Localization of bicoid (BCD) mRNA to the anterior and Oskar (OSK) mRNA to the posterior of the Drosophila oocyte is critical for embryonic patterning. exuperantia (exu) has been implicated in BCD mRNA localization, but its role in this process is not understood. Exu has been isolated and it has been shown that Exu is part of a large RNase-sensitive complex that contains at least seven other proteins. One of these proteins was identified as the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps), which binds directly to Exu and colocalizes with Exu in both the oocyte and nurse cells of the Drosophila egg chamber. Surprisingly, the Exu-Yps complex contains Oskar mRNA. This biochemical result led to a reexamination of the role of Exu in the localization of OSK mRNA. exu-null mutants are defective in OSK mRNA localization in both nurse cells and the oocyte. Furthermore, both Exu/Yps particles and OSK mRNA follow a similar temporal pattern of localization in which they transiently accumulate at the oocyte anterior and subsequently localize to the posterior pole. It is proposed that Exu is a core component of a large protein complex involved in localizing mRNAs both within nurse cells and the developing oocyte (Wilhelm, 2000).
To identify the protein components of the Exu complex, GFP-Exu was immunoprecipitated from whole fly extracts prepared from a GFP-Exu-expressing fly line using an anti-GFP antibody. The GFP tag does not impair Exu protein function, since the gfp-exu transgene fully complements a null allele of exu. Seven polypeptides of 57, 74, 76, 78, 82, 88, and 147 kD coimmunoprecipitate specifically with GFP-Exu. A similar set of polypeptides coimmunoprecipitates with GFP-Exu from an extract made from hand dissected ovaries, indicating that this complex is present within the female germ line. This same set of polypeptides also coimmunoprecipitates with Exu from wild-type fly extracts using an antibody directed against a COOH-terminal peptide of Exu. Of the coimmunoprecipitated proteins, the 57-, 74-, 76-, 78-, and 82-kD proteins are present in amounts comparable to that of GFP-Exu, whereas the 88- and 147-kD proteins are clearly substoichiometric. When the extract is extensively treated with RNase A, only the 57-kD protein remains associated with GFP-Exu. Taken together with gradient analysis, these results suggest that Exu and p57 are components of a 7-S RNase-resistant core complex; the other polypeptides (p74, p76, p78, p82, p88, and p147) all require the presence of RNA in order to associate with Exu (Wilhelm, 2000).
To confirm that the 57-kD polypeptide is a bona fide Exu-associated protein, a different purification strategy was used to isolate Exu complexes. Using flies that express Exu with an NH2-terminal GFP tag and a COOH-terminal His6 tag, extracts were subjected to a two-step purification consisting of binding to an Ni-NTA column, elution with imidazole, and then immunoprecipitation with the anti-GFP antibody. The 57-kD protein consistently copurifies stoichiometrically with GFP-Exu-His6 through this two-step affinity purification, confirming that it is a true Exu-associated polypeptide. The other polypeptides in the 74-82 kD range were identified in some of the preparations, but their presence and amount was highly variable, possibly due to RNA degradation during the procedure or instability of the complex during imidazole elution from the column (Wilhelm, 2000).
To characterize Yps, affinity purified antibodies were prepared against bacterially expressed Yps (amino acids 1-160). These antibodies recognized the 57-kD Yps protein in crude extracts by immunoblot. Using the Yps antibody, it was found that Yps comigrates with Exu in sucrose gradients and is distributed broadly in the 20-60 S size range. To rule out the possibility that Exu and Yps are components of distinct complexes of similar size, GFP-Exu was immunoprecipitated from individual gradient fractions and immunoblotted with the Yps antibody. This experiment shows that Exu and Yps coimmunoprecipitate together across the gradient, arguing strongly that Exu and Yps are part of the same complex (Wilhelm, 2000).
