Opposing roles for Egalitarian and Staufen in transport, anchoring and localization of oskar mRNA in the Drosophila oocyte.

Localization of oskar mRNA includes two distinct phases: transport from nurse cells to the oocyte, a process typically accompanied by cortical anchoring in the oocyte, followed by posterior localization within the oocyte. Signals within the oskar 3' UTR directing transport are individually weak, a feature previously hypothesized to facilitate exchange between the different localization machineries. We show that alteration of the SL2a stem-loop structure containing the oskar transport and anchoring signal (TAS) removes an inhibitory effect such that in vitro binding by the RNA transport factor, Egalitarian, is elevated as is in vivo transport from the nurse cells into the oocyte. Cortical anchoring within the oocyte is also enhanced, interfering with posterior localization. We also show that mutation of Staufen recognized structures (SRSs), predicted binding sites for Staufen, disrupts posterior localization of oskar mRNA just as in staufen mutants. Two SRSs in SL2a, one overlapping the Egalitarian binding site, are inferred to mediate Staufen-dependent inhibition of TAS anchoring activity, thereby promoting posterior localization. The other three SRSs in the oskar 3' UTR are also required for posterior localization, including two located distant from any known transport signal. Staufen, thus, plays multiple roles in localization of oskar mRNA.

Different roles for the adjoining and structurally similar A-rich and poly(A) domains of oskar mRNA: Only the A-rich domain is required for oskar noncoding RNA function, which includes MTOC positioning.

Drosophila oskar (osk) mRNA has both coding and noncoding functions, with the latter required for progression through oogenesis. Noncoding activity is mediated by the osk 3' UTR. Three types of cis elements act most directly and are clustered within the final ~120 nucleotides of the 3' UTR: multiple binding sites for the Bru1 protein, a short highly conserved region, and A-rich sequences abutting the poly(A) tail. Here we extend the characterization of these elements and their functions, providing new insights into osk noncoding RNA function and the makeup of the cis elements. We show that all three elements are required for correct positioning of the microtubule organizing center (MTOC), a defect not previously reported for any osk mutant. Normally, the MTOC is located at the posterior of the oocyte during previtellogenic stages of oogenesis, and this distribution underlies the strong posterior enrichment of many mRNAs transported into the oocyte from the nurse cells. When osk noncoding function was disrupted the MTOC was dispersed in the oocyte and osk mRNA failed to be enriched at the posterior, although transport to the oocyte was not affected. A previous study did not detect loss of posterior enrichment for certain osk mutants lacking noncoding activity (Kanke et al., 2015). This discrepancy may be due to use of imaging aimed at monitoring transport to the oocyte rather than posterior enrichment. Involvement in MTOC positioning suggests that the osk noncoding function may act in conjunction with genes whose loss has similar effects, and that osk function may extend to other processes requiring those genes. Further characterization of the cis elements required for osk noncoding function included completion of saturation mutagenesis of the most highly conserved region, providing critical information for evaluating the possible contribution of candidate binding factors. The 3'-most cis element is a cluster of A-rich sequences, the ARS. The close juxtaposition and structural similarity of the ARS and poly(A) tail raised the possibility that they comprise an extended A-rich element required for osk noncoding function. We found that absence of the poly(A) tail did not mimic the effects of mutation of the ARS, causing neither arrest of oogenesis nor mispositioning of osk mRNA in previtellogenic stage oocytes. Thus, the ARS and the poly(A) tail are not interchangeable for osk noncoding RNA function, suggesting that the role of the ARS is not in recruitment of Poly(A) binding protein (PABP), the protein that binds the poly(A) tail. Furthermore, although PABP has been implicated in transport of osk mRNA from the nurse cells to the oocyte, mutation of the ARS in combination with loss of the poly(A) tail did not disrupt transport of osk mRNA into the oocyte. We conclude that PABP acts indirectly in osk mRNA transport, or is associated with osk mRNA independent of an A-rich binding site. Although the poly(A) tail was not required for osk mRNA transport into the oocyte, its absence was associated with a novel osk mRNA localization defect later in oogenesis, potentially revealing a previously unrecognized step in the localization process.

Multiple cis-acting signals, some weak by necessity, collectively direct robust transport of oskar mRNA to the oocyte.

