BIOLOGICAL OVERVIEW

Staufen protein is required for the localization of multiple mRNA species during oogenesis and zygotic development. For two of these targets, Bicoid mRNA and Prospero mRNA, the direct binding of Staufen to these messengers is a requirement for their subcellular distribution. Staufen, identified as a posterior group protein, is also required for the localization of Oskar mRNA to the posterior pole of the oocyte, but Oskar mRNA does not associate with Staufen. This suggests that other proteins are required for the association of OSK mRNA with Stau (Farrandon, 1997 and references). Unexpectedly, there is a direct interaction between Osk protein and Stau, but the function of this interaction remains unknown (Breitwieser, 1996).

The localization of Bicoid mRNA is a multistep process that requires three known genes. Mutations in exuperantia and swallow disrupt the localization of BCD mRNA to the anterior cortex of the oocyte during oogenesis. This premature mislocalization leads to an almost homogeneous distribution (exuperantia), or to a shallow gradient (swallow) of BCD mRNA in the embryo. In embryos from staufen females, BCD mRNA is distributed in a steep anterior gradient, indicating that Stau is required after egg activation to anchor the BCD mRNA, after its release from the cortex, in the anterior cytoplasm of the egg (St Johnston, 1994). Stau is present in excess throughout the Drosophila egg but is associated specifically with BCD mRNA at the anterior pole (Ferrandon, 1997 and references).

Endogenous Stau associates with BCD mRNA 3'UTR when injected into the early embryo, resulting in the formation of characteristic RNA-protein particles. The STAU interaction requires a double-stranded conformation of the stems within the RNA localization signal. Two loops contact each other through base pairing. The two loops that base-pair with each other do so not within the same mRNA species but between two different mRNA molecules. This loop-loop interaction is intermolecular, not intra-molecular; thus dimers or multimers of the RNA localization signal must be associated with STAU protein in these particles (Ferrandon, 1997).

Staufen and Inscuteable play a complex role in the distribution of Prospero mRNA and protein during neurogenesis. Inscuteable protein localizes to the apical cortex of neuroblasts, the surface of the cell that is on the outside of the embryo. Inscuteable is known to play a critical role in the distribution of both Prospero and Numb proteins to the basal cortex of neuroblasts during mitosis, allowing these two proteins to be selectively partitioned to the ganglion mother cell that delaminates and migrates basally into the presumptive central nervous system (see Prospero, Numb and Inscuteable for further information and additional references).

Inscuteable and Prospero interact physically. The C-terminal 108 amino acids of Insc are sufficient to confer an interaction with Stau, while other residues of Insc appear to inhibit the interaction mediated by the C-terminal 108 amino acids. The C-terminal region (residues 769-1026) of Stau confers this specific interaction. Both Staufen and Inscuteable proteins are cortically localized in the apex of neuroblasts; the apical localization of Staufen protein requires the presence of Inscuteable (Li, 1997).

Paradoxically, Staufen is required for the basal localization of Prospero mRNA during mitosis. Prospero mRNA is localized to the apical cortex during interphase, however the change in PROS mRNA localization from the apical cortex at interpahse to the basal cortex at prophase fails to occur in animals that lack zygotic staufen. In staufen and inscuteable mutant neuroblasts, the PROS mRNA remains primarily on the apical cortex during mitosis, indicating that the apical cortical localization of PROS mRNA during interphase requires neither insc nor stau function. However, the basal cortical relocalization that takes place at prophase requires both insc and stau function. Since staufen mutation fails to affect either Inscuteable protein localization or mitotic spindle orientation in neuroblasts, it is concluded that stau acts downstream of inscuteable (Li, 1997).

Staufen binds to the 3' untranslated regions of Prospero mRNA, suggesting that Staufen's role in Prospero mRNA redistribution is mediated through this interaction. How then does Prospero mRNA get to the basal cortex during mitosis? It is assumed that the Inscuteable/Staufen-independent mechanism that operates to effect localization of PROS mRNA to the apical cortex during interphase is normally overridden by the Insc/Stau-mediated process during mitosis. There is strong evidence that suggests that during early development specific signals localized to the 3' UTR of Bicoid mRNA can recruit Staufen to form ribonucleoprotein particles that are subsequently transported in a process that requires intact microtubules (Ferrandon, 1994). It is therefore appealing to suggest that Stau might play a similar role in the neuroblast to transport PROS mRNA from the apical to basal cortex in the transition between interphase and mitosis. With respect to PROS mRNA localization, the role of Insc may be to facilitate Stau protein/PROS mRNA interaction with perhaps other components necessary for the transport of PROS mRNA (Li, 1997).

Drosophila Staufen protein is required for the localization of Oskar mRNA to the posterior of the oocyte, the anterior anchoring of Bicoid mRNA and the basal localization of Prospero mRNA in dividing neuroblasts. Analysis of an alignment of the Stau homologs reveals that the only regions of the protein to have been conserved during evolution are the five dsRBDs and a short region within an insertion that splits dsRBD2 into two halves. The M. domestica and D. melanogaster proteins show an average of 67% amino acid identity within the dsRBDs, but less than 15% in the rest of the protein. dsRBD2 and dsRBD5 were originally described as 'half domains' showing similarity to the dsRBD consensus only over the C-terminal portion of the domain. However, the conservation extends over a region corresponding to the length of a whole domain, and these domains should therefore be considered as complete, albeit divergent. The only other obvious homology between these proteins is a short region, one which is rich in proline and aromatic amino acids, within the insertion that splits dsRBD2. Since the regions of the protein essential for its activity are expected to be conserved during evolution, the dsRBDs and this proline-rich region are likely to mediate all of the functions of Stau, including its ability to bind both mRNA and the factors that localize Stau-mRNA complexes. dsRBDs 1, 3 and 4 bind dsRNA in vitro, but dsRBDs 2 and 5 do not, although dsRBD2 does bind dsRNA when the insertion is removed. Full-length Staufen protein lacking this insertion is able to associate with Oskar mRNA and activate its translation, but fails to localize the RNA to the posterior. In contrast, Staufen lacking dsRBD5 localizes Oskar mRNA normally, but does not activate its translation. Thus, dsRBD2 is required for the microtubule-dependent localization of OSK mRNA, and dsRBD5 is required for the derepression of Oskar mRNA translation, once localized. Since dsRBD5 has been shown to direct the actin-dependent localization of Prospero mRNA, distinct domains of Staufen mediate microtubule- and actin-based mRNA transport (Micklem, 2000).

