orb: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - orb

Synonyms - oo18 RNA-binding protein

Cytological map position - 94E11-13

Function - RNA binding protein

Keyword(s) - maternal - posterior group and dorsal group, regulation of translation, meiosis

Symbol - orb

FlyBase ID:FBgn0004882

Genetic map position -

Classification - RRM family

Cellular location - cytoplasmic

NCBI link: Entrez Gene

orb orthologs: Biolitmine

Recent literature
Norvell, A., Wong, J., Randolph, K. and Thompson, L. (2015). Wispy and Orb cooperate in the cytoplasmic polyadenylation of localized gurken mRNA. Dev Dyn [Epub ahead of print]. PubMed ID: 26214278
In Drosophila, the dorsal-ventral (D-V) axis of the oocyte is dependent on Gurken (Grk) protein distribution. This is achieved through the cytoplasmic localization of grk mRNA and regulation of its translation. As females carrying mutations in the gene encoding the CPEB protein Orb lay ventralized eggs due to insufficient Grk levels, it seemed likely that cytoplasmic polyadenylation of grk transcripts may play a role in their translational regulation. This study has found that grk is polyadenylated throughout oogenesis, with poly(A) tails of approximately 30-50 A residues. Hyperadenylated grk transcripts, with poly(A) tails of 50-90 As, are detected in late stage egg chambers, but they fail to accumulate in oocytes deficient in Orb or the poly(A) polymerase Wispy (Wisp). wisp females also lay weakly ventralized eggs, demonstrating that they produce inadequate amounts of Grk. Finally, unlocalized grk transcripts are also not appropriately hyperadenylated. It is concluded that localized cytoplasmic polyadenylation of grk mRNA by Wisp and Orb is necessary to achieve appropriate Grk protein accumulation in the D/A corner of the oocyte during mid to late oogenesis.