To provide further evidence for an Exu-Yps complex, GFP-Exu extracts were immunoprecipitated with anti-Yps polyclonal antibody. In agreement with immunoprecipitation results, immunoblots show that GFP-Exu specifically coimmunoprecipitates with Yps. However, immunoblots show only a weak GFP-Exu band in the Yps immunoprecipitate. The inefficient coimmunoprecipitation of GFP-Exu with Yps is probably due to the fact that the anti-Yps antibody may displace Exu from the complex, since it was raised against the Exu-binding region of Yps. The Yps immunoprecipitates also contain the same six proteins (p74, p76, p78, p82, p88, and p147) that strongly coimmunoprecipitate with GFP-Exu and are present in similar stoichiometries. The coimmunoprecipitation of these six proteins with Yps is diminished by RNase treatment, as is observed in Exu immunoprecipitation experiments. The ability of Yps and Exu antibodies to coimmunoprecipitate the same set of polypeptides argues that these proteins are bona fide components of the Exu-Yps complex (Wilhelm, 2000).
The coimmunoprecipitation of Exu and Yps after RNase A treatment suggests, but does not prove, that Exu and Yps bind directly to each other. To test this idea, the Exu-Yps interaction was examined in an in vitro translation reaction. Myc-tagged Yps was in vitro translated in the presence of [35S]methionine and then added to an unlabeled in vitro translation of HA-tagged Exu. Before mixing, each translation reaction was treated with RNase A to eliminate any residual RNA from the translation reaction. When HA-Exu was immunoprecipitated from the combined mixture with the anti-HA antibody, the 35S-labeled myc-Yps protein coimmunoprecipitates. The amount of myc-Yps that coimmunoprecipitated with HA-Exu was approximately half of the amount of myc-Yps that immunoprecipitated directly with the anti-myc antibody, showing that Yps is predominantly bound to Exu under these experimental conditions. These results demonstrate that the additional RNA and protein components of the native Exu complex are not required for Exu and Yps to associate stably with each other (Wilhelm, 2000).
To determine which region of Yps is important for binding to Exu, deletions of myc-Yps were assayed for their ability to bind HA-Exu in vitro. The NH2-terminal region (1-160 amino acids) of Yps, which contains the cold shock domain, binds to Exu at the same efficiency as the full-length protein. However, the minimal cold shock domain (56-151 amino acids) does not bind to Exu in this assay, suggesting that the sequences flanking the cold shock domain are likely to contribute to the Exu-binding site. The proline-rich COOH terminus of Yps presumably is not sufficient for binding. However, this could not be assessed experimentally, since this region did not stably express either in vitro or in bacteria (Wilhelm, 2000).
The shift in the size of the Exu-Yps complex after RNase treatment suggests that it might be directly involved in mRNA localization. To test this idea, either Exu or Yps was immunoprecipitated from extracts and the pellet was analyzed for the presence of various localized (BCD, OSK, NOS) messages. These transcripts were analyzed because each gene appears in the EST database at roughly equivalent frequencies, indicating that these messages are likely to be present in comparable amounts. Furthermore, BCD, OSK, and NOS are localized differently during oogenesis. BCD mRNA is localized to the anterior of the oocyte beginning at stage 7 of oogenesis; OSK mRNA is localized transiently to the anterior of the oocyte during stages 8/9 and is exclusively localized to the posterior by the end of stage 9; NOS mRNA is localized to the posterior of the oocyte during late oogenesis. When GFP-Exu immunoprecipitates were analyzed by RT-PCR, only OSK transcript was amplified by RT-PCR. The identical result was obtained when RT-PCR was performed on immunoprecipitations with the anti-Yps antibody. Therefore, the RT-PCR assay demonstrates that both Yps and Exu are associated with a complex that contains OSK mRNA. This was an unanticipated result, since previous genetic studies only reported a role for exu in the localization of BCD mRNA to the anterior of the oocyte. That the Exu-Yps complex may contain BCD mRNA cannot be ruled out, since the negative result could reflect technical difficulties, such as poor BCD mRNA stability. However, the RT-PCR results suggest the unanticipated possibility that exu is involved in posterior mRNA localization (Wilhelm, 2000).