Since Stau is also required for the anterior anchoring of BCD mRNA, it was of interest to determine whether the Stau lacking dsRBD5 and the RBD2 loop could rescue the BCD mRNA localization defect of a stau null mutation. Although both mutant proteins are expressed at similar levels to endogenous Stau, neither anchors BCD mRNA at the anterior. Surprisingly, both constructs almost completely rescue the stau head phenotype, even though they do not restore the wild-type localization of the BCD mRNA. Whereas 100% of the embryos laid by stau homozygous mutant females at 18°C lack all or part of the head skeleton, over two-thirds of the embryos laid by the transgenic stau minus females have wild-type heads, and the rest have much milder head defects than are found in stau mutants alone. This suggests that Stau plays a second role in the regulation of BCD mRNA expression that is independent of its function in localization. In contrast to its role in anchoring, this activity does not require dsRBD5 or the insertion in dsRBD2 (Micklem 2000).

It has previously been difficult to investigate the role of Stau in OSKmRNA translation for two reasons: (1) stau null mutations disrupt the localization of OSK mRNA, and it is not translated unless it is localized to the posterior pole; (2) it is difficult to distinguish between the effects of weak stau alleles on translation and anchoring, because Osk protein is required to anchor its own RNA, but the mRNA needs to be anchored at the posterior to be translated. However, Stau^DeltadsRBD5 seems to have a specific defect in OSK mRNA translation, since OSK mRNA is localized normally to the posterior at stage 10 in these ovaries, but no detectable Osk protein was produced. Furthermore, oskBRE^- RNA, lacking the Bruno response element, produces significant amounts of Osk activity in these ovaries, indicating that Stau^DeltadsRBD5 can function in the translation of derepressed OSK mRNA. Taken together, these results strongly suggest that dsRBD5 is required to relieve Bruno repression once the mRNA has reached the posterior. This requirement cannot be absolute, however, since some Osk protein must be present early in oogenesis to anchor Stau-osk mRNA complexes (Micklem 2000).

Since dsRBD5 does not bind RNA, it presumably mediates its function in Osk translation through protein-protein interactions. Although Miranda binds to this domain, this interaction is unlikely to play any role during oogenesis, since miranda null germline clones have no phenotype. Thus, dsRBD5 presumably interacts with other proteins to regulate OSK translation. A very similar translation defect is observed in osk transgenes that lack binding sites for 68 and 50 kDa proteins in the 5'UTR, whereas Stau is thought to associate with the localization signal in the 3'UTR . Thus, derepression is likely to involve cooperation between proteins bound to both ends of the RNA (Micklem 2000).

In addition to its role in derepressing OSK translation at the posterior, Stau is required for the efficient expression of derepressed oskBRE^- RNA. Since neither the insert in dsRBD2 nor dsRBD5 is necessary for this activity, it presumably depends on the dsRBDs that bind RNA. It is possible that these dsRBDs also interact with other proteins, since only one face of the domain contacts RNA, and several amino acids on the other faces of these domains have been conserved during evolution. Alternatively, the binding of Stau may enhance OSK mRNA translation indirectly, for example, by altering the folding of the RNA so that other factors can bind more efficiently (Micklem, 2000).

Both Stau^Deltaloop2 and Stau^DeltadsRBD5 partially rescue the stau head phenotype, even though they do not restore the wild-type localization of BCD mRNA. Thus, more Bcd activity must be produced from the mislocalized mRNA in the presence of these mutant proteins than in stau^D3 alone, indicating that they provide a function for Stau that is independent of its role in anchoring. A comparison of the phenotypes produced by vasa;exu and stau;exu double mutants also indicates that Stau has a second function in the regulation of BCD mRNA. exu mutants block the localization of BCD mRNA early in oogenesis, and result in a uniform distribution of the RNA along the anterior-posterior axis of the embryo, while both vasa and stau mutants prevent the formation of the pole plasm, and therefore lack Nanos activity, which represses BCD mRNA translation. Despite the identical distributions of BCD RNA in these genotypes, vasa;exu embryos develop anterior head structures everywhere, indicating that they contain high levels of Bcd activity, whereas stau;exu form only thoracic structures. Thus, the removal of Stau reduces the level of Bcd expression, in the absence of any effect on mRNA localization. Two explanations for this localization-independent function of Stau can be envisioned. Stau binding could protect BCD RNA from degradation, and therefore increase the total amount of RNA. Alternatively, Stau could enhance the efficiency of BCD translation, in much the same way as it does for OSK mRNA (Micklem, 2000 and references therein).

Since Stau has been conserved throughout animal evolution, it seems likely that the homologs will fulfil similar functions in mRNA localization and translational control in other organisms. In support of this view, recent evidence indicates that mammalian Stau mediates mRNA transport along microtubules in neurons. The mouse and human Stau genes share an extra region of homology (not found in the insect homologs) that resembles the microtubule-binding domain of MAP1B, and this region of HsStau binds to microtubules in vitro. It will therefore be interesting to see whether this domain or the insertion in dsRBD2 is required for the microtubule-dependent movement of Stau in neurons (Micklem, 2000).

