruitment of the EJC to osk mRNA or the subsequent activity of the EJC in switching from the anterior to the posterior localization pathway (Zimyanin, 2008).
Stau seems to function downstream of the EJC since osk mRNA is either trapped at the anterior or moves with a normal posterior bias toward the posterior pole. The results suggest that Stau regulates several aspects of osk mRNA behavior once it enters the oocyte. First, it seems to be required for the efficient release of the mRNA from the anterior. This may reflect a role of Staufen in the coupling of the mRNA to the kinesin-dependent posterior transport pathway. osk mRNA particles that escape the anterior move with a normal bias but a reduced frequency, suggesting that Stau is also required for full kinesin activity. Finally, Stau is essential for the activation of osk mRNA translation once the mRNA has reached the posterior pole. Thus, the in vivo analysis of osk mRNA dynamics reveals that different components of the osk mRNP complex are required for at least three distinct steps in the localization pathway, namely particle formation, uncoupling from the dynein/BicD pathway, and release from the anterior and coupling to the kinesin pathway, and this may begin to explain why so many trans-acting factors are required for the localization of this mRNA (Zimyanin, 2008).
The dynamics of osk mRNA particles have several features in common with the behavior of MS2-labeled mRNAs in mammalian cells. Fusco (2003) found that RNA particles undergo stochastic movements in COS cells, in which they switch between fast microtubule-dependent movements, diffusion, and stationary phases. Furthermore, an RNA containing the β-actin localization signal showed a 5-fold higher frequency of fast movements than a random RNA. This is very similar to the behavior of osk mRNA, which undergoes fast, direct movements 4-5 times more frequently in wild-type oocytes than in EJC, TmII, and stau mutants. MS2-GFP has also been used to image CamKIIα mRNA in the dendrites of cultured neurons and labels particles that show similar bidirectional movements to osk mRNA. Since dendrites contain microtubules of mixed orientations and Stau, Barentsz, and kinesin have been implicated in dendritic mRNA localization, it will be interesting to determine whether CamKIIα mRNA localizes by a biased random walk similar to osk mRNA (Zimyanin, 2008).
Wingless (Wg)/Wnt signaling is conserved in all metazoan animals and plays critical roles in development. The Wg/Wnt morphogen reception is essential for signal activation, whose activity is mediated through the receptor complex and a scaffold protein Dishevelled (Dsh). This study reports that the exon junction complex (EJC) activity is indispensable for Wg signaling by maintaining an appropriate level of Dsh protein for Wg ligand reception in Drosophila. Transcriptome analyses in Drosophila wing imaginal discs indicate that the EJC controls the splicing of the cell polarity gene discs large 1 (dlg1), whose coding protein directly interacts with Dsh. Genetic and biochemical experiments demonstrate that Dlg1 protein acts independently from its role in cell polarity to protect Dsh protein from lysosomal degradation. More importantly, human orthologous Dlg protein is sufficient to promote Dvl protein stabilization and Wnt signaling activity, thus revealing a conserved regulatory mechanism of Wg/Wnt signaling by Dlg and EJC (Liu, 2016).
The EJC is known to act in several aspects of posttranscriptional regulation, including mRNA localization, translation and degradation. After transcription, the pre-mRNA associated subunit eIF4AIII is loaded to nascent transcripts about 20-24 bases upstream of each exon junction, resulting in binding of Mago nashi (Mago)/Magoh and Tsunagi (Tsu)/Y14 proteins to form the pre-EJC core complex. The pre-EJC then recruits other proteins including Barentsz (Btz) to facilitate its diverse function). In vertebrates, the EJC is known to ensure translation efficiency as well as to activate nonsense-mediated mRNA decay (NMD). In Drosophila, however, the EJC does not contribute to NMD. It is instead required for the oskar mRNA localization to the posterior pole of the oocyte. Very recently, the pre-EJC has been shown to play an important role in alternative splicing of mRNA in Drosophila. Reduced EJC expression results in two forms of aberrant splicing. One is the exon skipping, which occurs in MAPK and transcripts that contain long introns or are located at heterochromatin (Ashton-Beaucage, 2010; Roignant, 2010). The other is the intron retention on piwi transcripts. Furthermore, transcriptome analyses in cultured cells indicates the role of EJC in alternative splicing is also conserved in vertebrates (Liu, 2016).