The biochemical studies linking Yps to Exu and OSK mRNA suggest that this RNA-binding protein plays a role in posterior mRNA localization. This assertion is further supported by immunofluorescence studies showing that Yps and OSK mRNA have strikingly similar localization patterns throughout oogenesis: both accumulate in the early oocyte, transiently localize to the oocyte anterior during stages 8 and 9, and then assume their final positions at the posterior pole during stages 9 and 10. What role might Yps play in the localization complex? Yps belongs to the cold shock domain family of RNA-binding proteins that have been implicated in regulating translation and mRNA secondary structure. A notable example is FRGY2, which is complexed with mRNAs in the Xenopus oocyte and is thought to be important for translational silencing. Yps may serve a similar role, since OSK mRNA is translationally repressed until it reaches the posterior pole. Interestingly, Yps must also serve a function without Exu, since yps is expressed broadly, whereas exu expression is limited to the germ line. It is possible that Yps is a component of the mRNA localization machinery outside the germ line, since other components of the oocyte mRNA localization machinery, such as Staufen, are also used for mRNA localization in somatic tissues. Determining the precise involvement in transport and/or translational regulation of Yps in the oocyte and other tissues will be resolved in the future by mutational studies (Wilhelm, 2000 and references therein).
The complex between Exuperantia and the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps) contains OSK mRNA. This biochemical result has led to a reexamination of the role of Exu in the localization of OSK mRNA. exu-null mutants are defective in OSK mRNA localization in both nurse cells and the oocyte. Furthermore, both Exu/Yps particles and OSK mRNA follow a similar temporal pattern of localization in which they transiently accumulate at the oocyte anterior and subsequently localize to the posterior pole. It is proposed that Exu is a core component of a large protein complex involved in localizing mRNAs, both within nurse cells and the developing oocyte (Wilhelm, 2000).
To identify the protein components of the Exu complex, GFP-Exu was immunoprecipitated from whole fly extracts prepared from a GFP-Exu-expressing fly line using an anti-GFP antibody. The GFP tag does not impair Exu protein function, since the gfp-exu transgene fully complements a null allele of exu. Seven polypeptides of 57, 74, 76, 78, 82, 88, and 147 kD coimmunoprecipitate specifically with GFP-Exu. A similar set of polypeptides coimmunoprecipitate with GFP-Exu from an extract made from hand dissected ovaries, indicating that this complex is present within the female germ line. This same set of polypeptides also coimmunoprecipitate with Exu from wild-type fly extracts using an antibody directed against a COOH-terminal peptide of Exu. Of the coimmunoprecipitated proteins, the 57-, 74-, 76-, 78-, and 82-kD proteins are present in amounts comparable to those of GFP-Exu, whereas the 88- and 147-kD proteins are clearly substoichiometric. When the extract is extensively treated with RNase A, only the 57-kD protein remains associated with GFP-Exu. Taken together with gradient analysis, these results suggest that Exu and p57 are components of a 7-S RNase-resistant core complex; the other polypeptides (p74, p76, p78, p82, p88, and p147) all require the presence of RNA in order to associate with Exu (Wilhelm, 2000).
To confirm that the 57-kD polypeptide is a bona fide Exu-associated protein, a different purification strategy was used to isolate Exu complexes. Using flies that express Exu with an NH2-terminal GFP tag and a COOH-terminal His6 tag, extracts were subjected to a two-step purification consisting of binding to an Ni-NTA column, elution with imidazole, and then immunoprecipitation with the anti-GFP antibody. The 57-kD protein consistently copurifies stoichiometrically with GFP-Exu-His6 through this two-step affinity purification, confirming that it is a true Exu-associated polypeptide. The other polypeptides in the 74-82 kD range were identified in some of these preparations, but their presence and amount is highly variable, possibly due to RNA degradation during the procedure or instability of the complex during imidazole elution from the column (Wilhelm, 2000).