Distinguishing direct from indirect roles for bicoid mRNA localization factors

Localization of bicoid mRNA to the anterior of the Drosophila oocyte is essential for patterning the anteroposterior body axis in the early embryo. bicoid mRNA localizes in a complex multistep process involving transacting factors, molecular motors and cytoskeletal components that remodel extensively during the lifetime of the mRNA. Genetic requirements for several localization factors, including Swallow and Staufen, are well established, but the precise roles of these factors and their relationship to bicoid mRNA transport particles remains unresolved. This study used live cell imaging, super-resolution microscopy in fixed cells and immunoelectron microscopy on ultrathin frozen sections to study the distribution of Swallow, Staufen, actin and dynein relative to bicoid mRNA during late oogenesis. Swallow and bicoid mRNA are shown to be transported independently and are not colocalized at their final destination. Furthermore, Swallow is not required for bicoid transport. Instead, Swallow localizes to the oocyte plasma membrane, in close proximity to actin filaments, evidence is presented that Swallow functions during the late phase of bicoid localization by regulating the actin cytoskeleton. In contrast, Staufen, dynein and bicoid mRNA form nonmembranous, electron dense particles at the oocyte anterior. These results exclude a role for Swallow in linking bicoid mRNA to the dynein motor. Instead a model is proposed for bicoid mRNA localization in which Swallow is transported independently by dynein and contributes indirectly to bicoid mRNA localization by organizing the cytoskeleton, whereas Staufen plays a direct role in dynein-dependent bicoid mRNA transport (Weil, 2010).

A direct role for Swa in either transport or anchoring of bcd mRNA predicts and requires that the protein be colocalized with bcd mRNA during transport or anchoring. This study tested this prediction conclusively in three ways. First, an OMX microscope with highly sensitive and rapid multi-channel imaging was used to study to movement of Swa and bcd RNA particles simultaneously. Second, advantage was taken of the increased resolution of the OMX microscope with fixed material to analyze the precise locations of Swa and bcd at the anterior oocyte cortex. Importantly, the results show conclusively that Swa and bcd mRNA move independently to the anterior and occupy distinct domains at the anterior cortex. Third, the subcellular distributions of Swa and bcd was determined at EM resolution, demonstrating that bcd is mostly in particles near the anterior cortex whereas Swa is mostly confined to the plasma membrane of the entire oocyte. How the membrane association of Swa, which does not contain a transmembrane domain and is not predicted to harbor lipid modifications, is mediated remains to be investigated (Weil, 2010).

At the plasma membrane, Swa is found in close proximity to the cortical actin cytoskeleton. Together with the defects in the cortical actin cytoskeleton observed in swa mutants, this indicates a role for Swa in organization of the actin cytoskeleton. Whereas the actin cytoskeleton is not required for anchoring bcd at the anterior cortex until the very end of oogenesis (Weil, 2008), it plays an indirect role in bcd localization by anchoring the anterior microtubules required for the continual transport of bcd during stages 11-13. The results indicate that, in swa mutants, these microtubules remain intact but are not properly attached to or organized at the cortex, such that bcd transport is non-productive (Weil, 2010).

Swa is not limited to the anterior, however, and can be detected along the entire cortex of the oocyte. Moreover, in swa mutants, the cortical actin cytoskeleton is disrupted throughout the entire oocyte. Accordingly, some swa alleles show defects in posterior localization of osk mRNA at late stages of oogenesis (Pokrywka, 2004). Since actin is required for osk mRNA anchoring, this phenotype could be a result of disruption of the actin cytoskeleton. Clues as to how Swa regulates the actin cytoskeleton are not readily apparent from the Swa protein sequence, and an understanding of this mechanism awaits further biochemical analysis of Swa (Weil, 2010).

IEM results provide direct evidence that bcd mRNA is packaged with Stau and dynein into RNPs that are enriched at, and presumably transported to, the anterior after stage 10b. Indeed, live imaging using the OMX system showed co-transport of bcd and Stau in the same dynamic particles. In addition to its role in bcd localization, Stau is a key component of osk RNPs and is required for kinesin-dependent transport of osk to the oocyte posterior. Thus, Stau is a component of two independent transport RNPs, each associated with a different motor protein for transport to distinct locations within the oocyte. Whether Stau, which contains five double-stranded RNA-binding domains, interacts directly with bcd and osk mRNAs or indirectly by association with sequence-specific mRNA-binding proteins, remains to be determined. However, structure/function analysis of Stau suggests that different Stau double-stranded RNA-binding domains may determine which transport factors are linked to each mRNA. Furthermore, as Stau functions in osk localization during stages 8-9 but is required for bcd localization only after stage 10b, the assembly of Stau/bcd transport complexes must be temporally regulated. Stau is also required during the oocyte-to-embryo transition, for the redistribution of bcd from its tight cortical distribution in the oocyte to its more diffuse anterior distribution in the early embryo. Stau has been shown to colocalize with bcd mRNA when it is anchored to the actin cytoskeleton at the latest stages of oogenesis and is retained by bcd particles in the early embryo (Weil, 2008). Thus, Stau remains an integral component of bcd RNPs as they are remodeled from transport to anchoring complexes and finally to their translationally active state in the embryo. In the future, detailed biochemical analysis combined with advanced imaging methods that permit detection of in vivo RNA-protein and protein-protein interactions will be necessary to ascertain how specificity is conferred on localization and anchoring (Weil, 2010).

Landskron, L., Steinmann, V., Bonnay, F., Burkard, T. R., Steinmann, J., Reichardt, I., Harzer, H., Laurenson, A. S., Reichert, H. and Knoblich, J. A. (2018). The asymmetrically segregating lncRNA cherub is required for transforming stem cells into malignant cells. Elife 7. PubMed ID: 29580384



The asymmetrically segregating lncRNA cherub is required for transforming stem cells into malignant cells

Tumor cells display features that are not found in healthy cells. How they become immortal and how their specific features can be exploited to combat tumorigenesis are key questions in tumor biology.This study describes the long non-coding RNA cherub (long non-coding RNA:CR43283) that is critically required for the development of brain tumors in Drosophila but is dispensable for normal development. In mitotic Drosophila neural stem cells, cherub localizes to the cell periphery and segregates into the differentiating daughter cell. During tumorigenesis, de-differentiation of cherub-high cells leads to the formation of tumorigenic stem cells that accumulate abnormally high cherub levels. cherub establishes a molecular link between the RNA-binding proteins Staufen and Syncrip. As Syncrip is part of the molecular machinery specifying temporal identity in neural stem cells, it is proposed that tumor cells proliferate indefinitely, because cherub accumulation no longer allows them to complete their temporal neurogenesis program (Landskron, 2018).