Localization of mRNAs can involve multiple steps, each with its own cis-acting localization signals and transport factors. How is the transition between different steps orchestrated? We show that the initial step in localization of Drosophila oskar mRNA - transport from nurse cells to the oocyte - relies on multiple cis-acting signals. Some of these are binding sites for the translational control factor Bruno, suggesting that Bruno plays an additional role in mRNA transport. Although transport of oskar mRNA is essential and robust, the localization activity of individual transport signals is weak. Notably, increasing the strength of individual transport signals, or adding a strong transport signal, disrupts the later stages of oskar mRNA localization. We propose that the oskar transport signals are weak by necessity; their weakness facilitates transfer of the oskar mRNA from the oocyte transport machinery to the machinery for posterior localization.

Region-specific activation of oskar mRNA translation by inhibition of Bruno-mediated repression.

A complex program of translational repression, mRNA localization, and translational activation ensures that Oskar (Osk) protein accumulates only at the posterior pole of the Drosophila oocyte. Inappropriate expression of Osk disrupts embryonic axial patterning, and is lethal. A key factor in translational repression is Bruno (Bru), which binds to regulatory elements in the osk mRNA 3' UTR. After posterior localization of osk mRNA, repression by Bru must be alleviated. Here we describe an in vivo assay system to monitor the spatial pattern of Bru-dependent repression, separate from the full complexity of osk regulation. This assay reveals a form of translational activation-region-specific activation-which acts regionally in the oocyte, is not mechanistically coupled to mRNA localization, and functions by inhibiting repression by Bru. We also show that Bru dimerizes and identify mutations that disrupt this interaction to test its role in vivo. Loss of dimerization does not disrupt repression, as might have been expected from an existing model for the mechanism of repression. However, loss of dimerization does impair regional activation of translation, suggesting that dimerization may constrain, not promote, repression. Our work provides new insight into the question of how localized mRNAs become translationally active, showing that repression of osk mRNA is locally inactivated by a mechanism acting independent of mRNA localization.

oskar RNA plays multiple noncoding roles to support oogenesis and maintain integrity of the germline/soma distinction.

The Drosophila oskar (osk) mRNA is unusual in that it has both coding and noncoding functions. As an mRNA, osk encodes a protein required for embryonic patterning and germ cell formation. Independent of that function, the absence of osk mRNA disrupts formation of the karyosome and blocks progression through oogenesis. Here we show that loss of osk mRNA also affects the distribution of regulatory proteins, relaxing their association with large RNPs within the germline, and allowing them to accumulate in the somatic follicle cells. This and other noncoding functions of the osk mRNA are mediated by multiple sequence elements with distinct roles. One role, provided by numerous binding sites in two distinct regions of the osk 3' UTR, is to sequester the translational regulator Bruno (Bru), which itself controls translation of osk mRNA. This defines a novel regulatory circuit, with Bru restricting the activity of osk, and osk in turn restricting the activity of Bru. Other functional elements, which do not bind Bru and are positioned close to the 3' end of the RNA, act in the oocyte and are essential. Despite the different roles played by the different types of elements contributing to RNA function, mutation of any leads to accumulation of the germline regulatory factors in the follicle cells.

RNA sequences required for the noncoding function of oskar RNA also mediate regulation of Oskar protein expression by Bicoid Stability Factor.

The Drosophila oskar (osk) mRNA is unusual in having both coding and noncoding functions. As an mRNA, osk encodes a protein which is deployed specifically at the posterior of the oocyte. This spatially-restricted deployment relies on a program of mRNA localization and both repression and activation of translation, all dependent on regulatory elements located primarily in the 3' untranslated region (UTR) of the mRNA. The 3' UTR also mediates the noncoding function of osk, which is essential for progression through oogenesis. Mutations which most strongly disrupt the noncoding function are positioned in a short region (the C region) near the 3' end of the mRNA, in close proximity to elements required for activation of translation. We show that Bicoid Stability Factor (BSF) binds specifically to the C region of the mRNA. Both knockdown of bsf and mutation of BSF binding sites in osk mRNA have the same consequences: Osk expression is largely eliminated late in oogenesis, with both mRNA localization and translation disrupted. Although the C region of the osk 3' UTR is required for the noncoding function, BSF binding does not appear to be essential for that function.

Multiple RNA binding domains of Bruno confer recognition of diverse binding sites for translational repression.

Bruno protein binds to multiple sites - BREs - in the oskar mRNA 3' UTR, thereby controlling oskar mRNA translation. Bruno also binds and regulates other mRNAs, although the binding sites have not yet been defined. Bruno has three RRM type RNA binding motifs, two near the amino terminus and an extended RRM at the C terminus. Two domains of Bruno, the first two RRMs (RRM1+2), and the extended RRM (RRM3+) - can each bind with specificity to the oskar mRNA regulatory regions; these and Bruno were used for in vitro selections. Anti-RRM3+ aptamers include long, highly constrained motifs, including one corresponding to the previously identified BRE. Anti-RRM1+2 aptamers lack constrained motifs, but are biased towards classes of short and variable sequences. Bruno itself selects for several motifs, including some of those bound by RRM3+. We propose that the different RNA binding domains allow for combinatorial binding, with extended Bruno binding sites assembled from sequences bound by the individual domains. Examples of such sites were identified in known targets of Bruno, and shown to confer Bruno-dependent translational repression in vivo. Other proteins with multiple RRMs may employ combinatorial binding to achieve high levels of specificity and affinity.

