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Gene name - trailer hitch
Synonyms - Cytological map position - 69C4 Function - RNA processing Keywords - trafficking of secreted proteins, endoplasmic reticulum to golgi transport, cytoplasmic mRNA processing |
Symbol - tral
FlyBase ID: FBgn0041775 Genetic map position - 3L Classification - N-terminal Sm-related domain, C-terminal FDF motif Cellular location - cytoplasmic |
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).
Localization of mRNAs is used by many polarized cells as a means of restricting the distribution of a protein to a particular cytoplasmic domain. This mechanism for protein targeting within the cytoplasm is critical for embryonic patterning, synapse formation, and cell migration. Three methods of localization have been described. Active transport of mRNA along either microtubules or actin filaments has been directly demonstrated for a number of transcripts, suggesting a role for cytoskeletal motor proteins in mRNA localization. Diffusion trapping involves the creation of a binding site for the message of interest at a particular subcellular location and allowing that site to passively trap target messages that it contacts by diffusion. Such a mechanism is used to localize nanos mRNA to the posterior pole of the developing Drosophila oocyte. Degradation protection involves selective stabilization of messages at the correct location while unlocalized messages are degraded. This type of mechanism is responsible for the posterior localization of a number of maternal messages during early embryogenesis in Drosophila (Wilhelm, 2005 and references therein).
One of the most extensively characterized systems for studying mRNA localization is the Drosophila oocyte. Oocytes develop as part of an egg chamber, which is composed of the oocyte and 15 nurse cells surrounded by a layer of somatic follicle cells. The oocyte is connected to the nurse cells by a network of cytoplasmic bridges called ring canals. Various mRNAs that are required for early embryogenesis are synthesized in the nurse cells and transported into the oocyte where some, such as oskar (osk) and gurken (grk), are localized to discrete subcellular locations (Wilhelm, 2005).
While genetic and biochemical approaches have identified an ever increasing group of proteins required for mRNA localization during Drosophila oogenesis, recent work in Drosophila and other systems has revealed unexpected connections between the factors that regulate mRNA localization, mRNA stability, and translation. For instance, Cup was identified as an eIF4E binding protein that is required to both translationally repress osk mRNA as well as to recruit the localization factor Barentsz. Similarly, the RNA helicase me31B has been shown to be required for translational repression of osk and Bicaudal-D in Drosophila, while its orthologs in yeast and humans are required for mRNA degradation. Most surprisingly, staufen, which is the archetypal mRNA localization factor in Drosophila and vertebrates, has recently been shown to regulate message stability via the same components that control nonsense-mediated mRNA decay. Consistent with these factors affecting multiple aspects of mRNA metabolism, the Drosophila proteins required for localization and translational control have long been known to reside in large cytoplasmic particles that are both static and exhibit microtubule-dependent transport. Furthermore, the yeast and human orthologs of one component of these particles, the translational repressor Me31B, have also been shown to be critical for the function of processing bodies (P bodies), large cytoplasmic particles that contain many of the regulators of mRNA stability (Cougot, 2004; Sheth, 2003). These results have suggested a model (Coller, 2004) in which mRNA localization, translational regulation, and stability are integrated functions of the P body (Wilhelm, 2005).
Because early patterning events in oogenesis are intimately connected to the proper regulation of cytoplasmic mRNA processing (i.e., translation, localization, and stability), existing insertional mutants were screened for defects in patterning in order to identify novel P body components. This screen identified a Drosophila gene, trailer hitch, that is required for efficient secretion of the TGF-α family member Grk and the vitellogenin receptor Yolkless (Yl). Biochemical and genetic analysis of Tral revealed an unexpected connection between cytoplasmic mRNA processing events and trafficking through the secretory pathway (Wilhelm, 2005).
A number of findings support a direct role for tral in promoting efficient trafficking through the secretory pathway: (1) tral is required for the efficient secretion of both Grk and Yl; (2) immunohistochemistry places the Tral complex on the ER in close association with a subset of ER exit sites in nurse cells, although further analysis at the EM level will be necessary to demonstrate this conclusively; (3) Tral protein is biochemically associated with the transcripts of two ER exit site components: sar1 and sec13; (4) tral is required for normal distribution of Sar1 protein, arguing that the sar1 message is a regulatory target of tral. Together, these results suggest that tral's role in secretion is to regulate the transcripts of ER exit site components on the surface of the ER (Wilhelm, 2005).