Throughout the animal kingdom, stem cells supply tissues with specialized cells. They can do this because they have the unique ability to both replicate themselves (an ability termed self-renewal) and to simultaneously generate other daughter cells with a more restricted developmental potential. Besides their role in tissue homeostasis, stem cells have also been linked to tumor formation. They can turn into so-called tumor stem cells that sustain tumor growth indefinitely. The mechanisms that endow tumor stem cells with indefinite proliferation potential are not fully understood (Landskron, 2018).

Most Drosophila brain tumors originate from the so-called type II neuroblasts (NBIIs). NBIIs divide asymmetrically into a larger cell that retains NB characteristics and a smaller intermediate neural progenitor (INP). Newly formed immature INPs (iINPs) go through a defined set of maturation steps to become transit-amplifying mature INPs (mINPs). After this, a mINP undergoes 3-6 divisions generating one mINP and one ganglion mother cell (GMC) that in turn divides into two terminally differentiating neurons or glial cells (Landskron, 2018).

During each NBII division, a set of cell fate determinants is segregated into the INP. Among those are the Notch inhibitor Numb and the TRIM-NHL protein Brain tumor (Brat). Loss of these cell fate determinants leads to the generation of ectopic NB-like cells at the expense of differentiated brain cells. Formation of malignant brain tumors has also been observed upon the depletion of downstream factors that normally maintain the INP fate (Landskron, 2018).

These features make Drosophila a model for the stepwise acquisition of tumor stem cell properties. When numb or brat are inactivated, the smaller NBII progeny fails to establish an INP fate and initially enters a long transient cell cycle arrest. Only after this lag period, the smaller cell regrows to a NB-sized cell that has acquired tumor stem cell properties and that it is therefore refered to as tumor neuroblast (tNB). NBIIs and ectopic tNBs are indistinguishable in terms of markers. Both cell populations are characterized by the expression of self-renewal genes and lack differentiation markers, but nevertheless behave differently. Shortly after entering pupal stages, NBs decrease their cell volumes successively with each NB division before they exit the cell cycle and differentiate. However, tNBs do not shrink during metamorphosis and continue to proliferate even in the adult fly brain. Moreover, in contrast to wild-type brains, the resulting tumor brains can be serially transplanted into host flies for years, indicating the immortality of these tumors (Landskron, 2018).

Similarly, mammalian homologues of numb and brat have been shown to inhibit tumor growth. Furthermore, the human brat homologue TRIM3 is depleted in 24% of gliomas and NUMB protein levels are markedly reduced in 55% of breast tumor cases. Therefore, results obtained in these Drosophila tumor models are highly relevant (Landskron, 2018).

This study used the Drosophila brat tumor model to investigate how tNBs differ from their physiological counterparts, the NBIIs. The results indicate that progression towards a malignant state is an intrinsic process in brat tNBs that does not correlate with stepwise acquisition of DNA alterations. Transcriptome profiling of larval NBs identified the previously uncharacterized long non-coding (lnc) RNA cherub as crucial for tumorigenesis, but largely dispensable for NB development. The data show that cherub is the first identified lncRNA to be asymmetrically segregated during mitosis into INPs, where the initial high cherub levels decrease with time. Upon the loss of brat, the smaller cherub-high cell reverts into an ectopic tNBs resulting in tumors with high cortical cherub. Molecularly, cherub facilitates the binding between the RNA-binding protein Staufen and the late temporal identity factor Syp and consequently tethers Syp to the plasma membrane. Depleting cherub in brat tNBs leads to the release of Syp from the cortex into the cytoplasm and represses tumor growth. These data provide insight into how defects in asymmetric cell division can contribute to the acquisition of tumorigenic traits without the need of DNA alterations (Landskron, 2018).

It is commonly assumed that cancer cells become malignant and gain replicative immortality by acquiring genetic lesions. Surprisingly, however, the current data indicate that brat tumors do not require additional genetic lesions for the transition to an immortal state. This is not a general feature of Drosophila tumors as genomic instability alone can induce tumors in Drosophila epithelial cells and intestinal stem cells. However, the current results are supported by previous experiments demonstrating that defects in genome integrity do not contribute to primary tumor formation in NBs. Similarly, tumors induced by loss of epigenetic regulators in Drosophila wing discs do not display genome instability. In addition, the short time it takes from the inactivation of brat to the formation of a fully penetrant tumor phenotype would most likely be insufficient for the acquisition of tumor-promoting DNA alterations. More likely, the enormous self-renewal capacity and fast cell cycle of Drosophila NBs requires only minor alterations for the adoption of malignant growth. Interestingly, epigenetic tumorigenesis has been described before in humans, where childhood brain tumors only harbor an extremely low mutation rate and very few recurrent DNA alterations. Comparable observations have been made for leukemia. The current results might help to understand mechanisms of epigenetic tumor formation, which are currently unclear in humans (Landskron, 2018).

cherub is the first lncRNA described to segregate asymmetrically during mitosis. Once cherub is allocated through binding to the RNA-binding protein Staufen into the cytoplasm of INPs, its levels decrease over time. The results show that the inability to segregate cherub into differentiating cells leads to its accumulation in tNBs. The increasing amount of tumor transcriptome data indicates that a vast number of lncRNAs show increased expression levels in various tumor types. Intriguingly, the mammalian homologue of cherub's binding partner Staufen has been also described to asymmetrically localize RNA in dividing neural stem cells. Hence, besides transcriptional upregulation, asymmetric distribution of lncRNAs between sibling cells might play a role in the accumulation of such RNAs in mammalian tumors (Landskron, 2018).

The data suggest a functional connection between cherub and proteins involved in temporal neural stem cell patterning. This study that tNBs retain the early temporal identity factor Imp even during late larval stages. However, IGF-II mRNA-binding protein (Imp) expression in brat mutants is heterogeneous and only a subpopulation of tNBs maintains young identity (Landskron, 2018).

