Gene name - exuperantia
Cytological map position - 57A8--10
Function - mRNA localization
Keywords - regulation of Bicoid mRNA localization, anterior class of maternal-effect segmentation genes
Symbol - exu
FlyBase ID: FBgn0000615
Genetic map position - 2-93
Classification - novel
Cellular location - cytoplasmic
|Lazzaretti, D., Veith, K., Kramer, K., Basquin, C., Urlaub, H., Irion, U. and Bono, F. (2016). The bicoid mRNA localization factor Exuperantia is an RNA-binding pseudonuclease. Nat Struct Mol Biol [Epub ahead of print]. PubMed ID: 27376588
Anterior patterning in Drosophila is mediated by the localization of bicoid (bcd) mRNA at the anterior pole of the oocyte. Exuperantia (Exu) is a putative exonuclease (EXO) associated with bcd and required for its localization. This study presents the crystal structure of Exu, which reveals a dimeric assembly with each monomer consisting of a 3'-5' EXO-like domain and a sterile alpha motif (SAM)-like domain. The catalytic site is degenerate and inactive. Instead, the EXO-like domain mediates dimerization and RNA binding. It was shown that Exu binds RNA directly in vitro, that the SAM-like domain is required for RNA binding activity and that Exu binds a structured element present in the bcd 3' untranslated region with high affinity. Through structure-guided mutagenesis, it was shown that Exu dimerization is essential for bcd localization. These data demonstrate that Exu is a noncanonical RNA-binding protein with EXO-SAM-like domain architecture that interacts with its target RNA as a homodimer.
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) is implicated in BCD mRNA localization, but its role in this process is not understood. Various studies have shown that localized messages are organized into particles, suggesting that a large protein complex may be involved in recognizing, transporting, and anchoring localized messages. Exu is part of a large RNase-sensitive complex that contains at least seven other proteins. One of these proteins is 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. The ExuYps complex also contains OSK mRNA. exu-null mutants are defective in OSK mRNA localization in both nurse cells and the oocyte. 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 and references therein).
Genetic screens have identified several mutants that have patterning defects due to the mislocalization of BCD and/or OSK mRNAs. Mutations in some genes, such as swallow and staufen, cause only partial disruption of BCD mRNA localization late in oogenesis. However, in exuperantia mutants, defects in BCD mRNA localization occur early in oogenesis and result in BCD mRNA being uniformly distributed in the mature oocyte (Berleth, 1988; St Johnston, 1989). Time-lapse confocal microscopy has shown that green fluorescent protein (GFP)-Exu forms particles that move in a microtubule-dependent manner and accumulate at the anterior and posterior of the oocyte (Theurkauf, 1998). Immunoelectron microscopy has also revealed that Exu is a component of large electron-dense structures called sponge bodies (Wilsch-Brauninger, 1997; Wilhelm, 2000 and references therein).
The pathways by which anterior- and posterior-localized mRNAs arrive at their destinations are poorly understood, although it is generally believed that these RNAs are recognized by different proteins and utilize distinct transport machineries. However, it is proposed that anterior- and posterior-localized mRNAs begin their localization process in the nurse cells using a similar complex, with Exu serving as a common core component. In this model, one of Exu's functions is as a component of an mRNA transport complex, since GFP-Exu particles have been observed to move in a microtubule-dependent manner (Theurkauf, 1998). Consistent with this idea, both OSK and BCD mRNA accumulate in apical patches within nurse cells, and exu mutants disrupt this localization pattern for both mRNAs (St Johnston, 1989; Pokrywka, 1995). It is also proposed that the Exu complex transports mRNAs from the nurse cells to the oocyte as well as within the oocyte, although these transport steps also can be achieved through other redundant mechanisms, such as nurse cell dumping and cytoplasmic streaming. Although the above model places Exu as part of a transport complex, it should be noted that Exu might contribute to the establishment of anchoring once mRNAs reach their final destination (Wilhelm, 2000).