This study has utilized the developing Drosophila wing as an in vivo model system to investigate new mode of regulation of Wg signaling. The pre-EJC was found to positively regulate Wg signaling through its effect on facilitating Wg morphogen reception. Further studies reveal that the basolateral cell polarity gene discs large 1 (dlg1) is an in vivo target of the pre-EJC in Wg signaling. Dlg1 acts independently from its role on cell polarity to stabilize Dsh protein, thus allowing Wg protein internalization required for signaling activation. Furthermore, it was demonstrated that human Dlg2 exhibits a similar protective role on Dvl proteins to enhance Wnt signaling in cultured human cells. Taken together, this study unveils a conserved regulatory mechanism of the EJC and Dlg in Wg/Wnt signaling (Liu, 2016).
In summary, this study uncovers a specific role of the RNA binding protein complex EJC in the Drosophila wing morphogenesis. Genetic and biochemical analyses demonstrate that the pre-EJC is necessary for Wg morphogen reception to activate the signal transduction. The identification of the cell polarity determinant dlg1 as one of the pre-EJC targets provides mechanistic basis for the pre-EJC regulation of the Wg signaling. Dlg1 controls the stability of the scaffold protein Dsh, which is the hub of the Wg signaling cascade. Importantly, this mode of regulation of Dvl by Dlg is conserved from flies to vertebrates (Liu, 2016).
The EJC as well as other RNA binding protein complexes are thought to function in a pleiotropic manner. However, the current data together with several recent studies argue that RNA regulatory machineries can act specifically on developmental signaling for pattern formation and organogenesis. It has been increasingly recognized that the production, transport or the location of mRNA are subject to precise regulation in Wg/Wnt signaling. For example, apical localization of wg RNA is essential for signal activation in epithelial cells. The specific role of RNA machineries on cell signaling is not limited to Wg/Wnt signaling. It has been reported that RNA-binding protein Quaking specifically binds to the 3'UTR of transcription factor gli2a mRNA to modulate Hedgehog signaling in zebrafish muscle development. RNA binding protein RBM5/6 and 10 could differentially control alternative splicing of a negative Notch regulator gene NUMB, thus antagonistically regulating the Notch signaling activity for cancer cell proliferation. Therefore, generally believed pleotropic RNA regulatory machineries emerge as important regulatory means to specifically control cell signaling and related developmental processes (Liu, 2016).
The most studied function of the EJC in development is to localize oskar mRNA to the posterior pole of the oocyte for oocyte polarity establishment and germ cell formation in Drosophila. Further study suggests that the proper oskar RNA localization relies on its mRNA splicing. In light of the current study of the EJC activity on dlg1 mRNA as well as the roles of EJC on mapk and piwi splicing, it is suspected that EJC might regulate oskar mRNA splicing to mediate its mRNA localization. RNA-seq analyses identified several hundreds of candidate mRNAs whose expression may be directly or indirectly subjected to EJC regulation. Apart from defects in Wg and MAPK signaling, however, altered wing patterning associated with other developmental signaling systems was not seen in EJC defective flies, arguing that EJC may primarily regulate Wg and MAPK signaling in patterning the developing wing (Liu, 2016).
Wg/Wnt signaling plays a fundamental role in development and tissue homeostasis in both flies and vertebrates. Its activation and maintenance rely on appropriate activity of the ternary receptor complex including Fz family proteins. In Drosophila, polarized localization of Fz and Fz2 proteins is essential for activation of non-canonical and canonical Wg signaling, respectively. Dsh, which acts as a hub mediating both canonical and non-canonical Wg signaling, however, is found at both the apical cell boundary and in the basal side of the cytoplasm. Thus, the polarized activity of Dsh must require distinct regulatory mechanisms at different sub-membrane compartments. The results provide the in vivo evidence suggesting that the basolateral polarity determinant Dlg1 may play a dominant role to control the Dsh abundance/activity in canonical Wg signaling (Liu, 2016).