Tumor heterogeneity has also been described for pros tumors, where only a subset of tNBs maintains expression of the early temporal factors Imp and Chinmo. Interestingly, it is this subpopulation that drives tumor growth in prospero tumors (Landskron, 2018).

Consistent with this, genetic experiments show that 'rejuvenating' tNBs enhances tumor growth and consequently increases the survival of tumor bearing flies, whereas 'aging' tNBs identity has the opposite effect. Although mammalian counterparts of Imp have not yet being shown to act as temporal identity genes, their upregulated expression has been implied in various cancer types. Therefore, temporal patterning of NBs has an essential role in brain tumor propagation in Drosophila (Landskron, 2018).

The subset of tNBs that retain early identity in tumors is lost in a cherub mutant background. This suggests that cherub itself might regulate temporal identity. In NBs and tNBs cherub regulates Syp localization by facilitating the binding of Syp to Staufen and thus recruiting it to the cell cortex. In tumors depleted of cherub, Syp localizes mainly to the cytoplasm and is no longer observed at the cortex. As the removal of Syp in tNBs leads to enhanced tumor growth and early lethality, those data suggest that cherub could control temporal NB identity by regulating the subcellular localization of Syp(Landskron, 2018).

How could cherub regulate the function of Syp? The RNA-binding protein Syp is a translational regulator and has been suggested to control mRNA stability. As mammalian SYNCRIP/hnRNP Q interacts with a lncRNA that suppresses translation, cherub might regulate Syp to inhibit or promote the translation of a subset of target mRNAs. In particular, in NBs Syp acts at two stages in NBs during development: Firstly, approximately 60 hr after larval hatching it represses early temporal NB factors, like Imp. Secondly, at the end of the NB lifespan Syp promotes levels of the differentiation factor prospero to facilitate the NB's final cell cycle exit. As cherub depletion in brat tumors leads to decreased tumor growth, it is possible that cherub inhibits the Syp-dependent repression of the early factor Imp, which this study shows to be required for optimal tumor growth. However, cherub mutant NBIIs do not show altered timing or expression of Imp during development. In accordance, brat tumors show high cortical cherub levels, but only a subset of NBs expresses Imp. Rather than rendering Syp completely inactive, it is suggested that cherub decreases the ability of Syp to promote factors important to restrict NB proliferation. As prospero is not expressed in NBIIs, it remains to be investigated which Syp targets are affected by cherub (Landskron, 2018).

Remarkably, cherub mutants are viable, fertile and do not affect NBII lineages. Neurons generated by NBIIs predominantly integrate into the adult brain structure termed central complex, which is important for locomotor activity. As cherub mutants show normal geotaxis, function of the lncRNA seems dispensable for NBIIs to generate their neural descendants (Landskron, 2018).

Nevertheless, the conserved secondary RNA structures of cherub and its conserved expression pattern in other Drosophila species suggest that it has a functional role. There are several possibilities why no phenotype is observed upon the loss of cherub. In wild-type flies cherub might confer robustness. A similar scenario was observed in embryonic NBs, in which Staufen segregates prospero mRNA into GMCs. The failure to segregate prospero mRNA does not result in a phenotype, but it enhances the hypomorphic prospero GMC phenotype. Thus segregation of prospero mRNA serves as support for Prospero protein to induce a GMC fate. Similarly, cherub could act as a backup to reliably establish correct Syp levels in NBIIs and in INPs. Alternatively, cherub might fine-tune the temporal patterning by regulating the cytoplasmic pool of Syp in the NBs. Increasing Syp levels have been suggested to determine distinct temporal windows, in which different INPs and ultimately neurons with various morphologies are sequentially born. Therefore, it cannot be excluded that changes in Syp levels lead to subtle alterations in the number of certain neuron classes produced by NBIIs that only reveal themselves in pathological conditions like tumorigenesis (Landskron, 2018).

This study illustrates how a lncRNA can control the subcellular localization of temporal factors. In addition to temporal NB identity, Syp regulates synaptic transmission and maternal RNA localization. While cherub is not expressed in ovaries or adult heads, Staufen has been implicated in these processes, suggesting that other RNAs might act similarly to cherub. Interestingly, the mammalian Syp homolog hnRNP Q binds the noncoding RNA BC200, whose upregulation is used as a biomarker in ovarian, esophageal, breast and brain cancer. In the future, it will be interesting to investigate whether the mechanism identified in Drosophila is involved in mammalian tumorigenesis as well (Landskron, 2018).

RNA-binding FMRP and Staufen sequentially regulate the coracle scaffold to control synaptic glutamate receptor and bouton development

Both mRNA-binding Fragile X Mental Retardation Protein (FMRP) and mRNA-binding Staufen regulate synaptic bouton formation and glutamate receptor (GluR) levels at the Drosophila neuromuscular junction (NMJ) glutamatergic synapse. This study tested whether these RNA-binding proteins (RBPs) act jointly in a common mechanism. Both dfmr1 and staufen mutants, and trans-heterozygous double mutants, were shown to display increased synaptic bouton formation and GluRIIA accumulation. With cell-targeted RNAi, a downstream Staufen role within postsynaptic muscle. With immunoprecipitation, this study showed that FMRP binds staufen mRNA to stabilize postsynaptic transcripts. Staufen is known to target actin-binding, GluRIIA anchor Coracle, and this study confirmed that Staufen binds to coracle mRNA. FMRP and Staufen were shown to act sequentially to co-regulate postsynaptic Coracle expression, and show Coracle, in turn, controls GluRIIA levels and synaptic bouton development. Consistently, this study found dfmr1, staufen and coracle mutants elevate neurotransmission strength. FMRP, Staufen and Coracle all suppress pMad activation, providing a trans-synaptic signaling linkage between postsynaptic GluRIIA levels and presynaptic bouton development. This work supports an FMRP-Staufen-Coracle-GluRIIA-pMad pathway regulating structural and functional synapse development (Song, 2022).