ER exit sites have traditionally been defined as localized foci of components of the COPII complex, which is required for ER exit. However, recent work has shown that proteins actually exit from the region of the ER surrounding these foci, rather than from the foci themselves (Mironov, 2003). Because these foci are quite stable, this result has led to a model where “ER exit sites” are actually storage sites to allow a high concentration of COPII components to be available to drive local vesicle formation (Wilhelm, 2005).
This model fits quite nicely with the current results. The observations that the Tral complex borders a subset of ER exit sites and is associated with sar1 mRNA suggest that the Tral complex regulates sar1 expression on the surface of the ER. Because recruitment of Sar1 is the first step in assembling the COPII complex on the membrane, Sar1 is an excellent target for regulating the size and distribution of ER exit sites. Consistent with this, tral mutants display abnormalities in both ER exit site morphology and secretion. Thus, the properly regulated assembly of COPII foci/ER exit sites is critical for normal ER-to-Golgi trafficking (Wilhelm, 2005).
Because many components of the secretory pathway are highly conserved, one might expect tral orthologs to also play a role in secretion. Genetic screens in Drosophila and C. elegans have suggested that this family of proteins does play a conserved role in the regulation of membrane trafficking. The yeast ortholog of tral, Scd6, was identified as a high copy suppressor of a deletion of the clathrin heavy chain locus, while the worm ortholog, car-1, was identified in RNAi screens for genes that are required for cytokinesis (Nelson, 1993 and Zipperlen, 2001). While there have been no additional studies of Scd6 to determine whether it acts directly or indirectly in the secretory pathway, abnormalities in membrane trafficking to the late cleavage furrow have been found in car-1-depleted embryos. Furthermore, it has also been found that disruption of car-1 leads to ER morphology defects similar to the foci of Grk and Yl that were observe in the Drosophila oocyte. The findings that Tral is associated with the ER and is required for efficient trafficking of secreted proteins are consistent with the phenotypes described in C. elegans and suggest that the role of the trailer hitch family in regulating membrane trafficking is conserved (Wilhelm, 2005).
Given these similarities, one might have anticipated that tral mutants would also have defects in cytokinesis. However, it is likely that none of the hypomorphic alleles of tral disrupts tral function sufficiently to display a cytokinesis defect. Consistent with this interpretation, it is clear that only small amounts of Tral protein are required for some of its functions. For instance, tral1 homozygotes display no defect in Yl trafficking, but tral1 hemizygotes do, even though tral1 homozygous or hemizygous ovaries express undetectable amounts of Tral. The identification of stronger alleles of tral will likely be necessary to determine whether the role of tral in cytokinesis is conserved between Drosophila and C. elegans (Wilhelm, 2005).
One of the most unexpected results of these studies was the identification of Tral as a component of an RNA-protein complex that is required for efficient membrane trafficking. Because cup and me31B have been implicated in various aspects of mRNA localization, mRNA stability, and translational control, this finding provided an unexpected link between cytoplasmic mRNA processing and the secretory pathway. While previous studies of cup and me31B did not describe any defect in secretion, such a role would likely have been missed, since the strongest alleles of cup and me31B cause egg chamber degeneration before either Grk or Yl secretion could be examined. Because the Tral complex localizes to the ER and sar1 mRNA is associated with Tral, it is proposed that Tral promotes proper ER exit site formation by local regulation of sar1 expression on the ER (Wilhelm, 2005).
The idea of a link between ER function and cytoplasmic mRNA processing is consistent with recent work in a number of other systems. In rice endosperm, prolamine mRNA, which encodes a storage protein used to support growth of the developing embryo, is targeted to discrete domains of the endoplasmic reticulum via its 3'UTR (Choi, 2000). This targeting is believed to be necessary for the formation of a specialized protein storage body derived from the ER. In Saccharomyces cerevisiae, the mRNA for the membrane protein Ist2p is localized to the cortical ER of daughter cells and this localization event allows Ist2p to be transported to the plasma membrane independent of the classic secretory pathway (Juschke, 2004). Work on tral suggests that the connection between cytoplasmic mRNA processing and ER function may be broader than these two isolated examples and that cytoplasmic mRNA processing regulates exit from the ER in addition to its previously established role in the translocation of prolamine and Ist2p into the ER. These findings raise exciting new possibilities for how cytoplasmic mRNA processing could interface with the classical secretory pathway to promote efficient protein trafficking in the cell (Wilhelm, 2005).