Altered Dvl production or activity has been linked with several forms of cancer. The stability of Dvl proteins can be controlled through regulated protein degradation both in vertebrates and in Drosophila as reported in this study. In HEK293T cells, Dapper1 induces whilst Myc-interacting zinc-finger protein 1 (MIZ1) antagonizes autophagic degradation of Dvl2 in lysosome. It is also reported that a tumor suppressor CYLD deubiquitinase inhibits the ubiquitination of Dvl. As Dlg1 prevents Dsh from degradation in Drosophila, it is important to investigate if Dlg1 participates in a posttranslational regulatory network of Dvl to integrate endocytosis and autophagy. Furthermore, upregulation of dvl2 and dlg2 expression has been found in various forms of cancer as shown in the COSMIC database. The study of the interaction between Dlg1 and Dsh may aid the development of novel approaches to prevent or treat relevant diseases. (Liu, 2016).
Dlg1 acts together with L(2)gl to form a basolateral complex in polarized epithelium. Dsh is known to interact with L(2)gl. On one hand, Dsh activity is required for correct localization of L(2)gl to establish apical-basal polarity in Xenopus ectoderm and Drosophila follicular epithelium. On the other hand, L(2)gl can regulate Dsh to maintain planar organization of the embryonic epidermis in Drosophila. Despite the complex interaction between L(2)gl and Dsh, not much is known about mutual regulation between Dlg1 and Dsh. A recent report suggests that Dsh binds to Dlg1 to activate Guk Holder-dependent spindle positioning in Drosophila. The current results unveil another side of the relationship in which Dlg1 controls the turnover of Dsh to ensure developmental signal propagation. Apart from its apical localization at the cell boundary, Dsh is also found in the basal side of the cytoplasm. It is likely that the interactions among Dsh, Dlg1 and L(2)gl may be dependent on their localization, and Dsh may serve as a bridge to connect cell signaling and polarity (Liu, 2016).
Developmental signaling and cell polarity intertwine to control a diverse array of cellular events. It is well known that Wg/Wnt signaling controls cell polarity in distinct manner. Non-canonical signaling acts through cytoskeletal regulators to establish planar cell polarity. Canonical signaling may also directly affect apical-basal cell polarity. On the other hand, disruption of epithelial cell polarity has a profound impact on protein endocytosis and recycling, both of which are essential regulatory steps for signal activation and maintenance. The current results add another layer of complexity by which polarity determinants could contribute to cell signaling independent of their conventional roles in polarity establishment and maintenance. Interestingly, this mode of regulation is also observed for other signaling processes. Loss of Dlg5 impairs Sonic hedgehog-induced Gli2 accumulation at the ciliary tip in mouse fibroblast cells that may not rely on cell polarity regulation. Similarly, L(2)gl regulates Notch signaling via endocytosis, independent of its role in cell polarity. It is believed that other cell polarity determinants may similarly participate in polarity-independent processes, however, the exact mechanism of how they cooperate to modulate developmental signaling awaits further investigation (Liu, 2016).
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).
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).
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).
A protein complex consisting of Mago Nashi and Tsunagi/Y14 is required to establish the major body axes and for the localization of primordial germ cell determinants during Drosophila oogenesis. The Mago Nashi:Tsunagi/Y14 heterodimer also serves as the core of the exon junction complex (EJC), a multiprotein complex assembled on spliced mRNAs. In previous studies reduced function alleles of mago nashi and tsunagi/Y14 were used to characterize the roles of the genes in oogenesis. This study investigated mago nashi and tsunagi/Y14 using null alleles and clonal analysis. Germline clones lacking mago nashi function divide but fail to differentiate. The mago nashi null germline stem cells produce clones over a period of at least 11 days, suggesting that mago nashi is not necessary for stem cell self-renewal. However, germline stem cells lacking tsunagi/Y14 function are indistinguishable from wild type. Additionally, in tsunagi/Y14 null germline cysts, centrosomes and oocyte-specific components fail to concentrate within a single cell and oocyte fate is not restricted to a single cell. Together, these results suggest not only that mago nashi is required for germline stem cell differentiation but that surprisingly mago nashi functions independently of tsunagi/Y14 in this process. In contrast, Tsunagi/Y14 is essential for restricting oocyte fate to a single cell and may function with mago nashi in this process (Parma, 2007).
This paper presents evidence that in addition to previously known roles in axis formation and oskar RNA anchoring/localization during late oogenesis, mago and tsu are also required in essential steps during early oogenesis. Mago is necessary for the entry of the GSC into the oogenic pathway. Unexpectedly, Mago functions independently from Tsu/Y14 to regulate this process. Tsu/Y14 is not required for the differentiation of GSCs. However, Tsu/Y14 is necessary to restrict oocyte fate to a single cell. Thus, these studies reveal a new role for Mago in GSC differentiation and a new role for Tsu/Y14 in oocyte fate specification (Parma, 2007).