This study reveals the mechanism of the established FMRP negative regulation of postsynaptic GluRIIA receptors and presynaptic bouton formation in the Drosophila FXS disease model. Specifically, the mRNA-binding FMRP-positive translational regulator binds to staufen mRNA as predicted, within the postsynaptic cell. Consequently, both dfmr1 and staufen mutants share the elevated GluRIIA level and bouton number phenotypes based on a common postsynaptic pathway function, and genetically interact as trans-heterozygotes to reproduce these phenotypes. Staufen acts as a dsRBP to bind coracle mRNA as predicted; both dfmr1 and staufen mutants exhibit elevated postsynaptic Coracle levels, and genetically interact as trans-heterozygotes to reproduce this phenotype. Coracle acts as a GluRIIA-binding anchoring scaffold within the postsynaptic domain to regulate local receptor accumulation (Chen, 2005). Consequently, dfmr1, staufen and coracle mutants all increase NMJ synaptic functional differentiation to elevate neurotransmission strength. Finally, the elevated postsynaptic GluRIIA levels mediate retrograde BMP receptor trans-synaptic signaling that induces pMad to drive new presynaptic bouton development. dfmr1, staufen and coracle mutants all exhibit elevated presynaptic pMad levels, thereby linking the postsynaptic GluRIIA accumulation and presynaptic supernumerary bouton formation defects shared by all of these mutants (Song, 2022).

The staufen mutant increased synaptic Coracle levels, GluRIIA levels and bouton number are all internally consistent. In a previous study, opposite phenotypes were measured in staufenHL/Df(2R)Pcl7B, which reduces another 14 genes in heterozygous deficiency, including loci involved in neuronal development (e.g. grh, nopo). Importantly, this study similarly found reduced synaptic protein levels and bouton number in staufenHL/Df(2R)Pcl7B, suggesting that heterozygosity of one or more of the neighboring genes impairs synaptic development. However, this study showed that a staufen RNAi that reduces transcript levels by ~90% replicates the staufen mutant NMJ phenotypes of increased GluRIIA levels and synaptic bouton numbers. This was also replicated with a second, independent staufen RNAi line. Moreover, this study showed that the effect is entirely restricted to postsynaptic muscle RNAi, with no effect from presynaptic neuron RNAi, consistent with restricted postsynaptic Staufen function. In addition, postsynaptic staufen rescue of the staufen mutant restored normal synaptic bouton formation, with OE reducing GluRIIA levels in staufen mutants and rescuing GluRIIA levels in dfmr1 mutants. Both staufen mutants and postsynaptic staufen RNAi also share the arrested supernumerary satellite bouton development characterizing dfmr1 null mutants. These many independent lines of evidence confirm the results, and are consistent with the known parallel FMRP role in restricting GluRIIA levels and synaptic bouton formation (Song, 2022).

To regulate Staufen, FMRP binds staufen mRNA and protects targeted staufen transcripts from degradation. FMRP contains at least three distinct RNA-binding domains (RBDs), and Staufen has five RBDs. Staufen reportedly binds a specific RNA hairpin structure formed by long 3' UTRs, but RIP shows that Staufen also binds mRNAs that are not predicted to generate this secondary structure. Although the decreased staufen mRNA levels in both dfmr1 mutants and muscle-targeted dfmr1 RNAi are predicted to be due to the lack of FMRP binding, it is also possible that other unregulated interactors cause the downregulated staufen mRNA expression (Shah et al., 2020). Localized labeling with an anti-Staufen antibody has been reported in the postsynaptic NMJ, which can be confirmed, but it was not possible to reduce labeling in staufen hypomorphic mutants. Therefore Staufen labeling was not shown in the current study. Moreover, western blots have been reported with the same anti-Staufen antibody; however, attempts were unsuccessful. Therefore qPCR was used to measure staufen mRNA levels. Staufen binds to coracle mRNA, but does so in a non-selective manner. This result is consistent with Staufen acting as a very broad spectrum dsRBP, and suggests that Staufen likely acts with a translational regulator partner to generate specificity. FMRP is very well established to partner with other RBPs to mediate the translational regulation of its target transcripts (Song, 2022). ------

The postsynaptic Coracle scaffold acts in a GluRIIA local anchoring mechanism, presumably to link the receptors to the underlying actin cytoskeleton (Chen, 2005). The jointly elevated Coracle and GluRIIA levels in both dfmr1 and staufen mutants are consistent with this scaffold function. Because the dfmr1/+; staufen/+ trans-heterozygotes share this correlated Coracle and GluRIIA upregulation in the postsynaptic domain, a single common signaling pathway is indicated. Coracle also restricts terminal branching development in peripheral sensory neurons. Both coracle mutants and sensory neuron-targeted coracle RNAi also display increased dendritic branch and termini numbers. These phenotypes are similar to the expanded NMJ terminals and increased synaptic bouton development reported in this study. Importantly, both coracle loss of function (mutants and muscle-targeted RNAi) and gain of function (muscle-targeted OE) increase postsynaptic GluRIIA levels and generate supernumerary boutons. Likewise, the knockdown and OE of many other similar scaffolds are known to cause phenocopying defects. Some examples include the muscle chaperone UNC-45, the tight junction scaffold zonula occludens-1 (ZO-1) and synaptic UNC-13. Indeed, both coracle loss and OE similarly cause increased dendritic crossing in Drosophila sensory neurons, similar to the phenocopy of developmental defects reported in this study. Combining the roles of postsynaptic FMRP-Staufen-Coracle in GluRIIA clustering, it was reasoned that this pathway must be a regulatory determinant of synaptic functional development (Song, 2022).

Removing FMRP, Staufen and Coracle strongly enhances functional synaptic differentiation and NMJ neurotransmission strength. This is consistent with expectations from the postsynaptic GluRIIA accumulation in all of these mutants. Elevated GluRIIA levels are well known to be associated with increased evoked functional responses and prolonged channel open times. A GluRIIA pore sequence (MQQ) critically required for the Drosophila channel Ca2+ permeability is conserved in mammalian receptors. This selectivity allows Ca2+-dependent participation in spontaneous (mEJC) and evoked (EJC) neurotransmission. Although enhanced evoked EJC amplitudes are typically accompanied by mEJC alterations, this study found that mEJC amplitude and frequency are unchanged in both the staufen and coracle mutants, and show only minimal changes in the dfmr1 mutants. Classically, both evoked and spontaneous neurotransmission were thought to be mediated by the same vesicles; however, more recent evidence has indicated that spontaneous and evoked neurotransmission have distinct machinery and vesicle pools. Postsynaptic receptors can be segregated into different compartments that are activated by either spontaneous or evoked release. This work supports this growing body of evidence for differential regulation. Importantly, GluRIIA has unique functions, modulating both presynaptic glutamate release and presynaptic bouton development (Song, 2022).