Davidson, A., Parton, R. M., Rabouille, C., Weil, T. T. and Davis, I. (2016). Localized translation of gurken/TGF-α mRNA during axis specification is controlled by access to Orb/CPEB on processing bodies. Cell Rep 14: 2451-2462. PubMed ID: 26947065
In Drosophila oocytes, gurken/TGF-α mRNA is essential for establishing the future embryonic axes. gurken remains translationally silent during transport from its point of synthesis in nurse cells to its final destination in the oocyte, where it associates with the edge of processing bodies. This study shows that, in nurse cells, gurken is kept translationally silent by the lack of sufficient Orb/CPEB, its translational activator. Processing bodies in nurse cells have a similar protein complement and ultrastructure to those in the oocyte, but they markedly less Orb and do not associate with gurken mRNA. Ectopic expression of Orb in nurse cells at levels similar to the wild-type oocyte dorso-anterior corner at mid-oogenesis is sufficient to cause gurken mRNA to associate with processing bodies and translate prematurely. It is proposed that controlling the spatial distribution of translational activators is a fundamental mechanism for regulating localized translation.
Wu, J. K., Tai, C. Y., Feng, K. L., Chen, S. L., Chen, C. C. and Chiang, A. S. (2017). Long-term memory requires sequential protein synthesis in three subsets of mushroom body output neurons in Drosophila. Sci Rep 7(1): 7112. PubMed ID: 28769066
Creating long-term memory (LTM) requires new protein synthesis to stabilize learning-induced synaptic changes in the brain. In the fruit fly, Drosophila melanogaster, aversive olfactory learning forms several phases of labile memory to associate an odor with coincident punishment in the mushroom body (MB). It remains unclear how the brain consolidates early labile memory into LTM. This study surveyed 183 Gal4 lines containing almost all 21 distinct types of MB output neurons (MBONs) and showed that sequential synthesis of learning-induced proteins occurs at three types of MBONs. Downregulation of oo18 RNA-binding proteins (ORBs) in any of these MBONs impaired LTM. And, neurotransmission outputs from these MBONs are all required during LTM retrieval. Together, these results suggest an LTM consolidation model in which transient neural activities of early labile memory in the MB are consolidated into stable LTM at a few postsynaptic MBONs through sequential ORB-regulated local protein synthesis.
Ote, M. and Yamamoto, D. (2018). The Wolbachia protein TomO interacts with a host RNA to induce polarization defects in Drosophila oocytes. Arch Insect Biochem Physiol: e21475. Pubmed ID: 29851149
Wolbachia is an endosymbiont prevalent in arthropods. To maximize its transmission thorough the female germline, Wolbachia induces in infected hosts male-to-female transformation, male killing, parthenogenesis, and cytoplasmic incompatibility, depending on the host species and Wolbachia strain involved. However, the molecular mechanisms underlying these host manipulations by Wolbachia remain largely unknown. The Wolbachia strain wMel, an inhabitant of Drosophila melanogaster, impairs host oogenesis only when transplanted into a heterologous host, for example, Drosophila simulans. Egg polarity defects induced by wMel infection in D. simulans can be recapitulated in the natural host D. melanogaster by transgenic overexpression of a variant of the Wolbachia protein Toxic manipulator of oogenesis (TomO), TomOwMel(HS) , in the female germline. RNA immunoprecipitation assays demonstrated that TomO physically associates with orb mRNA, which, as a result, fails to interact with the translation repressor Cup. This leads to precocious translation of Orb, a posterior determinant, and thereby to the misspecification of oocytes and accompanying polarity defects. It is proposed that the ability of TomO to bind to orb mRNA might provide a means for Wolbachia to enter the oocyte located at the posterior end of the egg chamber, thereby accomplishing secure maternal transmission thorough the female germline.
Barr, J., Charania, S., Gilmutdinov, R., Yakovlev, K., Shidlovskii, Y. and Schedl, P. (2019). The CPEB translational regulator, Orb, functions together with Par proteins to polarize the Drosophila oocyte. PLoS Genet 15(3): e1008012. PubMed ID: 30865627
orb is a founding member of the CPEB family of translational regulators and is required at multiple steps during Drosophila oogenesis. Previous studies showed that orb is required during mid-oogenesis for the translation of the posterior/germline determinant oskar mRNA and the dorsal-ventral determinant gurken mRNA. This study reports that orb also functions upstream of these axes determinants in the polarization of the microtubule network (MT). Prior to oskar and gurken translational activation, the oocyte MT network is repolarized. The MT organizing center at the oocyte posterior is disassembled, and a new MT network is established at the oocyte anterior. Repolarization depends upon cross-regulatory interactions between anterior (apical) and posterior (basal) Par proteins. Repolarization of the oocyte also requires orb and orb is needed for the proper functioning of the Par proteins. orb interacts genetically with aPKC and cdc42 and in egg chambers compromised for orb activity, Par-1 and aPKC protein and aPKC mRNA are mislocalized. Moreover, like cdc42-, the defects in Par protein localization appear to be connected to abnormalities in the cortical actin cytoskeleton. These abnormalities also disrupt the localization of the spectraplakin Shot and the microtubule minus-end binding protein Patronin. These two proteins play a critical role in the repolarization of the MT network.
Barr, J., Gilmutdinov, R., Wang, L., Shidlovskii, Y. and Schedl, P. (2019). The Drosophila CPEB protein Orb specifies oocyte fate by a 3'UTR dependent autoregulatory loop. Genetics. PubMed ID: 31594794
orb encodes one of the two fly CPEB proteins. These widely conserved proteins bind to the 3'UTRs of target mRNAs and activate or repress their translation. This study shows that a positive autoregulatory loop driven by the orb gene propels the specification of oocyte identity in Drosophila egg chambers. Oocyte fate specification is mediated by a 3'UTR dependent mechanism that concentrates orb mRNAs and proteins in one of the two pro-oocytes in the 16 cell germline cyst. When the orb 3'UTR is deleted, orb mRNA and protein fail to localize and all sixteen cells become nurse cells. In WT, the oocyte is specified when orb and other gene products concentrate in a single cell in region 2b of the germarium. A partially functional orb 3'UTR replacement delays oocyte specification until the egg chambers reach stage 2 of oogenesis. Prior to this point, orb mRNA and protein are unlocalized, as are other markers of oocyte identity, and the oocyte is not specified. After stage 2, approximately 50% of the chambers successfully localize orb in a single cell, and this cell assumes oocyte identity. In the remaining chambers, the orb autoregulatory loop is not activated and no oocyte is formed. Finally, maintenance of oocyte identity requires continuous orb activity.
Hervas, R., Del Carmen Fernandez-Ramirez, M., Galera-Prat, A., Suzuki, M., Nagai, Y., Bruix, M., Menendez, M., Laurents, D. V. and Carrion-Vazquez, M. (2021). Divergent CPEB prion-like domains reveal different assembly mechanisms for a generic amyloid-like fold. BMC Biol 19(1): 43. PubMed ID: 33706787
Amyloids are ordered, insoluble protein aggregates, characterized by a cross-β sheet quaternary structure in which molecules in a β-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. This study describes the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compares them to those of the Drosophila ortholog, Orb2 PLD. Using in vitro single-molecule and bulk biophysical methods, this study found transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. These results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.
Fang, J. and Lerit, D. A. (2022). Orb-dependent polyadenylation contributes to PLP expression and centrosome scaffold assembly. Development 149(13). PubMed ID: 35661190
As the microtubule-organizing centers of most cells, centrosomes engineer the bipolar mitotic spindle required for error-free mitosis. Drosophila Pericentrin-like protein (PLP) directs formation of a pericentriolar material (PCM) scaffold required for PCM organization and microtubule-organizing center function. This study investigated the post-transcriptional regulation of Plp mRNA. Conserved binding sites were identified for cytoplasmic polyadenylation element binding (CPEB) proteins within the Plp 3'-untranslated region and examine the role of the CPEB ortholog Oo18 RNA-binding protein (Orb) in Plp mRNA regulation. The data show that Orb interacts biochemically with Plp mRNA to promote polyadenylation and PLP protein expression. Loss of orb, but not orb2, diminishes PLP levels in embryonic extracts. Consequently, PLP localization to centrosomes and its function in PCM scaffolding are compromised in orb mutant embryos, resulting in genomic instability and embryonic lethality. Moreover, this study found that PLP overexpression restores centrosome scaffolding and rescues the cell division defects caused by orb depletion. These data suggest that Orb modulates PLP expression at the level of Plp mRNA polyadenylation and demonstrates that the post-transcriptional regulation of core, conserved centrosomal mRNAs is crucial for centrosome function.
Fang, J. and Lerit, D. A. (2022). Orb-dependent polyadenylation contributes to PLP expression and centrosome scaffold assembly. Development 149(13). PubMed ID: 35661190
As the microtubule-organizing centers of most cells, centrosomes engineer the bipolar mitotic spindle required for error-free mitosis. Drosophila Pericentrin-like protein (PLP) directs formation of a pericentriolar material (PCM) scaffold required for PCM organization and microtubule-organizing center function. This study investigated the post-transcriptional regulation of Plp mRNA. Conserved binding sites were identified for cytoplasmic polyadenylation element binding (CPEB) proteins within the Plp 3'-untranslated region and examine the role of the CPEB ortholog Oo18 RNA-binding protein (Orb) in Plp mRNA regulation. The data show that Orb interacts biochemically with Plp mRNA to promote polyadenylation and PLP protein expression. Loss of orb, but not orb2, diminishes PLP levels in embryonic extracts. Consequently, PLP localization to centrosomes and its function in PCM scaffolding are compromised in orb mutant embryos, resulting in genomic instability and embryonic lethality. Moreover, this study found that PLP overexpression restores centrosome scaffolding and rescues the cell division defects caused by orb depletion. These data suggest that Orb modulates PLP expression at the level of Plp mRNA polyadenylation and demonstrates that the post-transcriptional regulation of core, conserved centrosomal mRNAs is crucial for centrosome function.
Kozlov, E. N., Deev, R. V., Tokmatcheva, E. V., Tvorogova, A., Kachaev, Z. M., Gilmutdinov, R. A., Zhukova, M., Savvateeva-Popova, E. V., Schedl, P. and Shidlovskii, Y. V. (2023). 3'UTR of mRNA Encoding CPEB Protein Orb2 Plays an Essential Role in Intracellular Transport in Neurons. Cells 12(13). PubMed ID: 37443751
Intracellular trafficking plays a critical role in the functioning of highly polarized cells, such as neurons. Transport of mRNAs, proteins, and other molecules to synaptic terminals maintains contact between neurons and ensures the transmission of nerve impulses. Cytoplasmic polyadenylation element binding (CPEB) proteins play an essential role in long-term memory (LTM) formation by regulating local translation in synapses. This study shows that the 3'UTR of the Drosophila CPEB gene orb2 is required for targeting the orb2 mRNA and protein to synapses and that this localization is important for LTM formation. When the orb2 3'UTR is deleted, the orb2 mRNAs and proteins fail to localize in synaptic fractions, and pronounced LTM deficits arise.WYhe phenotypic effects of the orb2 3'UTR deletion were rescued by introducing the 3'UTR from the orb, another Drosophila CPEB gene. In contrast, the phenotypic effects of the 3'UTR deletion were not rescued by the 3'UTR from one of the Drosophila α-tubulin genes. These results show that the orb2 mRNAs must be targeted to the correct locations in neurons and that proper targeting depends upon sequences in the 3'UTR.