Establishing where Mago functions within the network of pathways regulating GSC differentiation is fundamental to understanding Mago's mechanism of action. This network includes the Bmp, Nanos/Pumilio and Bam/Bgcn pathways (Parma, 2007).
Bmp, produced within the somatic cells of the GSC niche, is an extracellular signaling molecule that regulates self-renewal and asymmetric division of GSCs. A signal transduction pathway within GSCs, activated by Bmp, produces a complex containing two Smad family member proteins. The complex translocates to the nucleus, binds a transcriptional silencer within the bam promoter and prevents transcription of bam (Parma, 2007).
The nanos (nos) and pumilio (pum) genes function intrinsically to regulate GSC self-renewal and asymmetric division. Based on the observation that Pum and Nos form a heterodimer that binds and inhibits translation of hunchback mRNA in embryos, it has been proposed that in GSCs Pum:Nos suppresses the translation of mRNAs encoding proteins essential for cystoblast differentiation (Parma, 2007).
The bam (encoding a novel protein) and bgcn (encoding a DExH-box RNA-binding protein) genes play key roles in GSC differentiation. While Bmp signaling silences bam transcription in GSCs and pre-cystoblasts, bam transcription is upregulated within cystoblasts. Within cystoblasts, Bam is synthesized and forms a complex with Bgcn, resulting in differentiation of the cystoblast. It has been proposed that the Bam:Bgcn complex acts by antagonizing the Pum:Nos complex, thus relieving translational repression and inducing the synthesis of cystoblast differentiation factors (Parma, 2007).
Although GSCs that are null for mago divide, the expression of a bam transcriptional reporter is not detected, suggesting that these cells are blocked in a GSC or in a pre-cystoblast state and accounting for the epistatic relationship between mago and bgcn. Within the network of pathways regulating GSC differentiation, there are two candidate sites where Mago may function. First, Mago may negatively regulate the Bmp signal transduction pathway within GSCs or a redundant pathway. In this case, transcriptional silencing of bam would be maintained, even outside of the niche due to stability of the negative regulatory signal. Alternatively, Mago may be required for transcriptional activation of bam. In the absence of Mago, there would be insufficient Bam:Bgcn to displace Pum:Nos and consequently translational repression of cystoblast differentiation factors would continue. Future research will help to distinguish between these alternatives. In addition, since Mago's function in GSC differentiation is independent of Tsu/Y14, it will be of interest to elucidate Mago's mechanism of action during this process (Parma, 2007).
Oocyte selection in Drosophila can be divided into the following three major steps: (a) fusome formation, (b) concentration of determinants first within the pro-oocytes and finally within the oocyte, and (c) oocyte maintenance. Asymmetry during oogenesis is first apparent during fusome formation, when the cystoblast inherits a third of the fusome from the GSC. Subsequent divisions result in the acquisition of a larger amount of the fusome material by the two pro-oocytes than other cells of the cyst. Without functional fusomes, microtubule polarity within a cyst is aberrant and an oocyte is not specified. A polarized microtubule cytoskeleton is critical for establishing the intra-cyst asymmetry that results in the selective concentration of specific RNAs and proteins (e.g., Org, Egl, BicD and Par-1), first within the two pro-oocytes and ultimately within the definitive oocyte. Accumulation of proteins/RNAs is required either for oocyte determination or for maintenance of oocyte fate. Mutations in genes required for oocyte determination disrupt the accumulation of proteins/RNAs within a single cell within the cyst. However, mutations in genes required for oocyte maintenance do not interfere with the initial accumulation of oocyte-specific factors within a single cell but disrupt the translocation of these components from the anterior to the posterior of the oocyte. Prior to this analysis of tsu, only two genes were known to be required for the accumulation of both oocyte determination and maintenance factors within the presumptive oocyte (Parma, 2007).