The dfmr1, staufen and coracle mutants all showed upregulated presynaptic pMad correlated with postsynaptic activated GluRIIA accumulation. GluRIIA activation triggers presynaptic pMad signaling via BMP receptors surrounding active zones, which, in turn, stabilizes GluRIIA receptors in the postsynaptic domains. This trans-synaptic signaling mechanism induces new presynaptic bouton development. The targeted postsynaptic RNAi for all three genes confirms this intercellular link. Synaptic BMP signaling involves both the type I serine/threonine kinase receptors and the type II receptor Wit. Although BMP ligand Glass bottom boat (Gbb) signaling via Wit presynaptic receptors is well established at the NMJ to modulate synaptogenesis, the mechanism of presynaptic bouton formation induced by activated GluRIIA signaling does not involve canonical BMP signaling via Gbb. In the dfmr1 mutants, it is suggested that postsynaptic GluRIIA accumulation induces presynaptic bouton development via non-canonical GluRIIA-Wit trans-synaptic retrograde signaling. Similarly, the muscle postsynaptic glypican Dally-like protein (Dlp) negatively regulates NMJ synaptic development by inhibiting this same non-canonical BMP pathway through decreased activated GluRIIA expression. Postsynaptic GluRIIA clustering can thus trigger presynaptic bouton formation, although supernumerary boutons do not always induce reciprocal GluRIIA changes. It is concluded that an FMRP-Staufen-Coracle-GluRIIA-pMad pathway regulates intertwined structural and functional glutamatergic synapse development (Song, 2022).

An RNA-based feed-forward mechanism ensures motor switching in oskar mRNA transport

Regulated recruitment and activity of motor proteins is essential for intracellular transport of cargoes, including messenger ribonucleoprotein complexes (RNPs). This study shows that orchestration of oskar RNP transport in the Drosophila germline relies on interplay between two double-stranded RNA-binding proteins, Staufen and the dynein adaptor Egalitarian (Egl). Staufen antagonizes Egl-mediated transport of oskar mRNA by dynein both in vitro and in vivo. Following delivery of nurse cell-synthesized oskar mRNA into the oocyte by dynein, recruitment of Staufen to the RNPs results in dissociation of Egl and a switch to kinesin-1-mediated translocation of the mRNA to its final destination at the posterior pole of the oocyte. This study additionally showed that Egl associates with staufen (stau) mRNA in the nurse cells, mediating its enrichment and translation in the ooplasm. These observations identify a novel feed-forward mechanism, whereby dynein-dependent accumulation of stau mRNA, and thus protein, in the oocyte enables motor switching on oskar RNPs by downregulating dynein activity (Gaspar, 2023).

The activity of motor molecules is essential for establishing proper intracellular localization of mRNA molecules, which in turn underlies spatiotemporal restriction of protein function. The dynein and kinesin microtubule motors play key roles in positioning of mRNAs in many systems, including by acting sequentially on the same RNP species. However, it is unclear how the opposing activities of these motors are coordinated during RNP trafficking (Gaspar, 2023).

This study used the tractable Drosophila egg chamber to reveal a mechanism for spatiotemporal control of dynein and kinesin-1 activity. Central to this system are two dsRBPs, Staufen, and Egl. Genetic interaction experiments have previously shown that these proteins have opposing activities in the context of oskar mRNA localization in the oocyte and anteroposterior patterning, and both proteins were shown to complex with oskar mRNA. Recent work (Mohr, 2021) further corroborated the ability of Egl to antagonize localization of oskar to the posterior of the oocyte. Mohr (2021) also showed that Egl binds in vitro to a double-stranded region in the oskar 3' UTR that can enrich the mRNA at the anterior cortex of the oocyte, which they termed the Transport and Anchoring Signal (TAS). The TAS partially overlaps with one of the Staufen Recognized Structures (SRS) which are important for Staufen binding in vitro and for posterior oskar mRNA localization in the oocyte. These data suggest that Staufen could antagonize Egl function by interfering with binding of the latter protein to oskar mRNA. However, Mohr (2021) did not directly test this possibility. This study found that knocking down Staufen increases the association of Egl with the mRNA, demonstrating the role of Staufen in antagonizing association of Egl with oskar mRNA. The results of current in vitro motility assays are also consistent with this scenario, as RNA binding to Egl is a prerequisite for full dynein activity within this minimal RNP. It is conceivable that the transport and anchoring functions ascribed to the TAS are two facets of the same underlying molecular mechanism, as increased dynein activity in the absence of Staufen would drive enrichment of oskar RNPs at microtubule minus ends that are nucleated at the anterior cortex (Gaspar, 2023).

This study additionally reveals, through smFISH-based colocalization analysis in egg chambers, how the interactions of Staufen and Egl with oskar mRNA are orchestrated in time and space. When Staufen levels are low, such as in early oogenesis, amounts of Egl per RNP scale with oskar mRNA content. During stage 9, this scaling is lost in the oocyte and the relative amount of Egl on oskar RNPs decreases as Staufen is recruited to oskar. The data indicate that Staufen-mediated displacement of Egl is a critical step in switching to kinesin-1-based trafficking of oskar mRNA to the posterior pole. This switch is also likely to involve the activities of exon junction complex components, which are required for posterior localization of oskar. This study provides a framework for understanding how the activities of these factors are coordinated with that of Staufen (Gaspar, 2023).

Intriguingly, whilst an excess of Staufen can dissociate Egl from oskar RNPs, increasing Egl concentration does not overtly affect Staufen recruitment to these structures. This observation might be due to the five-to-one excess of SRSs over the TAS in each oskar molecule (Mohr, 2021), which could mask the loss of Staufen binding to the TAS-proximal SRS (Gaspar, 2023).