The contents of the Drosophila egg form a highly organized ensemble of protein systems, ready to abandon the quietude of the oocyte for the actively developing zygote once fertilization has occurred. Four maternal systems exist in the pre-fertilized egg. They will eventually determine the anterior-posterior axis and the dorsoventral axis in the developing embryo. The gene orb is involved in two of these four systems and the processes they govern.

orb is considered a gene of both the posterior group and the dorsal group. It functions in the transport of mRNAs coding for two essential proteins that govern polarity in the egg: Oskar (anterior/posterior) and Gurken (dorsal/ventral). ORB protein contains two RNA recognition motifs characteristic of proteins that bind the 3' messenger RNA. The correct distribution of both Oskar and Gurken mRNAs is dependent on ORB during the middle phase of oogenesis, stages 8-10. Oskar and Gurken are crucial to appropriate anterior-posterior and dorso-ventral patterning. Osk protein recruits other posterior group gene products involved in the formation of pole plasm and in the localization and regulation of the posterior determinant, Nanos. During oogenesis ORB protein is enriched in the cortical region that underlies the oocyte plasma membrane, especially in the anterior dorsal region and at the posterior pole of the oocyte. Gurken mRNA is usually localized to the anterior dorsal region of the egg during this period, while Oskar mRNA is localized to the posterior pole. orb mutation interfers with both localization processes. Gurken acts both early and late in the oocyte assmblage process. Consequently, since orb is involved in both phases of Gurken mRNA localization, orb also acts both early and late in oocyte assembly (Christerson, 1994).