To identify the 57-kD Exu-associated protein, three tryptic peptides from the purified protein were microsequenced. The sequence from two of the three peptides match a previously identified protein, the product of the yps gene. Yps is a member of the cold shock family of RNA-binding domain proteins and was identified as part of a degenerate PCR screen to identify cold shock domain containing genes from Drosophila melanogaster (Thieringer, 1997). However, the third peptide only matched the Yps sequence in a reading frame other than the published open reading frame. To rule out the possibility that yps expression is subject to ribosomal frameshifting or RNA editing, six independent yps ESTs (Berkeley Drosophila Genome Project) were obtained and sequenced. The original yps sequence was shown to contain several sequencing errors and the correct open reading frame contains all three microsequenced peptides. The cold shock domain of Yps shows extensive sequence identity to other cold shock domain proteins. This domain has been shown in several studies to bind RNA, although its ability to recognize specific substrates remains uncertain. Beyond the cold shock domain, Yps exhibits no significant homology to any other protein except YB-1, a cold shock domain protein from Drosophila silvesteris. Since the YB-1 protein is 70% identical to Yps across the entire length of the protein, it is likely to be a true ortholog of Yps. No function was assigned to either YB-1 or Yps in these prior studies (Wilhelm, 2000).
To further characterize Yps, affinity purified antibodies were prepared against bacterially expressed Yps (amino acids 1-160). These antibodies recognize the 57-kD Yps protein in crude extracts by immunoblot. Using the Yps antibody, Yps was found to comigrate with Exu in sucrose gradients and is distributed broadly in the 20-60 S size range. To rule out the possibility that Exu and Yps are components of distinct complexes of similar size, GFP-Exu was immunoprecipitated from individual gradient fractions and immunoblotted with the Yps antibody. This experiment shows that Exu and Yps coimmunoprecipitate together across the gradient, and argues strongly that Exu and Yps are part of the same complex (Wilhelm, 2000).
To provide further evidence for an Exu-Yps complex, GFP-Exu extracts were immunoprecipitated with anti-Yps polyclonal antibody. Immunoblots show that GFP-Exu specifically coimmunoprecipitates with Yps. However, immunoblots show only a weak GFP-Exu band in the Yps immunoprecipitate. The inefficient coimmunoprecipitation of GFP-Exu with Yps is probably due to the fact that the anti-Yps antibody may displace Exu from the complex, since it was raised against the Exu-binding region of Yps. The Yps immunoprecipitates also contain the same six proteins (p74, p76, p78, p82, p88, and p147) that strongly coimmunoprecipitate with GFP-Exu and are present in similar stoichiometries. The coimmunoprecipitation of these six proteins with Yps is diminished by RNase treatment, as is observed in Exu immunoprecipitation experiments. The ability of Yps and Exu antibodies to coimmunoprecipitate the same set of polypeptides argues that these proteins are bona fide components of the Exu-Yps complex (Wilhelm, 2000).
The coimmunoprecipitation of Exu and Yps after RNase A treatment suggests, but does not prove, that Exu and Yps bind directly to each other. To test this idea, the Exu-Yps interaction was examined in an in vitro translation reaction. Myc-tagged Yps was in vitro translated in the presence of [35S]methionine and then added to an unlabeled in vitro translation of HA-tagged Exu. Before mixing, each translation reaction was treated with RNase A to eliminate any residual RNA from the translation reaction. When HA-Exu was immunoprecipitated from the combined mixture with the anti-HA antibody, the 35S-labeled myc-Yps protein was coimmunoprecipitated. The amount of myc-Yps that coimmunoprecipitated with HA-Exu was approximately half of the amount of myc-Yps that was immunoprecipitated directly with the anti-myc antibody, showing that Yps is predominantly bound to Exu under these experimental conditions. These results demonstrate that the additional RNA and protein components of the native Exu complex are not required for Exu and Yps to associate stably with each other (Wilhelm, 2000).
To determine which region of Yps is important for binding to Exu, deletions of myc-Yps were assayed for their ability to bind HA-Exu in vitro. The NH2-terminal region (1-160 amino acids) of Yps, which contains the cold shock domain, binds to Exu at the same efficiency as the full-length protein. However, the minimal cold shock domain (56-151 amino acids) does not bind to Exu in this assay, suggesting that the sequences flanking the cold shock domain are likely to contribute to the Exu-binding site. The proline-rich COOH terminus of Yps presumably is not sufficient for binding. However, this could not be assessed experimentally, since this region does not stably express either in vitro or in bacteria (Wilhelm, 2000).