This also found that dissociation of Egl from oskar RNPs in the stage 9 oocyte does not detectably alter the amount of dynein on these structures. This work lends in vivo support to the notion that additional proteins or RNA sequences can recruit dynein and dynactin in an inactive state to RNPs. Presumably, Egl recruits only a small fraction of the total number of dynein complexes on oskar RNPs or activates the motility of complexes that are linked to the RNA by other factors. Unexpectedly, this study found that the bulk association of BicD with oskar RNPs is also not dependent on Egl, pointing to an additional mechanism for recruiting BicD, presumably in the autoinhibited state. While the relative amount of the dynein machinery associated with oskar RNPs is greater in the oocyte than the nurse cells, association of the motor complex with oskar RNPs in the oocyte does not scale proportionally with RNA content. How the amount of dynein recruited to these RNPs could be limited is unclear. However, this mechanism could be a means to prevent sequestration of this multi-functional motor to a very abundant cargo (~0.5-1 million copies of oskar mRNA per oocyte) (Gaspar, 2023).

Whilst the current data build a strong case that a key function of Staufen in oskar mRNA localization is to limit dynein activity by displacing Egl, several lines of evidence suggest that this is not its only role in this context. It was found that Staufen had a partial inhibitory effect on minus-end-directed motility of purified dynein-dynactin complexes activated in an Egl-independent manner by a constitutively active truncation of a BicD protein. Indeed, polarity defects observed in oocytes strongly overexpressing Staufen may be due to direct interference with dynein function. Moreover, Mohr (2021) found that the SRSs that are not proximal to the TAS in the oskar sequence also contribute to posterior localization of the mRNA in a Staufen-dependent manner. Although it is possible that these elements are close enough to the binding site for Egl-BicD-dynein-dynactin in the folded RNA molecule to interfere directly with the assembly or activity of the complex, they could also regulate oskar mRNA distribution through an independent mechanism. Other observations in this study do hint at other roles of Staufen. It was observed that when Staufen is depleted, motile oskar RNPs tend to have a lower RNA content, suggesting an additional function of the protein in RNA oligomerization. Furthermore, whilst the magnitude of the effect was much smaller than for minus-end-directed motion, there was increased plus-end-directed velocity of a subset of oskar RNPs in ex vivo motility assays when Staufen was disrupted. Although this observation could reflect dynein's ability to promote kinesin-1 activity, it is also possible that Staufen directly tunes the activity of the plus-end-directed motor (Gaspar, 2023).

An important question raised by this analysis of Staufen's effects on oskar mRNA transport was how the timing and location of this process is controlled. Evidence is provided that this is based on another mRNA localization process in which Egl, as part of stau RNPs, is responsible for the enrichment of stau mRNA in the developing oocyte. It is proposed that this mechanism constitutes a feed-forward type of switch, whereby the activity of the dynein-mediated transport machinery deploys its own negative regulator to a distant location. The resultant increase in Staufen levels in the ooplasm prevents dynein-mediated transport of oskar to the anterior of the oocyte, while the almost complete absence of the protein from the nurse cells is likely to be important for uninterrupted transport of oskar RNPs into the oocyte by dynein during early- and mid-oogenesis. Presumably, stau translation is suppressed during transit into the oocyte or the protein is translated en route but only builds up to meaningful levels where the RNA is concentrated in the oocyte. Staufen protein might also modulate its own localization in the ooplasm by antagonizing the association of stau mRNA with Egl, and thereby its minus-end-directed motility. Consistent with this notion, the level of Egl on stau RNPs declines at a similar stage to when Staufen inhibits the association of Egl with oskar RNPs. As Staufen controls localization and translation of other mRNAs in the maturing oocyte, mRNA-based regulation of the protein distribution in the egg chamber is likely to have functions that extend beyond orchestrating oskar mRNA localization. Given the functional conservation of Staufen protein and the observation that the mRNA encoding mStau2 is localized in dendrites of mammalian neurons, it is plausible that the feed-forward loop established by stau RNA localization during Drosophila oogenesis is an evolutionarily conserved process that controls RNA trafficking and protein expression in polarized cells (Gaspar, 2023).

GENE STRUCTURE

Bases in 5' UTR -274

Bases in 3' UTR - 1885

PROTEIN STRUCTURE

Amino Acids - 1026

Structural Domains

The protein is fairly basic (9.9% lysine and arginine) and rich in proline (9.3%) (St Johnston, 1991).

NMR spectroscopy has been used to determine the secondary structure of one of the double-stranded RNA binding domains from the Drosophila protein Staufen. The domain has an alpha beta beta beta alpha arrangement of secondary structure, with the beta strands forming an antiparallel beta sheet. The secondary structure differs from that found in the RNP RNA binding domain (Bycroft, 1995a).

The double-stranded RNA binding domain (dsRBD) is an approximately 65 amino acid motif that is found in a variety of proteins that interact with double-stranded (ds) RNA, such as Escherichia coli RNase III and the dsRNA-dependent kinase, PKR. Drosophila Staufen protein contains five copies of this motif, and the third of these binds dsRNA in vitro. Using multinuclear/multidimensional NMR methods, it has been determined that Staufen dsRBD3 forms a compact protein domain with an alpha-beta-beta-beta-alpha structure in which the two alpha-helices lie on one face of a three-stranded anti-parallel beta-sheet. This structure is very similar to that of the N-terminal domain of a prokaryotic ribosomal protein S5. Furthermore, the consensus derived from all known S5p family sequences shares several conserved residues with the dsRBD consensus sequence, indicating that the two domains share a common evolutionary origin. Using in vitro mutagenesis, several surface residues have been identified which are important for the RNA binding of the dsRBD, and these all lie on the same side of the domain. Two residues that are essential for RNA binding, F32 and K50, are also conserved in the S5 protein family, suggesting that the two domains interact with RNA in a similar way (Bycroft, 1995b).

date revised:  25 October 2023 

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.