Two other genes, cappuccino and spire, are also required for the proper determination of both axes. There is little doubt that proper RNA localization requires a large protein complex to shuttle transcripts to specific sites. In the egg, RNA localization represents a macrocosm of similar phenomena that must occur in normal cells in the process of determining cell polarity and alternative fates for cells during mitosis. The fly egg provides developmental biology a peek at these phenomena. It remains to be seen if the same processes function during normal cell division to generate intracellular mRNA asymmetries, comparable to the localizations occurring in the egg.

The critical step in the targeting of posterior determinants during the establishment oocyte polarity axes is the localization of Oskar (OSK) mRNA to the pole and its on-site translation. The role of the Drosophila CPEB homolog, the orb gene, in the OSK mRNA localization pathway has been analyzed. The expression of Osk protein is dependent upon the orb gene. In strong orb mutants, Osk protein expression is undetectable, while in the hypomorphic mutant, orbmel, little or no on-site expression of Osk protein at the posterior pole is observed. The defects in Osk protein accumulation in orb mutant ovaries are correlated with a reduction in the length of the OSK poly(A) tails. OSK mRNA is found in immunoprecipitable complexes with Orb protein in ovaries. The OSK 3' UTR can be UV cross-linked to Orb protein in ovarian extracts. These data suggest that Orb is required to activate the translation of OSK mRNA and that this may be accomplished by a mechanism similar to that used by the Xenopus CPEB protein to control translation of 'masked' mRNAs (Chang, 1999).

What role does Orb protein play in the translation of OSK mRNA localized at the posterior pole in vitellogenic oocytes? One model that could potentially account for the current findings posits that translationally masked OSK mRNA would be deposited from the nurse cells into the anterior of the oocyte and then transported to the posterior pole by the Stauffen protein. Good candidates for ensuring that OSK mRNA is not translated in the nurse cells or at the anterior of the oocyte would be the Bruno protein and p50 protein. When the masked OSK mRNA arrives at the posterior pole, it would be bound by an Orb protein complex that is already anchored in the posterior cytoskeleton. Orb protein would then activate OSK mRNA translation, most probably by promoting polyadenylation (Chang, 1999 and references therein).

Orb plays an early role during oogenesis in controlling entry into meiosis. The oocyte is the only cell in Drosophila that goes through meiosis with meiotic recombination, but several germ cells in a 16-cell cyst enter meiosis and form synaptonemal complexes (SC) before one cell is selected to become the oocyte. Using an antibody that recognises a component of the SC or the synapsed chromosomes, an analysis was carried out of how meiosis becomes restricted to one cell, in relation to the other events in oocyte determination. Although Bicaudal-D and egalitarian mutants both cause the development of cysts with no oocyte, they have opposite effects on the behavior of the SC: none of the cells in the cyst form SC in BicD null mutants, whereas all of the cells do in egl and orb mutants. Furthermore, unlike all cytoplasmic markers for the oocyte, the SC still becomes restricted to one cell when the microtubules are depolymerised, even though the BicD/Egl complex is not localised. These results have lead to the proposal of a model in which BicD, Egl and Orb control entry into meiosis by regulating translation (Huynh, 2000).

In the course of a study on the role of inscuteable (insc) during oogenesis, it has been found that an anti Insc antibody recognizes a nuclear structure that is present in some of the germ cells in regions 2a to 3 of the germarium. However, this staining does not disappear in germline clones of protein null allele insc 22, indicating that it is due to a cross-reaction of the antibody. Nevertheless, the staining pattern is very reminiscent of that expected for a component of the synaptonemal complex (SC), and therefore the staining was analyzed further, since this would be the first marker identified for the SC structure in Drosophila. Several lines of evidence indicate that the antiserum does indeed label the SC or a component associated with its formation. (1) The nuclear staining colocalizes with DNA, and has a morphology that corresponds exactly with the observed behaviour of the SC in electron micrographs. The staining is dotty in very early region 2a when the SC starts to form, becomes more thin and thread-like when the chromosomes are fully synapsed, and then becomes more compact in region 3, when the meiotic chromosomes condense to form the karyosome. (2) This structure first appears at the stage when the cysts enter into meiosis. The mitotic cysts in region 1 of the germarium express Bam protein, but this disappears after the final division when the cysts move from region 1 to region 2a of the germarium. The nuclear staining is only detectable in cysts that no longer show any Bam expression, indicating that it labels a postmitotic structure. (3) The spatial distribution of the signal within the cyst precisely follows that described for the SC at the EM level. The signal first appears in two cells in early region 2a and spreads to four cells per cyst in the middle of 2a, before it is restricted to two cells, and finally to one cell in region 2b. Ovaries from females that are mutant for C(3)G were examined, since these are the only characterized mutants that completely abolish the formation of the SC at the electron microscope level. C(3)G encodes the fly homolog of yeast Zip1 and mammalian SCP1, components of the transverse filament of the SC, and the effects of the C(3)G mutation on the SC are therefore likely to be direct (Szauter and Hawley, personal communication to Huynh, 2000). The nuclear structure stained by this antibody is absent in C(3)G mutant cysts, even though the localization of Orb protein to the oocyte occurs normally. Thus, the antibody acts as a marker for the formation of the SC, although the molecular nature of the epitope recognized is not known (Huynh, 2000).