Translational control of localized messenger mRNAs (mRNAs) is critical for cell polarity, synaptic plasticity, and embryonic patterning. While progress has been made in identifying localization factors and translational regulators, it is unclear how broad a role they play in regulating basic cellular processes. Drosophila trailer hitch (tral) has been identified as required for the proper secretion of the dorsal-ventral patterning factor Gurken, as well as the vitellogenin receptor Yolkless. Surprisingly, biochemical purification of Tral reveals that it is part of a large RNA-protein complex that includes the translation/localization factors Me31B and Cup as well as the mRNAs for endoplasmic reticulum (ER) exit site components, that regulate exit of proteins from the ER. This complex is localized to subdomains of the ER that border ER exit sites. Furthermore, tral is required for normal ER exit site formation. These findings raise exciting new possibilities for how the mRNA localization machinery could interface with the classical secretory pathway to promote efficient protein trafficking in the cell (Wilhelm, 2005).
In order to better understand the role of Tral in regulating membrane trafficking, the identification of Tral-associated proteins was attempted by immunoprecipitating Tral from Drosophila embryo extract using Tral antibody. By colloidal blue staining, three major bands were found that specifically coimmunoprecipitated with Tral: p147, p70, and p50. Using mass spectrometry, p147 was identified as the eIF4E binding protein Cup, and p70 as poly(A) binding protein (PABP). p50 was found to be a mixture of the RNA binding protein Ypsilon Schactel (Yps) and the RNA helicase Me31B. To confirm the identities of the Tral-associated proteins, Tral was immunoprecipitated from ovarian extracts and immunoblotted for Cup, Yps, and Me31B. Me31B, Yps, and Cup all specifically coimmunoprecipitate with Tral, indicating that these proteins are bona fide components of the complex. Because Me31B, Yps, and Cup have been previously shown to be part of an RNA-protein complex, the ability of each protein to coimmunoprecipitate with Tral was tested in RNase-treated ovarian extracts. It was found that while the association of Tral with Me31B, Yps, and Cup is RNase resistant, the association of Yps with Cup is sensitive to RNase treatment, indicating the presence of RNA in the complex (Wilhelm, 2005).
Previous work has shown that Me31B, Cup, and Yps colocalize in vivo. In order to demonstrate that Tral is part of the Me31B-Cup-Yps complex in vivo, egg chambers were immunoprecipitated for Tral and Me31B as well as Tral and Cup. The particulate staining in nurse cells showed a high degree of overlap for both the Tral/Cup and Tral/Me31B double-labeled egg chambers. Furthermore, the temporal-spatial pattern of Tral localization within the oocyte is identical to that previously described for Cup, Me31B, and Yps. These results, together with the previously demonstrated colocalization of Me31B, Cup, and Yps, indicate that Tral, Cup, Me31B, and Yps all exist as a complex in vivo (Wilhelm, 2005).
Because Tral is present on discrete domains of the ER, it was next asked whether other components of the complex were also present on the ER. Colocalization studies of GFP-KDEL with either Me31B or Cup showed that Me31B and Cup are both present on discrete ER subdomains. This observation, together with the biochemical analysis of the Tral complex, demonstrates that Tral is part of an RNA-protein complex that is associated with the ER (Wilhelm, 2005).
Because mutations in tral have such striking effects on morphology of COPII foci, attempts were made to define the relationship between these foci and components of the Tral complex. Using GFP-Sar1 as a marker for COPII complex formation, it was found that while some COPII sites are not associated with the Tral complex, a number of sites either colocalize with or are bordered by the Tral complex. These observations are highly suggestive of a direct role in regulating exit site function, as recent work has implicated the regions around COPII sites in exit from the ER (Wilhelm, 2005).
Developmental expression of the yps gene was investigated using an RNase protection assay (RPA). The yps transcript was detected in all stages of development that were tested, including 2-3 h embryos, 14-22 h embryos, third instar larvae and adult flies. The highest expression was found in adult flies, with about a 3-fold increase over other stages tested (Thieringer, 1997).
It was of interest to test the possibility that this gene may be cold-shock inducible, due to the fact that three of the bacterial homologs are cold-shock inducible. The question of cold induciblity of the eukaryotic Y-box proteins has not been previously addressed. This was investigated in the Kc cell line, an embryonic cell line that has endogenous yps expression. After both 30 min and 4 h incubation at 10°C the amount of the YPS mRNA did not change appreciably. There is a slight increase in the relative amount of the message during cold shock. However, these values were obtained as relative amounts against rp49 expression, which was slightly decreased at 10°C (Thieringer, 1997).