After arriving in the oocyte, BCD- and OSK-containing RNPs must be sorted so that BCD becomes anchored at the anterior, whereas OSK is transported to the posterior pole. Since Yps, Exu, BCD mRNA, and OSK mRNA all first colocalize at the anterior, it is proposed that this sorting decision occurs at the anterior of the oocyte. Evidence for this anterior sorting model comes from genetic studies of staufen (stau) and tropomyosin II (TmII) that show that these proteins do not interfere with anterior localization but rather block the release and transport of OSK transcripts to the posterior. The molecular basis for this sorting decision is unclear, but may involve modifications to the transport machinery or the recruitment of additional factors (Wilhelm, 2000 and references therein).
Whereas Exu and Yps associate with one another independently of mRNA, another component of the ribonucleoprotein complex, the DEAD-box protein, Me31B, associates with Exu and Yps in a RNase-sensitive manner. Me31B is dispensable for the transport of the associated mRNA and proteins molecules to oocytes. Exu, OSK and BicaudalD mRNAs can be transported to the oocyte even in the absence of Me31B. Nevertheless, Me31B is essential for the translational silencing of OSK and BicaudalD mRNAs during their transport to the oocyte. This suggests that Me31B and the Exu-Yps complex bind different regions of the same RNA molecule. These data lead to the speculation that the assembly of a cytoplasmic RNP complex is achieved by binding of functionally different proteins to discrete regions of an oocyte-localizing RNA (Nakamura, 2001).
The Ser/Thr kinase Par-1 is required for cell polarisation in diverse organisms such as yeast, worms, flies and mammals. During Drosophila oogenesis, Par-1 is required for several polarisation events, including localisation of the anterior determinant bicoid. To elucidate the molecular pathways triggered by Par-1, a genome-wide, high-throughput screen for Par-1 targets was carried out. Among the targets identified in this screen was Exuperantia (Exu), a mediator of bicoid mRNA localisation. Exu is a phosphoprotein whose phosphorylation is dependent on Par-1 in vitro and in vivo. Two motifs were identifed in Exu that are phosphorylated by Par-1; their mutation abolishes bicoid mRNA localisation during mid-oogenesis. Interestingly, exu mutants in which Exu phosphorylation is specifically affected can to some extent recover from these bicoid mRNA localisation defects during late oogenesis. These results demonstrate that Par-1 establishes polarity in the oocyte by activating a mediator of bicoid mRNA localisation. Furthermore, this analysis reveals two phases of Exu-dependent bicoid mRNA localisation: an early phase that is strictly dependent on Exu phosphorylation and a late phase that is less phosphorylation dependent (Riechmann, 2004).
Par-1 has two distinct functions in bicoid mRNA localisation. Par-1 is necessary for the release of bicoid mRNA from the oocyte cortex. Genetic epistasis experiments indicate that the exu independent function of par-1 acts at a step upstream of exu in bicoid mRNA localisation. By generating mutants that abolish Exu phosphorylation, two phases of Exu dependent bicoid mRNA localisation could be further distinguished; an early phase, in which bicoid mRNA localisation is abolished when Exu is unphosphorylated and a late phase, in which the requirement for Exu phosphorylation is less stringent. Thus, these results show that bicoid mRNA localisation is a multi-step process, and that redundant mechanisms are used to ensure the anterior accumulation of bicoid mRNA (Riechmann, 2004).