A detailed analysis of the behavior of the SC in comparison to that of cytoplasmic markers for oocyte determination, such as Orb and Bic-D proteins, reveals a number of distinct steps in the restriction of oocyte fate to one cell. The SC first appears in early region 2a cysts in the nuclei of two cells, which are presumably the pro-oocytes. The punctate appearance of the SC suggests that they are at the zygotene stage of meiotic prophase 1. The next one or two cysts per germarium have four cells in synapsis. Two of these cells have four ring canals (the pro-oocytes) and contain an almost continuous SC, typical of the pachytene stage, while the two cells on either side, presumably the cells with three ring canals, contain a zygotene-like SC. In the middle of region 2a, the SC disappears from the two cells with three ring canals, but the two pro-oocytes still have complete SCs, and accumulate Orb and Bic-D proteins. Soon afterwards, Orb and Bic-D become concentrated in only one of these cells, providing the first sign that this pro-oocyte has been selected to become the oocyte. However, the SC still appears identical in both pro-oocytes at this stage. The SC disappears from one pro-oocyte as the cyst enters region 2b, and the cell that remains in meiosis is always the one that has already accumulated Orb or Bic-D. Finally, SC becomes more compact in region 3 and a hole forms in its middle, before it disappears soon after the cyst leaves the germarium. This comparison of the behavior of nuclear and cytoplasmic markers for the oocyte reveals two important features about how oocyte fate becomes restricted to one cell. (1) The two pro-oocytes are already different from the other 14 cells in the cyst in early region 2a, as they both start to form SC at this stage. BicD and Orb only accumulate in these cells in mid 2a, about two cysts further down the germarium. (2) Orb and Bic-D become restricted to the oocyte before any sign of oocyte identity can be deduced from the behavior of the SC (Huynh, 2000).

A cyst can progress through the normal pattern of SC localization to one cell in the presence of high concentrations of colcemid, suggesting restriction of SC to one cell is not mediated by microtubules. Unlike the microtubules, BicD, orb and egl mutations disrupt all steps in the restriction of the SC to one cell, and this leads to two important conclusions: (1) BicD and Egl must have a function that is independent of microtubules, even though they are required for the establishment or maintenance of the MTOC in the oocyte; (2) this function of BicD, Egl and Orb does not depend on their own localisation to the oocyte, since all three proteins are completely delocalized after colcemid treatments, yet the SC still becomes restricted to one cell. Although both BicD and egl mutations give rise to cysts in which all 16 cells appear identical, they have different effects on the behavior of the SC itself. In BicD null germline clones, none of the cells form a detectable SC, whereas all cells reach the full pachytene stage in egl mutants (Huynh, 2000).

BicD and Egl are part of the same protein complex, and it is therefore surprising that they have opposite phenotypes. It is suggested that BicD and Egl may have different functions. BicD is required to enhance SC formation in the cells that normally enter meiosis, whereas Egl functions to repress SC formation in the other cells of the cyst. The strongest mutations in orb have a very similar effect on SC formation as do egl mutants, suggesting that Orb protein is also involved in this repression. Given the colocalization of Orb with Egl and BicD, it will be interesting to determine whether it is part of the same protein complex (Huynh, 2000).

The discovery that the restriction of SC to one cell requires neither microtubules nor the localization of BicD, Egl and Orb raises the question of how this asymmetry arises. It has previously been suggested that BicD and Egl function in the transport of meiosis promoting factors and oocyte determinants from the future nurse cells into the oocyte. Although this could still be the case if this transport occurs either very early in region 2a or along some non-microtubule cytoskeletal network, such as actin, this model cannot easily explain why BicD and egl mutations have opposite effects on SC formation. An alternative model is preferred in which BicD, Egl and Orb are required to interpret a pre-existing asymmetry that is set up in region 1 (Huynh, 2000 and references therein).

The divisions that give rise to the cyst are asymmetric with respect to the fusome, and recent data strongly suggest that this structure, or some factor associated with it, somehow marks the future oocyte. If this is correct, this unidentified mark could regulate the BicD/Egl complex, so that it performs different functions in the different cells of the cyst. For example, the Egl-dependent activity of the complex could repress SC in the cells that do not inherit the factor, and the BicD-dependent activity could enhance its formation in the cells that do, thereby explaining the different phenotypes of the null mutations in the two genes. It is interesting to note that BicD protein is phosphorylated, and that mutations that disrupt this phosphorylation give rise to egg chambers with 16 nurse cells. Thus, this post-translational modification could be responsible for the spatial regulation of the activity of the BicD/Egl complex (Huynh, 2000).