Sequence analysis of tral revealed that it is a member of a highly conserved family of proteins that is present in virtually all eukaryotes (Albrecht, 2004; Anantharaman, 2004). The Trailer Hitch family of proteins contains several conserved sequence features, including an amino-terminal Sm-related domain and a carboxy-terminal FDF motif. The canonical Sm domain is a motif common to a number of proteins that regulate various aspects of RNA metabolism, including splicing and mRNA stability (Bouveret, 2000; Mayes, 1999; Tharun, 2000). Thus, the identification of a divergent Sm domain within tral suggests that it may regulate some aspect of RNA metabolism. The identification of an FDF motif in tral also supports a role for tral in posttranscriptional gene regulation. The FDF motif was originally defined as a motif conserved within the EDC3 family of proteins (Anantharaman, 2004). EDC3p in Drosophila has been shown to promote mRNA decay by enhancing removal of the 5' cap, indicating that the FDF motif is also likely to play a role in RNA recognition or stability (Kshirsagar, 2004). In addition to the sequence analysis, the newt ortholog of tral, Rap55, has been shown to be part of a cytoplasmic RNA-protein complex (Lieb, 1998). Together, these observations suggest that the Trailer Hitch family may regulate some aspect of RNA metabolism, such as stability, localization, or translation (Wilhelm, 2005).
Two conserved features of oogenesis are the accumulation of translationally quiescent mRNA, and a high rate of stage-specific apoptosis. Little is understood about the function of this cell death. In C. elegans, apoptosis occurring through a specific 'physiological' pathway normally claims about half of all developing oocytes. The frequency of this germ cell death is dramatically increased by a lack of the RNA helicase CGH-1, orthologs of which are involved in translational control in oocytes and decapping-dependent mRNA degradation in yeast processing (P) bodies. A predicted RNA-binding protein, CAR-1 (Drosophila homolog, Trailer hitch), associates with CGH-1 and Y-box proteins within a conserved germline RNA-protein (RNP) complex, and in cytoplasmic particles in the gonad and early embryo. The CGH-1/CAR-1 interaction is conserved in Drosophila oocytes. When car-1 expression is depleted by RNA interference (RNAi), physiological apoptosis is increased, brood size is modestly reduced, and early embryonic cytokinesis is abnormal. Surprisingly, if apoptosis is prevented car-1(RNAi) animals are characterized by a progressive oogenesis defect that leads rapidly to gonad failure. Elevated germ cell death similarly compensates for lack of the translational regulator CPB-3 (CPEB), orthologs of which function together with CGH-1 in diverse organisms. It is concluded that CAR-1 is of critical importance for oogenesis, that the association between CAR-1 and CGH-1 has been conserved, and that the regulation of physiological germ cell apoptosis is specifically influenced by certain functions of the CGH-1/CAR-1 RNP complex. It is proposed that this cell death pathway facilitates the formation of functional oocytes, possibly by monitoring specific cytoplasmic events during oogenesis (Boag, 2005).
The mRNA processing body (P-body) is a cellular structure that has an important role in mRNA degradation. P-bodies have also been implicated in RNAi-mediated post-transcriptional gene silencing. The objective of this study was to identify and characterize novel components of the mammalian P-body. Approximately 5% of patients with the autoimmune disease primary biliary cirrhosis have antibodies directed against this structure. Serum from one of these patients was used to identify a cDNA encoding RAP55, a 463-amino acid protein. RAP55 colocalizes P-body components DCP1a and Ge-1. RAP55 contains an N-terminal Sm-like domain and two C-terminal RGG-rich domains separated by an FDF motif. The two RGG domains and the FDF domain are necessary and sufficient to target the protein to P-bodies. A fragment of RAP55 consisting of the FDF and the second RGG domains did not localize to P-bodies, but was able to displace other P-body components from this structure. After cells were subjected to arsenite-induced stress, RAP55 was detected in TIA-containing stress granules. The second RGG domain is necessary and sufficient for stress granule localization. siRNA-mediated knock-down of RAP55 results in loss of P-bodies, suggesting that RAP55 acts prior to the 5'-decapping step in mRNA degradation. The results of this study show that RAP55 is a component of P-bodies in cells at rest and localizes in stress granules in arsenite-treated cells. RAP55 may serve to shuttle mRNAs between P-bodies and stress granules (Yang, 2006).