Knock down analysis reveals critical phases for specific oskar noncoding RNA functions during Drosophila oogenesis.

The oskar transcript, acting as a noncoding RNA, contributes to a diverse set of pathways in the Drosophila ovary, including karyosome formation, positioning of the microtubule organizing center (MTOC), integrity of certain ribonucleoprotein particles, control of nurse cell divisions, restriction of several proteins to the germline, and progression through oogenesis. How oskar mRNA acts to perform these functions remains unclear. Here, we use a knock down approach to identify the critical phases when oskar is required for three of these functions. The existing transgenic shRNA for removal of oskar mRNA in the germline targets a sequence overlapping a regulatory site bound by Bruno1 protein to confer translational repression, and was ineffective during oogenesis. Novel transgenic shRNAs targeting other sites were effective at strongly reducing oskar mRNA levels and reproducing phenotypes associated with the absence of the mRNA. Using GAL4 drivers active at different developmental stages of oogenesis, we found that early loss of oskar mRNA reproduced defects in karyosome formation and positioning of the MTOC, but not arrest of oogenesis. Loss of oskar mRNA at later stages was required to prevent progression through oogenesis. The noncoding function of oskar mRNA is thus required for more than a single event.

Bicaudal C and trailer hitch have similar roles in gurken mRNA localization and cytoskeletal organization.

Bicaudal C and trailer hitch are both required for dorsoventral patterning of the Drosophila oocyte. Each mutant produces ventralized eggs, a phenotype typically associated with failure of the oocyte to provide a dorsalization signal--the Gurken protein--to the follicle cells. Bicaudal C and trailer hitch are both implicated in post-transcriptional gene regulation. Bicaudal C acts in recruiting a deadenylase to specific mRNAs, leading to translational repression. The role of trailer hitch is less well defined, but mutants have defects in protein secretion, and show aberrant distribution of an endoplasmic reticulum exit site marker whose mRNA is associated with Trailer hitch protein. We show that Bicaudal C and trailer hitch interact genetically. Mutants of these two genes have shared defects in localization of gurken and other anteriorly-localized mRNAs, as well as altered microtubule organization which may underlie the mRNA localization defects. Bicaudal C and trailer hitch mutants also share a syndrome of actin-related abnormalities, including the formation of ectopic actin cages near the anterior of the oocyte. The cages sequester Gurken protein, blocking its secretion and thus interfering with signaling of the follicle cells to specify dorsal fate.

Bruno protein contains an expanded RNA recognition motif.

The RNA recognition motif (or RRM) is a ubiquitous RNA-binding module present in approximately 2% of the proteins encoded in the human genome. This work characterizes an expanded RRM, which is present in the Drosophila Bruno protein, and targets regulatory elements in the oskar mRNA through which Bruno controls translation. In this Bruno RRM, the deletion of 40 amino acids prior to the N-terminus of the canonical RRM resulted in a significantly decreased affinity of the protein for its RNA target. NMR spectroscopy showed that the expanded Bruno RRM contains the familiar RRM fold of four antiparallel beta-strands and two alpha-helices, preceded by a 10-residue loop that contacts helix alpha(1) and strand beta(2); additional amino acids at the N-terminus of the domain are relatively flexible in solution. NMR results also showed that a truncated form of the Bruno RRM, lacking the flexible N-terminal amino acids, forms a stable and complete canonical RRM, so that the loss of RNA binding activity cannot be attributed to disruption of the RRM fold. This expanded Bruno RRM provides a new example of the features that are important for RNA recognition by an RRM-containing protein.

Recognition of a bicoid mRNA localization signal by a protein complex containing Swallow, Nod, and RNA binding proteins.

Localization of mRNAs, a process essential for embryonic body patterning in Drosophila, requires recognition of cis-acting signals by cellular components responsible for movement and anchoring. We have purified a large multiprotein complex that binds a minimal form of the bicoid mRNA localization signal in a manner both specific and sensitive to inactivating mutations. Identified complex components include the RNA binding proteins Modulo, PABP, and Smooth, the known localization factor Swallow, and the kinesin family member Nod. We demonstrate that localization of bcd mRNA is defective in modulo mutants. The presence of three required localization components (Swallow, Modulo, and specific RNA binding activity) within the recognition complex strongly implicates it in mRNA localization.