Mutations in genes encoding fusome-associated components that are required for restricting oocyte specification to a single cell within the 16-cell cyst are expected to disrupt the accumulation of oocyte determination and maintenance factors within the presumptive oocyte. One such fusome component is the Drosophila spectraplakin protein, Short stop (Shot), a cytoskeletal linker protein that contains a domain capable of bundling and stabilizing microtubules (GAS2). Germline cells lacking shot function contain fusomes, but components required to specify and maintain oocyte fate fail to accumulate in a single cell. Consequently, all 16 cells of the cyst develop as nurse cells. Furthermore, the centrosomes, which normally migrate into the oocyte along fusomes, fail to accumulate within a single cell in shot mutant germline cells (Parma, 2007).
Dynein heavy chain 64C is another fusome-associated protein. Dynein heavy chain 64C (Dhc64C) mutant germline cells contain abnormal fusomes. As observed in shot mutants, oocyte-specific components (e.g., BicD) fail to accumulate in a single cell and centrosome migration is aberrant. Par-1 (a serine/threonine kinase) is a third component of the fusomes. However, unlike shot and Dhc64C mutant germline cells, in par-1 mutant germline cells, centrosome migration is normal. Oocyte-specific components accumulate in a single cell but fail to translocate from the anterior to the posterior of the oocyte. Thus, fusomes are important for oocyte determination (Shot and Dhc64C) and for maintenance (Par-1) of oocyte fate (Parma, 2007).
Two proteins not known to be associated with fusomes, BicD and Egl, are also essential for the accumulation of oocyte-specific components within a single cell. However, mutations in these genes do not disrupt the migration of centrosomes into a single cell, even though oocyte specification fails. Migration of centrosomes into the oocyte occurs in the presence of colchicine, suggesting that migration of centrosomes into the oocyte does not occur along dynamic microtubules. Consistent with this observation, acetylated tubulins (a form of tubulin found in stable microtubules), are found in a population of microtubules associated with fusomes. Based on the presence of acetylated tubulins in microtubules associated with fusomes, the ability of Shot's GAS2 domain to stabilize microtubules and the shot mutant germline phenotype, it has been suggested that centrosomes migrate along stable microtubules (Parma, 2007).
Utilizing the synaptonemal complex formation, Orb accumulation and centrosome migration, this study monitored the ability of wild-type and tsunull germline cysts to select a single cell of the 16-cell cyst as the definitive oocyte. The synaptonemal complex marks progression of individual cells of the cyst through meiosis. In tsu mutant germline cysts, four cells enter meiosis in germarial region 2a and remain in meiosis into region 3, suggesting that selection of the oocyte is abnormal. Microtubule depolymerizing drugs do not interfere with the formation the synaptonemal complex but the SC is not maintained in germarial region 3, indicating its maintenance at this stage is dependent on dynamic microtubules. Given that the synaptonemal complex persists in tsu mutant germline cysts in germarial region 3, it is concluded that dynamic microtubules are not disrupted in tsu mutant cells (Parma, 2007).
Previously, only shot and Dhc64C had been demonstrated to be essential for the polarized transport of all oocyte-specific components. This study has show that Tsu/Y14 is an additional factor regulating the polarized transport of centrosomes and all other oocyte-specific components. This suggests that, like Shot and Dhc64C, Tsu/Y14 functions upstream of BicD and Egl to restrict oocyte fate to a single cell. Reduced function alleles of mago exhibit phenotypes very similar to those observed in tsunull germline cells, suggesting that Mago and Tsu/Y14 function together to restrict oocyte fate to a single cell. Further studies will be necessary to establish the position of Tsu/Y14 in the pathway relative to Shot and Dhc64C (Parma, 2007).
The data presented in this paper, considered with previous studies of the role of Tsu/Y14 in the localization/transport of RNAs, provides evidence that Tsu/Y14 is involved in an early step in the polarized transport of oocyte-specific RNAs/proteins. Future studies will determine the position of Tsu/Y14 within the oocyte specification pathway and reveal additional components involved in polarized transport during early oogenesis (Parma, 2007).