Although these results suggest that BicD and Egl have functions that are independent of the microtubules, the nature of this activity is unclear. However, a number of lines of evidence suggest that these proteins may be involved in translational control. (1) BicD was originally identified because two single amino acid changes in the gene produce a dominant bicaudal phenotype in which Oskar mRNA is mis-expressed at the anterior of the oocyte. Since Oskar translation is normally repressed unless the RNA is localized to the posterior pole, these mutant BicD proteins must not only trap Oskar RNA at the anterior, but also relieve translational repression. Mutations in egl suppress the BicD gain-of-function phenotype, while extra copies of egl enhance it, indicating that the ectopic translation of Oskar mRNA requires the formation of the BicD/Egl complex. The second argument for a role of BicD and Egl in translational control comes from the discovery that orb null mutations give a very similar phenotype to egl mutants. Orb protein, which contains two RNA-binding motifs, has recently been shown to associate with the 3'UTR of Oskar mRNA, and is required for its efficient translation. Similarly, the Xenopus Orb homolog, CPEB, binds to elements in the 3'UTRs of a number of mRNAs, and induces the polyadenylation and translational activation of these mRNAs during oocyte maturation. Furthermore, the Spisula solidissima (clam) homolog plays a role not only in translational activation, but also in repression, since it binds to masking elements in the 3'UTRs of cyclin mRNAs to prevent their translation before fertilization. Thus, Orb functions as a regulator of translation, and can act as both a repressor and an activator in other species. This raises the possibility that the BicD/Egl complex exerts different effects in the cells of the cyst by controlling the inhibitory and activating functions of Orb. For example, Orb could repress the translation of factors required for SC formation in the future nurse cells, and activate their translation in the pro-oocytes and oocyte. If this model is correct, the selection of the oocyte would occur by a similar mechanism to the other asymmetries that are generated later in oogenesis, which are also all based on the translational regulation of asymmetrically localized mRNAs, such as Bicoid, Gurken and Oskar (Huynh, 2000 and references therein).

The behavior of the SC indicates that the determination of the oocyte occurs in two steps. The two pro-oocytes must have been selected by early region 2a, because they already behave differently from the other 14 cells of the cyst at this stage, but the development of the cyst remains symmetric until the end of 2a, when BicD and Orb disappear from the losing pro-oocyte. It has been proposed that the choice between the two pro-oocytes could depend on competition between these cells as they progress through meiosis, with the cell that is more advanced becoming the oocyte and then inhibiting its neighbor. However, the results presented here argue against this model: (1) cytoplasmic factors, such as BicD and Orb, are concentrated in one cell before there is any visible difference between the SCs in the two pro-oocytes; (2) the cytoplasmic aspects of oocyte determination occur normally in C(3)G mutants, which completely lack the SC, and in meiW68 mutants, which fail to initiate meiotic recombination. Thus, any competition between these two cells must be independent of SC formation and recombination (Huynh, 2000 and references therein).

Although meiosis is not required for oocyte determination, it can clearly influence this process, as demonstrated by the results on the spn genes. Several lines of evidence indicate that mutations in spnB, C and D disrupt the repair of dsDNA breaks during meiotic recombination, activating a checkpoint pathway that inhibits Gurken mRNA translation and the formation of the karyosome. The results presented here strongly suggest that this checkpoint also inhibits the determination of the oocyte, since the SC becomes restricted to one cell much later than in wild type in these mutants. This phenotype also allows the time when recombination occurs to be narrowed down. This process cannot begin until the SC forms in early region 2a, but the double-strand DNA breaks have to be repaired before the two cells with three ring canals exit meiosis, since this stage is delayed in spnC mutants, indicating that the checkpoint pathway has already been activated (Huynh, 2000).

Activation of the meiotic checkpoint causes a change in the mobility of Vasa protein, leading to the suggestion that the patterning defects seen in spn mutants result from the inhibition of Vasa by this pathway. The results presented here show that the SC becomes restricted to one cell at the normal time in most vasa mutant cysts. Thus, the delay in oocyte determination in spn mutants cannot be a consequence of the inhibition of Vasa, suggesting that the checkpoint pathway has additional targets that control oocyte selection (Huynh, 2000).

One problem in the study of cyst development in region 2 has been the difficulty in ordering the various developmental events that occur in this region. Using this marker for the SC, the behavior of this structure relative to the localization of cytoplasmic factors like Orb and BicD could be followed, and these could be correlated with the data from EM studies on the behavior of the SC, and the centrioles. On the basis of this comparison, a number of distinct stages in the restriction of oocyte fate to one cell can be distinguished: (1) The first cyst in region 2a shows no sign of SC, but Bam protein has already disappeared. (2) The two pro-oocytes reach the zygotene stage of meiosis in early region 2a, and start to form SC. (3) Soon afterwards, the two cells with three ring canals also form SC. The SC in the pro-oocytes has reached its maximum length, indicating that they have reached the pachytene stage. The dsDNA breaks generated during recombination must have already been repaired, since the meiotic checkpoint can arrest the pattern of SC staining at this stage. EM data also suggest that intracellular transport begins at this point, since the first signs of the migration of the centrioles towards the pro-oocytes can be seen when the two cells with three ring canals are in meiosis, and this may correlate with the first appearance of a focus of microtubules in the cyst in the middle of region 2a. (4) The SC disappears from the two cells with three ring canals in the middle of region 2a, but the two pro-oocytes still have complete SCs. Orb and Bic-D start to accumulate in the pro-oocytes at this stage. The centrioles have migrated to either side of the largest ring canal, which connects the two pro-oocytes, and the first signs of 'nutrient streaming' appear, since elongated mitochondria can be seen inside the ring canals in electron micrographs. (5) All of the steps in cyst development so far are symmetric, relative the largest ring canal, and the first asymmetry becomes evident in cysts numbers 5 and 6, when Orb and Bic-D become concentrated in one cell. The centrioles also start to move into the oocyte, and the largest ring canal is presumably open, because mitochondria can now be seen inside it. However, both pro-oocytes still contain an identical intact SC at this stage. (6) As the cyst enters region 2b, one pro-oocyte loses its SC and reverts to the nurse cell pathway of development. The pro-oocyte that remains in meiosis and becomes the oocyte is always the cell that has already accumulated Orb and Bic-D. The cytoplasm of the oocyte now contains all of the centrioles, BicD and Orb proteins, and an obvious MTOC, which nucleates microtubules that extend into the other 15 cells of the cyst. Thus, both the nucleus and cytoplasm of the oocyte are clearly different from the other cells of the cyst by this stage. Immediately afterwards, the oocyte starts to behave differently from the other cells in the cyst, as it moves to the posterior during the transition between region 2b and region 3. At the same time, the karyosome forms, and the SC becomes more compact, before disappearing soon after the cyst leaves the germarium (Huynh, 2000).