Localization-dependent oskar protein accumulation; control after the initiation of translation.

The appearance of Oskar protein occurs coincident with localization of oskar mRNA to the posterior pole of the Drosophila oocyte, and earlier accumulation of the protein is prevented by translational repression. We find that the nascent polypeptide-associated complex (NAC) is required for correct localization of oskar mRNA. The timing of the defects suggests that, if NAC acts directly via an interaction with nascent Oskar protein, oskar mRNA should be undergoing translation prior to its localization. Polysome analysis confirms that oskar mRNA is associated with polysomes even in the absence of localization of the mRNA or accumulation of Oskar protein. Thus, the mechanisms that prevent accumulation of Oskar protein until it can be secured at the posterior pole of the oocyte include regulated degradation or inhibition of translational elongation.

Imp associates with squid and Hrp48 and contributes to localized expression of gurken in the oocyte.

Localization and translational control of Drosophila melanogaster gurken and oskar mRNAs rely on the hnRNP proteins Squid and Hrp48, which are complexed with one another in the ovary. Imp, the Drosophila homolog of proteins acting in localization of mRNAs in other species, is also associated with Squid and Hrp48. Notably, Imp is concentrated at sites of gurken and oskar mRNA localization in the oocyte, and alteration of gurken localization also alters Imp distribution. Imp binds gurken mRNA with high affinity in vitro; thus, the colocalization with gurken mRNA in vivo is likely to be the result of direct binding. Imp mutants support apparently normal regulation of gurken and oskar mRNAs. However, loss of Imp activity partially suppresses a gurken misexpression phenotype, indicating that Imp does act in control of gurken expression but has a largely redundant role that is only revealed when normal gurken expression is perturbed. Overexpression of Imp disrupts localization of gurken mRNA as well as localization and translational regulation of oskar mRNA. The opposing effects of reduced and elevated Imp activity on gurken mRNA expression indicate a role in gurken mRNA regulation.

Recognition of the bcd mRNA localization signal in Drosophila embryos and ovaries.

The process of mRNA localization, often used for regulation of gene expression in polarized cells, requires recognition of cis-acting signals by components of the localization machinery. Many known RNA signals are active in the contexts of both the Drosophila ovary and the blastoderm embryo, suggesting a conserved recognition mechanism. We used variants of the bicoid mRNA localization signal to explore recognition requirements in the embryo. We found that bicoid stem-loop IV/V, which is sufficient for ovarian localization, was necessary but not sufficient for full embryonic localization. RNAs containing bicoid stem-loops III/IV/V did localize within the embryo, demonstrating a requirement for dimerization and other activities supplied by stem-loop III. Protein complexes that bound specifically to III/IV/V and fushi tarazu localization signals copurified through multiple fractionation steps, suggesting that they are related. Binding to these two signals was competitive but not equivalent. Thus, the binding complexes are not identical but appear to have some components in common. We have proposed a model for a conserved mechanism of localization signal recognition in multiple contexts.

Two distinct domains of Bruno bind specifically to the oskar mRNA.

Selective deployment of Oskar protein at the posterior pole of the Drosophila oocyte relies on localization of oskar mRNA, combined with translational regulation to ensure that only the localized mRNA produces protein. The Bruno protein binds to Bruno Response Elements (BREs) in the oskar mRNA, and prevents translation of unlocalized oskar mRNA. Bruno contains three copies of the RNA Recognition Motif (RRM), a protein motif that often binds directly to RNA. Either of two nonoverlapping parts of Bruno--RRMs 1 and 2, and RRM 3 and 42 flanking amino acids--can bind specifically to BRE-containing RNA, but both domains are required for maximal binding. When expressed in Drosophila ovaries, Bruno proteins with a single RNA binding domain mutated have reduced repressive activity, while mutation of both binding domains largely eliminates this activity. Notably, the same proteins expressed as fusions to GFP accumulate in nuclei, with the most severe mislocalization occurring when both RNA binding domains are mutated. A similar mislocalization of endogenous Bruno occurs when mRNA export is blocked. Thus, Bruno shuttles between the nucleus and cytoplasm, and may first bind oskar mRNA in the nucleus.

Translational control: a cup half full.

Repression of translation of oskar and nanos mRNAs prior to their posterior localization in the egg and embryo is essential for body patterning in Drosophila. The Cup protein is now found to have an important role in repression of both mRNAs, and apparently does so in a manner similar to the action of the Xenopus Maskin protein.