Alexandrov, A., Colognori, D., Shu, M. D. and Steitz, J. A. (2012). Human spliceosomal protein CWC22 plays a role in coupling splicing to exon junction complex deposition and nonsense-mediated decay. Proc Natl Acad Sci U S A 109(52): 21313-21318. PubMed ID: 23236153
Andersen, C. B., et al. (2006). Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA. Science 313(5795): 1968-72. Medline abstract: 16931718
Ashton-Beaucage, D., Udell, C. M., Lavoie, H., Baril, C., Lefrancois, M., Chagnon, P., Gendron, P., Caron-Lizotte, O., Bonneil, E., Thibault, P. and Therrien, M. (2010). The exon junction complex controls the splicing of MAPK and other long intron-containing transcripts in Drosophila. Cell 143: 251-262. PubMed ID: 20946983
Barbosa, I., Haque, N., Fiorini, F., Barrandon, C., Tomasetto, C., Blanchette, M. and Le Hir, H. (2012). Human CWC22 escorts the helicase eIF4AIII to spliceosomes and promotes exon junction complex assembly. Nat Struct Mol Biol 19(10): 983-990. PubMed ID: 22961380
Bono, F., et al. (2004). Molecular insights into the interaction of PYM with the Mago-Y14 core of the exon junction complex. EMBO Rep. 5(3): 304-10. Medline abstract: 14968132
Bono, F., Ebert, J., Lorentzen, E. and Conti, E. (2006). The crystal structure of the exon junction complex reveals how it maintains a stable grip on mRNA.
Cell 126(4): 713-25. Medline abstract: 16923391
Boswell, R. E., Prout, M. E. and Steichen, J. C. (1991). Mutations in a newly identified Drosophila melanogaster gene, mago nashi, disrupt
germ cell formation and result in the formation of mirror-image symmetrical double abdomen embryos. Development 113(1): 373-384. PubMed Citation: 1765008
Fribourg, S., Gatfield, D., Izaurralde, E. and Conti, E. (2003). A novel mode of RBD-protein recognition in the Y14-Mago complex. Nat Struct Biol 10: 433-439. Medline abstract: 12730685
Fusco, D., et al. (2003). Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr. Biol. 13: 161-167. PubMed Citation: 12546792
Hachet, O. and Ephrussi, A. (2001). Drosophila Y14 shuttles to the posterior of the oocyte and is required for oskar mRNA transport. Cur. Bio. 11: 1666-1674. 11696323
Hachet, O. and Ephrussi, A. (2004). Splicing of oskar RNA in the nucleus is coupled to its cytoplasmic localization. Nature 428(6986): 959-63. PubMed Citation: 15118729
Kataoka, N., et al. (2001). Magoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing-dependent exon-exon junction complex. EMBO J. 20(22): 6424-6433. 11707413
Kawano, T., Kataoka, N., Dreyfuss, G. and Sakamoto, H. (2004). Ce-Y14 and MAG-1, components of the exon-exon junction complex, are required for embryogenesis and germline sexual switching in Caenorhabditis elegans. Mech. Dev. 121(1): 27-35. Medline abstract: 14706697
Kim, V. N., Yong, J., Kataoka, N., Abel, L., Diem, M. D. and Dreyfuss, G. (2001a). The Y14 protein communicates to the cytoplasm the position of exon-exon junctions. EMBO J. 20: 2062-2068. 11296238
Kim, V. N., Kataoka, N. and Dreyfuss, G. (2001b). Role of the nonsense-mediated decay factor hUPF3 in the splicing-dependent exon-exon junction complex. Science 293: 1832-1836. 11546873
Lau, C. K., Diem, M. D., Dreyfuss, G. and Van Duyne, G. D. (2003). Structure of the Y14-Magoh core of the exon junction complex. Curr Biol 13: 933-941. Medline abstract: 12781131
Le Hir, H., Moore, M. J. and Maquat, L. E. (2000a). Pre-mRNA splicing alters mRNP composition: evidence for stable association of proteins at exon-exon junctions. Genes Dev. 14: 1098-1108. 10809668
Le Hir, H., Izaurralde, E., Maquat, L. E. and Moore, M. J. (2000b). The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions. EMBO J. 19: 6860-6869. 11118221
Le Hir, H., Gatfield, D., Izaurralde, E. and Moore, M. J. (2001a). The exon-exon junction complex provides a binding platform for factors involved in mRNA export and NMD. EMBO J. 20: 4987-4997. 11532962
Le Hir, H., Gatfield, D., Braun, I. C., Forler, D. and Izaurralde, E. (2001b). The protein Mago provides a link between splicing and mRNA localization.