Exu protein is an essential mediator of bicoid mRNA localisation. Par-1 kinase phosphorylates Exu, and this phosphorylation is necessary for anterior localisation of bicoid mRNA during mid-oogenesis. Exu phosphorylation does not affect Exu localisation, its ability to form mobile particles, or its colocalisation with bicoid mRNA. How then might Par-1 phosphorylation enable Exu to mediate bicoid mRNA localisation? Experiments in which fluorescently labelled bicoid mRNA was microinjected into living egg chambers have revealed that Exu is required in the nurse cells for anterior localisation of bicoid mRNA within the oocyte. These experiments have led to a model whereby Exu associates in the nurse cells with bicoid mRNA and mediates the recruitment of additional nurse cell factors required for targeting of bicoid mRNA to the anterior of the oocyte. The finding that mutation of Exu phosphorylation sites results in a phenotype that is, during mid-oogenesis, indistinguishable from that of exu-null mutants suggests that Exu phosphorylation is involved in the recruitment of these anterior-targeting factors in the nurse cells. Phosphorylation might increase the binding affinity of Exu for these nurse cell factors, promoting their association with bicoid mRNA. The colocalisation of Exu-GFP, Par-1 and bicoid mRNA in patches in the nurse cells suggests that this is where the bicoid RNP complexes assemble (Riechmann, 2004).
The consequences of exu and par-1 mutations on bicoid mRNA localisation are distinct. Although loss of exu function results in diffuse bicoid mRNA distribution in the ooplasm, a reduction in par-1 function causes cortical localisation of the mRNA. An Exu protein has been generated that localises bicoid mRNA independent of phosphorylation by Par-1 and rescues exu mutants, but that is unable to rescue bicoid mRNA localisation in par-1 mutants. Therefore, the cortical mislocalisation of bicoid mRNA in par-1 mutant oocytes is independent of Exu function. What might be the other function of Par-1 in localisation of bicoid mRNA? The fact that bicoid localisation requires the microtubule cytoskeleton, together with the report that oocyte microtubules are improperly polarised in par-1 mutants, suggests that cortical localisation in the mutants is caused by a microtubule defect. It has been proposed that microtubules of different qualities may nucleate from different regions of the oocyte cortex. A simple explanation for the aberrant localisation of bicoid mRNA in par-1 oocytes would be that the subset of microtubules nucleating from the anterior corners of the oocyte and serving as tracks for anterior transport of bicoid mRNA are not restricted to the anterior corners, but spread along the cortex, resulting in the lateral cortical localisation of bicoid mRNA. However, this model is not supported by the genetic epistasis experiments, which indicate that the exu independent function of par-1 acts at a step upstream of exu in bicoid mRNA localisation. Therefore, a different model is favored, in which in wild-type oocytes bicoid mRNA first localises cortically preceding its targeted transport along microtubules. In this model, most of the bicoid mRNA entering the oocyte moves in a nonpolar fashion, either passively or by active transport, to the oocyte cortex. Only after this cortical localisation does the targeted transport of bicoid mRNA to the anterior corners of the oocyte commence. In par-1 mutants, the improperly organised microtubule cytoskeleton prevents release of the mRNA from the cortex to the (anterior-targeting) microtubules and the mRNA remains cortically localised. In exu mutants, the polarity of the microtubules is normal and bicoid mRNA is released from the cortex. However, its targeted transport to the anterior is impaired and the mRNA is diffusely distributed in the ooplasm (Riechmann, 2004).
The requirement for Exu phosphorylation in bicoid mRNA localisation decreases during the later stages of oogenesis. This is revealed by the partial recovery of bicoid mRNA localisation in exu mutants that abolish phosphorylation. These mutants are indistinguishable from exu-null mutants through stage 10b of oogenesis, but during early embryogenesis two-thirds of the mutants localise enough bicoid mRNA at the anterior to support formation of a Bicoid protein. This indicates that the mechanism of bicoid mRNA localisation changes after stage 10b of oogenesis, from an early phase that is strictly dependent on Exu phosphorylation, to a late phase that is less dependent on phosphorylation. Stage 10b is the stage at which ooplasmic streaming commences, providing a possible mechanism for localisation of bicoid mRNA in mutants in which Exu phosphorylation cannot occur. Before stage 10b, anterior targeting of bicoid mRNA could be mediated solely by directed transport of bicoid mRNA complexes along microtubules, a process that is strictly dependent on Exu phosphorylation. After stage 10b, this directed transport might be complemented or replaced by a passive trapping mechanism, which has also been postulated for the localisation of oskar and nanos mRNAs during late oogenesis. This mechanism relies on the movements generated by ooplasmic streaming, which could bring bicoid mRNA complexes into contact with the anterior cortex of the oocyte, where the mRNA could be trapped by localised anchoring molecules. This change in the mechanism of bicoid mRNA localisation would occur at the time of assembly of the anterior MTOC that is essential in the late phase of bicoid mRNA localisation, suggesting that the MTOC might be involved in the trapping mechanism. Such a trapping mechanism would be differentially affected in Exu-null mutants and in mutants that specifically abolish Exu phosphorylation. It is possible that Exu provides bicoid mRNA not only with factors required for anterior targeting, but also with factors required for anchoring of bicoid mRNA. Unphosphorylated Exu might be inactive in recruiting the factors for anterior targeting, but be competent for binding of factors required for anchoring (Riechmann, 2004).