Community effects in regulation of translation.

Certain forms of translational regulation, and translation itself, rely on long-range interactions between proteins bound to the different ends of mRNAs. A widespread assumption is that such interactions occur only in cis, between the two ends of a single transcript. However, certain translational regulatory defects of the Drosophila oskar (osk) mRNA can be rescued in trans. We proposed that inter-transcript interactions, promoted by assembly of the mRNAs in particles, allow regulatory elements to act in trans. Here we confirm predictions of that model and show that disruption of PTB-dependent particle assembly inhibits rescue in trans. Communication between transcripts is not limited to different osk mRNAs, as regulation imposed by cis-acting elements embedded in the osk mRNA spreads to gurken mRNA. We conclude that community effects exist in translational regulation.

Prediction and verification of microRNA targets by MovingTargets, a highly adaptable prediction method.

BACKGROUND: MicroRNAs (miRNAs) mediate a form of translational regulation in animals. Hundreds of animal miRNAs have been identified, but only a few of their targets are known. Prediction of miRNA targets for translational regulation is challenging, since the interaction with the target mRNA usually occurs via incomplete and interrupted base pairing. Moreover, the rules that govern such interactions are incompletely defined. RESULTS: MovingTargets is a software program that allows a researcher to predict a set of miRNA targets that satisfy an adjustable set of biological constraints. We used MovingTargets to identify a high-likelihood set of 83 miRNA targets in Drosophila, all of which adhere to strict biological constraints. We tested and verified 3 of these predictions in cultured cells, including a target for the Drosophila let-7 homolog. In addition, we utilized the flexibility of MovingTargets by relaxing the biological constraints to identify and validate miRNAs targeting tramtrack, a gene also known to be subject to translational control dependent on the RNA binding protein Musashi. CONCLUSION: MovingTargets is a flexible tool for the accurate prediction of miRNA targets in Drosophila. MovingTargets can be used to conduct a genome-wide search of miRNA targets using all Drosophila miRNAs and potential targets, or it can be used to conduct a focused search for miRNAs targeting a specific gene. In addition, the values for a set of biological constraints used to define a miRNA target are adjustable, allowing the software to incorporate the rules used to characterize a miRNA target as these rules are experimentally determined and interpreted.

Genetic interactions of Drosophila melanogaster arrest reveal roles for translational repressor Bruno in accumulation of Gurken and activity of Delta.

arrest mutants have pleiotropic phenotypes, ranging from an early arrest of oogenesis to irregular embryonic segmentation defects. One function of arrest is in translational repression of oskar mRNA; this biochemical activity is presumed to be involved in other functions of arrest. To identify genes that could provide insight into how arrest contributes to translational repression or that may be targets for arrest-dependent translational control, we screened deficiency mutants for dominant modification of the arrest phenotype. Only four of the many deficiencies tested, which cover approximately 30% of the genome, modified the starting phenotype. One enhancer, identified fortuitously, is the Star gene. Star interaction with arrest results in excess Gurken protein, supporting the model that gurken is a target of repression. Two modifiers were mapped to individual genes. One is Lk6, which encodes a protein kinase predicted to regulate the rate-limiting initiation factor eIF4E. The second is Delta. The interaction between arrest and Delta mimics the phenotype of homozygous Delta mutants, suggesting that arrest could positively control Delta activity. Indeed, arrest mutants have significantly reduced levels of Delta protein at the interface of germline and follicle cells.

Translational activation of oskar mRNA: reevaluation of the role and importance of a 5' regulatory element [corrected].

Local translation of oskar (osk) mRNA at the posterior pole of the Drosophila oocyte is essential for axial patterning of the embryo, and is achieved by a program of translational repression, mRNA localization, and translational activation. Multiple forms of repression are used to prevent Oskar protein from accumulating at sites other than the oocyte posterior. Activation is mediated by several types of cis-acting elements, which presumably control different forms of activation. We characterize a 5' element, positioned in the coding region for the Long Osk isoform and in the extended 5' UTR for translation of the Short Osk isoform. This element was previously thought to be essential for osk mRNA translation, with a role in posterior-specific release from repression. From our work, which includes assays which separate the effects of mutations on RNA regulatory elements and protein coding capacity, we find that the element is not essential, and conclude that there is no evidence supporting a role for the element only at the posterior of the oocyte. The 5' element has a redundant role, and is only required when Long Osk is not translated from the same mRNA. Mutations in the element do disrupt the anchoring function of Long Osk protein through their effects on the amino acid sequence, a confounding influence on interpretation of previous experiments.