Rasputin functions as a positive regulator of orb in Drosophila oogenesis

The determination of cell fate and the establishment of polarity axes during Drosophila oogenesis depend upon pathways that localize mRNAs within the egg chamber and control their on-site translation. One factor that plays a central role in regulating on-site translation of mRNAs is Orb. Orb is a founding member of the conserved CPEB family of RNA-binding proteins. These proteins bind to target sequences in 3' UTRs and regulate mRNA translation by modulating poly(A) tail length. In addition to controlling the translation of axis-determining mRNAs like grk, fs(1)K10, and osk, Orb protein autoregulates its own synthesis by binding to orb mRNA and activating its translation. Previous studies have shown that Rasputin (Rin), the Drosophila homologue of Ras-GAP SH3 Binding Protein (G3BP), associates with Orb in a messenger ribonucleoprotein (mRNP) complex. Rin is an evolutionarily conserved RNA-binding protein believed to function as a link between Ras signaling and RNA metabolism. This study shows that Orb and Rin form a complex in the female germline. Characterization of a new rin allele shows that rin is essential for oogenesis. Co-localization studies suggest that Orb and Rin form a complex in the oocyte at different stages of oogenesis. This is supported by genetic and biochemical analyses showing that rin functions as a positive regulator in the orb autoregulatory pathway by increasing Orb protein expression. Tandem Mass Spectrometry analysis shows that several canonical stress granule proteins are associated with the Orb-Rin complex suggesting that a conserved mRNP complex regulates localized translation during oogenesis in Drosophila (Costa, 2013).

Previous studies have shown that orb autoregulation promotes localized accumulation of Orb protein in subcellular compartments where its activity is required. To investigate the mechanisms underlying orb autoregulation this study attempted to identify proteins that physically associate with Orb and thus potentially help regulate its expression and/or activity. It has been previously shown that the Drosophila Fragile-X protein (dFMR1) is found in complexes with Orb in Drosophila ovaries and functions to negatively regulate orb accumulation and activity. This study shows that Rin, the Drosophila G3BP homologue, is also associated with Orb in ovaries. However, in contrast to dFMR1, Rin functions as a positive regulatory factor, helping to promote Orb accumulation and activity (Costa, 2013).

The results show that Rin associates with Orb as part of an RNase-resistant complex. The RNase-resistance aspect of this interaction suggests that their association is mediated by protein-protein interactions rather than, or in addition to, binding to the same mRNA species. While it is possible that Orb and Rin interact directly with each other, an equally plausible scenario is that their association is mediated by one or more proteins found in both Orb and Rin immunoprecipitates. For example, mammalian G3BP has been shown to interact directly with Caprin-1, and the fly Caprin protein is found in both Rin and Orb immunoprecipitates. Two other findings would also seem to argue in favor of an indirect, rather than a direct interaction. First the overlap between Rin and Orb, especially in vitellogenic chambers is quite limited. Second, only a small subset of the proteins associated with Rin or Orb are common to both. It is also possible that the initial association between Orb and Rin could depend upon binding to the same target mRNAs and their subsequent interaction could depend upon a short stretch of RNA that is hidden in the complex and protected from RNase activity. In this case, the limited colocalization observed in egg chambers would imply that only a subset of their mRNA targets are in common (Costa, 2013).