It is proposed that in the first phase of bicoid mRNA localisation, the mRNA is transported to the anterior corners of the oocyte, resulting in a ring-like distribution. This targeted transport requires the formation of RNP complexes that contain bicoid mRNA and specific anterior-targeting factors that allow the RNPs to identify those microtubules that nucleate from the anterior corners of the oocyte. Assembly of this complex takes place in the nurse cells and requires the phosphorylation of Exu by Par-1. Upon entry of the complex into the oocyte, a specific proportion of the RNP complexes encounter the microtubules that nucleate from the anterior corners, and these complexes are directly transported to their final destination. However, a large proportion of the complexes does not find these microtubules directly, and moves first to the oocyte cortex. The transfer of these cortically localised complexes to microtubules nucleating from the anterior corners solely requires a properly polarised microtubule network. Only at this stage can the nurse cell factors assembled on the mRNA act to transport the cortically localised complexes to the anterior corners of the oocyte. During the second phase of bicoid mRNA localisation, the ring-shaped distribution changes to a disc-shaped distribution and a MTOC forms at the anterior of the oocyte. The third phase of bicoid mRNA localisation begins after the onset of ooplasmic streaming. In this late phase, the mechanism of bicoid mRNA localisation changes from targeted transport to passive trapping, mediated by ooplasmic streaming, and the mRNA is anchored at the anterior margin. The generation of exu mutants that abolish phosphorylation allows distinguishing between early and the late mechanisms of bicoid mRNA localisation, since the two mechanisms differ in their sensitivity to Exu phosphorylation (Riechmann, 2004).
It has also been shown that Par-1 controls posterior patterning by phosphorylating Oskar. In addition, Par-1 regulates anterior patterning by phosphorylating Exu. Although Oskar is an intrinsically unstable protein whose stability is increased by Par-1 phosphorylation, Par-1 phosphorylation does not affect Exu stability but does affect its ability to mediate bicoid mRNA localisation. Thus, Par-1 uses at least two different mechanisms to generate polarity within the same cell. Interestingly, these two Par-1 substrates, Oskar and Exu, are unique to Diptera, showing that during evolution Par-1 gained fly-specific mediators of cell polarisation as substrates. Par-1 is therefore flexible in the mechanisms and in the targets by which it mediates cell polarisation. This is in striking contrast to the PDZ-containing proteins Par-3 and Par-6, which appear to establish polarity by the assembly of a conserved protein complex (Riechmann, 2004).
How highly conserved are the mechanisms of mRNA localization, a process crucial to Drosophila body patterning? Two components are involved in that process: the exuperantia gene, required for an early step in localization, and the cis-acting signal that directs BCD mRNA localization. The cloned D. melanogaster exu gene has been used to identify the exu genes from D. virilis and D. pseudoobscura. Surprisingly, D. pseudoobscura has two closely related exu genes, while D. melanogaster and D. virilis have only one each. When expressed in D. melanogaster ovaries, the D. virilis exu gene and one of the D. pseudoobscura exu genes can substitute for the endogenous exu gene in supporting localization of BCD mRNA, demonstrating that function is conserved (Luk, 1994).
date revised: 30 September 2001
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