EMBO Rep. 2(12): 1119-24. 11743026
Li, W., Boswell, R. and Wood, W. B. (2000). mag-1, a nomolog of Drosophila mago nashi, regulates hermaphrodite germ-line sex
determination in Caenorhabditis elegans. Dev. Biol. 218: 172-182. PubMed Citation: 10656761
Liu, M., Li, Y., Liu, A., Li, R., Su, Y., Du, J., Li, C. and Zhu, A. J. (2016). The exon junction complex regulates the splicing of cell polarity gene dlg1 to control Wingless signaling in development. Elife 5:e17200. PubMed ID: 27536874
Micklem, D. R., et al. (1997). The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila. Curr. Biol. 7(7): 468-478. PubMed Citation: 9210377
Mingot, J. M., Kostka, S., Kraft, R., Hartmann, E. and Gorlich, D. (2001). Importin 13: a novel mediator of nuclear import and export. EMBO J. 20: 3685-3694. 11447110
Mohr, S. E., Dillon, S. T. and Boswell, R. E. (2001). The RNA-binding protein Tsunagi interacts with Mago Nashi to establish polarity and localize oskar mRNA during Drosophila oogenesis. Genes Dev. 15: 2886-2899. 11691839
Newmark, P. A. and Boswell, R. E. (1994). The mago nashi locus encodes an essential product required for germ plasm assembly
in Drosophila. Development 120(5): 1303-1313. PubMed Citation: 8026338
Newmark, P. A., et al. (1997). mago nashi mediates the posterior follicle cell-to-oocyte signal to organize axis formation in Drosophila. Development 124(16): 3197-3207. PubMed Citation: 9272960
Obrdlik, A., Lin, G., Haberman, N., Ule, J. and Ephrussi, A. (2019). The transcriptome-wide landscape and modalities of EJC binding in adult Drosophila. Cell Rep 28(5): 1219-1236. PubMed ID: 31365866
Palacios, I. M., Gatfield, D., St Johnston, D. and Izaurralde, E. (2004). An eIF4AIII-containing complex required for mRNA localization and nonsense-mediated mRNA decay. Nature 427(6976): 753-7. Medline abstract: 14973490
Parma, D. H., Bennett, P. E. and Boswell, R. E. (2007). Mago Nashi and Tsunagi/Y14, respectively, regulate Drosophila germline stem cell differentiation and oocyte specification. Dev. Biol. 308(2): 507-19. PubMed Citation: 17628520
Roignant, J. Y. and Treisman, J. E. (2010). Exon junction complex subunits are required to splice Drosophila MAP kinase, a large heterochromatic gene. Cell 143: 238-250. PubMed ID: 20946982
Sheehan, C. J., McMahon, J. J., Serdar, L. D. and Silver, D. L. (2020). Dosage-dependent requirements of Magoh for cortical interneuron generation and survival. Development 147(1). PubMed ID: 31857347
Shi, H. and Xu, R. M. (2003). Crystal structure of the Drosophila Mago nashi-Y14 complex. Genes Dev. 17(8): 971-6. Medline abstract: 12704080
Steckelberg, A. L., Altmueller, J., Dieterich, C. and Gehring, N. H. (2015). CWC22-dependent pre-mRNA splicing and eIF4A3 binding enables global deposition of exon junction complexes. Nucleic Acids Res 43(9): 4687-4700. PubMed ID: 25870412
Theurkauf, W. E., et al. (1992). Reorganization of the cytoskeleton during Drosophila oogeneis: implication for axis specification and intercellular transport. Development 115: 923-936. PubMed Citation: 1451668
van der Weele, C. M., Tsai, C. W. and Wolniak, S. M. (2007). Mago nashi is essential for spermatogenesis in Marsilea. Mol. Biol. Cell 18(10): 3711-22. PubMed Citation: 17634289
Wolfgang, W. J. and Forte, M. (1995). Posterior localization of the Drosophila Gi alpha protein during early embryogenesis requires a subset of the posterior group genes. Int. J. Dev. Biol. 39(4): 581-586. PubMed Citation: 8619956
Zhao, X. F., et al. (1998). The mammalian homologue of mago nashi encodes a serum-inducible protein. Genomics 47(2): 319-22
Zhao, X. F., et al. (2000). MAGOH interacts with a novel RNA-binding protein. Genomics 63(1): 145-148. PubMed Citation: 10662555
mago nashi:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
| References
date revised: 3 January 2020
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.