GB3Ps in mammals are thought to have two functions. The first is repressing the translation of target mRNAs by mechanisms that depend upon their helicase and RNase activities, while the second is in the assembly of stress granules under conditions of environmental stress such as heat shock or drug treatment. For this reason, it is anticipated that rin, like dfmr1, would function to negatively regulate orb activity and/or expression. However, exactly the opposite result was observed. Instead of suppressing the DV polarity defects in eggs from dominant negative orb, orb mutant (Hd19G orb343/+) females, reducing rin activity increases the frequency of DV defects. Conversely, DV polarity defects are suppressed by providing excess rin activity. While it is clear from the phenotypic effects seen in mutants that rin has multiple functions in oogenesis (and these will be independent of orb), the most plausible explanation for the effects of decreasing and increasing rin activity on DV polarity in Hd19G orb343/+ females is that they arise because of changes in the expression/accumulation of Orb protein. Consistent with this explanation, it was found that the level of Orb protein can be changed by manipulating rin activity. If orb is wild type, reducing the rin dose by half has little if any effect on Orb protein accumulation. However, when orb activity is compromised by the Hd19G orb343/+ combination, heterozygosity for rin results in Orb protein levels that are less than 5% that of wild type. Conversely, increasing rin activity in a wild type background elevates Orb protein levels almost two-fold over wild type, while in background compromised by the Hd19G orb343/+ combination adding extra rin restores Orb protein levels to that of wild type. Taken together, these findings argue that rin functions as a positive regulator of orb. At this point it is not clear how rin might control the accumulation of Orb protein. Since Rin and Orb are associated with each other, the simplest model is that Rin helps activate the translation of orb mRNA and thus functions as a co-factor in orb autoregulation. The substantial reduction in Orb protein levels evident in rin3 ovaries is consistent with this idea. This view would also be supported by the DV polarity defects evident in eggs laid by rin mutant females. However, other less direct models (e.g., rin represses the translation of some factor that inhibits orb mRNA translation or rin is required to stabilize Orb protein) can't be excluded at this time. Likewise, rin may have other targets besides orb in the establishment of DV polarity (Costa, 2013).

Potentially arguing in favor of a role for Rin in the translation of orb mRNA and/or in the activity of Orb protein is the fact that mutations in genes encoding several of the other proteins found in both Orb and Rin immunoprecipitates also show genetic interactions with orb. Thus, dfmr1 and parp suppress the DV polarity defects in eggs laid by Hd19G orb343/+ females, while parp and lig enhance the polarity defects. Previous studies have shown that dfmr1 also exerts its effects, at least in part, by altering the expression of Orb protein. Further connecting the effects of rin and dfmr1 to translation, it was also found that Orb, dFMR1 and Rin fractionate with polysomes in sucrose gradients. If the proteins detected in Orb and Rin immunoprecipitates are part of the same complex rather than different complexes, then the gene dose effects that were observed would suggest that they act coordinately to regulate Orb expression and that the relative balance between positive (e.g., Rin) and negative (e.g., dFMR1) factors in the egg chamber helps set the level of Orb accumulation. Further studies will be required to understand precisely how rin influences Orb protein accumulation and how some of the other factors associated with Rin and Orb like dFMR1, Lig and PABP function in this process (Costa, 2013).

Although these studies implicate rin as a positive regulator of orb, this is clearly not the only role for rin in the ovary. Instead, the phenotypic effects of rin mutations point to a potentially diverse array of functions, not only in gem cells but also in the surrounding somatic follicle cells. For example, the fragmentation of ring canals, the failure to properly disperse the endoreplicated nurse cell chromosomes, and the dumpless phenotype are not observed in orb mutants. Moreover, ring canal and chromatin dispersal defects suggest that rin has functions in nurse cells, which is a compartment that has only little Orb protein. Likewise, the encapsulation defects could be of somatic origin where rin, but not orb is expressed. Further studies will be required to identify the rin regulatory targets in these and potentially other processes (Costa, 2013).

The cytoplasmic polyadenylation element binding protein (CPEB), Orb, is important for dendrite development and neuron fate specification in Drosophila melanogaster

Cytoplasmic polyadenylation element binding proteins (CPEBs) are widely conserved proteins that regulate the length of poly(A) tails in the cytoplasm, regulate translation, and regulate mRNA transport. While CPEBs are best known for regulating maternal messages in oocytes, CPEBs also have roles in many other cell types including neurons. This study extends the knowledge of the roles of CPEBs in neurons by showing that the Drosophila CPEB-encoding gene, orb, is required for proper dendrite development in larval sensory dendritic arborization neurons. Furthermore, evidence is provided that orb is important for neuron cell fate specification (Olesnicky, 2020).


orb has two transcripts, each initiating from a different promoter. The ovarian and early embryonic transcript is 4.5 kb in length and contains four exons. The male specific transcript is 3.2 kb in length and consists of three exons. The second and third exon of the male specific transcript correspond to the third and fourth exons of the ovarian transcript, except that the transcription of the male third exon terminates early (Lantz, 1992).

cDNA clone length - 4737 for ovarian

Bases in 5' UTR -782 for ovarian; 318 for testicular

Exons (ovarian) - four

Exons (testicular) - three

Bases in 3' UTR - 1104 for ovarian; 216 for testicular


Amino Acids - 915

Structural Domains

The predicted proteins contain two regions with similarity to the RRM family of RNA-binding proteins. In the N-terminal portion of the protein there is a glycine/ulanine rich region of about 100 amino acids located between two opa repeats (Lantz, 1992).

Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 15 December 2023

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