The Balbiani body or mitochondrial cloud is a large distinctive organelle aggregate found in developing oocytes of many species, but its presence in the mouse has been controversial. Using confocal and electron microscopy, this study reports that a Balbiani body does arise in mouse neonatal germline cysts and oocytes of primordial follicles but disperses as follicles begin to grow. The mouse Balbiani body contains a core of Golgi elements surrounded by mitochondria and associated endoplasmic reticulum. Because of their stage specificity and perinuclear rather than spherical distribution, these clustered Balbiani body mitochondria may have been missed previously. The Balbiani body also contains Trailer hitch, a widely conserved member of a protein complex that associates with endoplasmic reticulum/Golgi-like vesicles and transports specific RNAs during Drosophila oogenesis. These results provide evidence that mouse oocytes develop using molecular and developmental mechanisms widely conserved throughout the animal kingdom (Pepling, 2007).
The presence of a Balbiani body in diverse species of young oocytes suggests that it is associated with a conserved function. The strongest candidate for such a role is in the transport and localization of organelles and RNAs within oocytes. Consequently, conserved proteins known to function in these processes within oocytes of other species were examined for presence in the mouse Balbiani body. Recently, specific ribonucleoprotein (RNP) complexes have been characterized that are required to transport and localize oskar RNA, the key germinal granule component in Drosophila. One component of these complexes is Trailer hitch, which is thought to directly interact with other components of the RNP complex, including Me31B and Cup. The trailer hitch (tral) gene is highly conserved in eukaryotes with the highest homology in two regions, the Sm and FDF domains. Sm domains are found in proteins involved in RNA metabolism such as splicing. FDF domains are found in a family of proteins involved in regulation of mRNA decay (Pepling, 2007).
In addition to its role in oocytes, Trailer hitch is likely to be involved in RNA localization in other cells types and for general cellular functions such as ER exit-site formation, which is postulated to involve RNA localization. The mouse genome contains a single trailer hitch gene, but its function has not yet been characterized. However, the human Trailer hitch protein, RAP55, localizes to processing bodies (P bodies). P bodies are cytoplasmic structures involved in mRNA degradation that have been described in both yeast and human cells. siRNA knockdown of RAP55 results in the loss of P bodies and suggests that RAP55 plays a role in mRNA degradation by promoting assembly of P bodies or by delivering mRNAs to P bodies. The mammalian homologue of cup, 4E-T, also localizes to P bodies and siRNA knockdown of 4E-T results in loss of P bodies and decreases mRNA stability. In addition, the mouse 4E-T, Clast4, is localized to the cytoplasm of developing oocytes and may play a role in mRNA degradation during female germ-cell development (Pepling, 2007).
Drosophila and Mouse Tral proteins are 59% identical and 74% similar within in their N-terminal Sm-like domain. To develop a specific antibody that recognizes mouse Trailer hitch, whether an antibody generated against the Drosophila Tral Sm domain would recognize mouse Tral was investigated. By Western blot, a band with a nominal molecular weight of ~60 kDa was detected in extracts prepared from mouse ovaries and testes. This is slightly larger than the predicted size of 50 kDa suggesting posttranslational modification. An antibody generated against the human Trailer hitch protein, RAP55, was investigated. RAP55 was expressed in bacteria and found to be detected by using the Drosophila Tral antibody. Thus, the Drosophila Tral antibody recognizes mammalian Tral protein (Pepling, 2007).
Whether Tral protein is enriched in the Drosophila Balbiani body was investigated. Using immunofluorescence and confocal microscopy, it was observed that the anti-Tral antibody labels organelle clusters in late Drosophila cysts and the large anterior Balbiani body that is present in newly forming follicles. Trailer hitch protein distribution becomes perinuclear and on the nuclear envelope in the nurse cells throughout oogenesis and is localized within the oocyte to the posterior pole (Pepling, 2007).
The expression and localization of Tral during the early stages of mouse oogenesis were very similar to its expression in Drosophila ovaries. Using whole-mount immunocytochemistry in developing embryonic and neonatal gonads, Tral was not detected at 13.5 days of development. However, it is detected at 14.5 days in developing ovaries. At this time, there is a low level of Tral in all cells of ovaries, but expression appears stronger in the germ cells. Tral becomes progressively stronger in the cytoplasm of oocytes over the next several days, whereas expression in somatic cells becomes weaker. In addition, Tral is highly enriched in a circular structure in the cytoplasm reminiscent of the Golgi. To verify that Tral is localized in mouse oocytes within the Balbiani body-associated Golgi, ovaries were double-labeled with antibodies specific for GM130 and Tral. PND1 ovaries were exposed to both GM130 and Tral antibodies, and GM130 and Tral were detected in the same circular structure. Thus, the mouse Balbiani body contains Trailer hitch, a component of a conserved complex that is involved in regulating RNAs in multiple species (Pepling, 2007).