In order to further investigate the expression of the yps gene during development, in situ hybridization of whole mount embryos was performed. The yps transcript is clearly present in the stage 2 embryo, which implies that it is maternally loaded into the egg cytoplasm prior to fertilization. Between stages 2 and 4 the egg cytoplasm concentrates peripherally in the so-called cortex. At stage 4 a region of staining in the periphery of the embryo is observed, which is explained by the fact that the cytoplasm has now migrated to this position. By stage 6 the somatic cells have incorporated most of the egg cytoplasm, and within these cells, the nuclei are situated to the outside and the cytoplasm inside. This picture is particularly clear at stage 6. At stage 8, as germ band elongation has begun, dark staining is clearly present along the mesodermal layer of cells. Staining is also seen above the cephalic furrow, most likely in the cephalic mesoderm, or anterior midgut primordium. At the latest stage no staining can be seen in the outer epidermal tissues (Thieringer, 1997).
To learn more about the in vivo role of Yps in RNA localization, Drosophila ovaries were labeled with affinity purified antibodies to the NH2 terminus (1-160 amino acids) of Yps. Immunofluorescence staining revealed a strong Yps signal in both the germ cells and follicle cells of developing egg chambers. Examination of different stage egg chambers revealed that Yps accumulates in the oocyte during stages 1-7. This signal is stronger at the posterior of the oocyte in early stages, although it is also present throughout much of the oocyte cytoplasm. In both early and midstage egg chambers, Yps exhibits a particulate staining that is frequently concentrated around the nurse cell nuclei. At stages 8 and 9, faint anterior localization is sometimes apparent in the oocyte, and during stages 9 and 10 Yps accumulates at the posterior of the oocyte (Wilhelm, 2000).
The localization of Yps during oogenesis is very similar to the observed distribution of GFP-Exu. To compare the distributions of Exu and Yps directly, Yps was immunostained in egg chambers expressing GFP-Exu. Individual particles containing both proteins are detected in the nurse cells. The early accumulation of Yps in the oocyte (stages 1-7), its accumulation at the oocyte anterior (stages 8 and 9), and its later localization to the posterior pole (stages 9 and 10) all coincide with the localization of Exu protein and OSK mRNA. The agreement between the biochemical and in vivo localization studies supports a role for the Exu-Yps complex in the localization of OSK mRNA (Wilhelm, 2000).
Evdokimova, V., et al. (2001). The major mRNA-associated protein YB-1 is a potent 5' cap-dependent mRNA stabilizer. EMBO J. 20(19): 5491-502. 11574481
Mansfield, J. H., Wilhelm, J. E. and Hazelrigg, T. (2002). Ypsilon Schachtel, a Drosophila Y-box protein, acts antagonistically to Orb in the oskar mRNA localization and translation pathway. Development 129: 197-209. 11782413
Moss, E. G., Lee, R. C. and Ambros, V. (1997). The cold shock domain protein LIN-28 controls developmental timing in C. elegans and is regulated by the lin-4 RNA. Cell 88: 637-646. 9054503
Ruzanov, P. V., Evdokimova, V. M., Korneeva, N. L., Hershey, J. W. and Ovchinnikov, L. P. (1999). Interaction of the universal mRNA-binding protein, p50, with actin: a possible link between mRNA and microfilaments. J. Cell Sci. 112: 3487-3496. 10504297
Thieringer, H. A., Singh, K., Trivedi, H. and Inouye, M. (1997). Identification and developmental characterization of a novel Y-box protein from Drosophila melanogaster. Nucleic Acids Res. 25: 4764-4770. 9365254
Wilhelm, J. E., et al. (2000). Isolation of a ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes. J. Cell Biol. 148: 427-440.
Wilhelm, J. E., Buszczak, M. and Sayles S. (2005). Efficient protein trafficking requires trailer hitch, a component of a ribonucleoprotein complex localized to the ER in Drosophila. Dev. Cell 9(5): 675-85. 16256742
date revised: 25 March 2006
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