Nuage-like structures have been observed within the Balbiani body of young mouse oocytes in electron micrographs. Nuage has been best characterized during mouse development in spermatocytes and developing spermatids, where it is found in the chromatoid body. Consequently, mouse seminiferous tubules wee stained with anti-Tral antibodies and they were examined using confocal microscopy. Strong specific labeling of a perinuclear body morphologically similar to the chromatoid body was observed in pachytene spermatocytes and round spermatids. This labeling appeared similar to labeling with an antibody to Vasa and Tudor, mouse proteins previously found to be localized to the mouse chromatoid body. However, double labeling of seminiferous tubules with antibodies against these proteins showed they do not overlap. Several other nuage-containing structures have been described in male germ cells, but a cytological marker exists for only one of these, the RNF17 granule. Therefore, localization of Tral to the nuage-containing RNF17 granule was tested, but Tral protein did not label this granule either. Thus, the mouse Tral protein is not a component of the nuage-containing chromatoid body or the RNF17 granule. Tral protein may be a component of another nuage-containing body in male germ cells, but lack of cytological markers for these structures makes addressing this difficult (Pepling, 2007).
Identification of Trailer hitch as a Balbiani body constituent strongly supports the view that this structure is related to universal molecular mechanisms of RNA metabolism that may be present in most or all cells. The yeast homologue of mTral, Scd6, was identified as a high-copy suppressor of a deletion of the clathrin heavy-chain locus, suggesting it may play a role in the secretory pathway. RNAi of the C. elegans Trailer hitch homologue, CAR-1, results in increased germ cell death in hermaphrodites as well as cytokinesis defects and lethality of embryos. In Drosophila, P element insertions in tral result in female sterility. These mutants are defective in the secretion of Gurken, which is required for proper dorsal ventral patterning of the embryo. Null alleles of tral have not yet been described in Drosophila (Pepling, 2007). Previously, the Drosophila Tral protein was shown to be part of an RNP complex involved in mRNA localization and translational regulation in Drosophila oogenesis. This complex consists of at least six other proteins, including Me31B (DEAD box helicase), Orb [Cytoplasmic Polyadenylation Element Binding Protein (CPEB)], Yps (Y-box), eIF4E, cup (eIF4E binding), and Exuperentia. Complexes containing at least a subset of these proteins exist in C. elegans and Xenopus. In C. elegans, CAR-1 localizes to the P granules along with CHG-1, the Me31B homologue (Pepling, 2007).
P bodies are cytoplasmic structures involved in mRNA degradation that have been described in both yeast and human cells. In human cells, these P bodies, also called dcp1 bodies, contain dcp1 and dcp2, proteins involved in decapping RNAs as well as Sm domain-containing proteins. The P bodies also have several components in common with the Drosophila RNP complex, including Rck (Me31B homologue), CPEB, 4E-T (Cup homologue), and RAP55 (Trailer hitch homologue). Knockdown of 4E-T or RAP55 causes loss of P bodies, suggesting a role for these proteins in P body assembly and in regulating mRNA decay. In the Drosophila ovary, cup mutants also affect RNP particle assembly of the mRNA localization complex. The similarity of components in P bodies and the Drosophila mRNA localization complex suggests these are related structures. The human Trailer hitch protein, RAP55, is localized to P bodies. In addition, chromatoid bodies and P bodies also exhibit similarity in their molecular nature (Pepling, 2007).
Drosophila and Xenopus oocytes are highly polar and contain localized RNAs and other components that mediate the patterning of the early embryo. Mammalian oocytes, in contrast, are often viewed as completely symmetrical and nonpolar. Embryonic polarity is not thought to be established until implantation, although this view has been challenged. The current experiments have shown there is not a simple relationship between the presence of a Balbiani body and egg polarity. It is proposed that all oocytes that grow to a larger size than normal cells may require large amounts of the machinery used normally to move and store cytoplasmic constituents. Whether this activity actually leads to the localization of patterning RNAs or germ-cell determinants late in oogenesis may be determined simply later and may vary from species to species. Thus, both patterned and unpatterned eggs may be built using largely conserved processes of organelle and RNA metabolism (Pepling, 2007).
date revised: 20 March 2006
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