InteractiveFly: GeneBrief

belle: Biological Overview | References

BIOLOGICAL OVERVIEW

Belle (Bel), the Drosophila homolog of the yeast DEAD-box RNA helicase DED1 and human DDX3, has been shown to be required for oogenesis and female fertility. This study reports a novel role of Bel in regulating the expression of transgenes. Abrogation of Bel by mutations or RNAi induces silencing of a variety of P-element-derived transgenes. This silencing effect depends on downregulation of their RNA levels. Genetic studies have revealed that the RNA helicase Spindle-E (Spn-E), a nuage RNA helicase that plays a crucial role in regulating RNA processing and PIWI-interacting RNA (piRNA) biogenesis in germline cells, is required for loss-of-bel-induced transgene silencing. Conversely, Bel abrogation alleviates the nuage-protein mislocalization phenotype in spn-E mutants, suggesting a competitive relationship between these two RNA helicases. Additionally, disruption of the chromatin remodeling factor Mod(mdg4) or the microRNA biogenesis enzyme Dicer-1 (Dcr-1) also alleviates the transgene-silencing phenotypes in bel mutants, suggesting the involvement of chromatin remodeling and microRNA biogenesis in loss-of-bel-induced transgene silencing. Finally genetic inhibition of Bel function was shown to lead to de novo generation of piRNAs from the transgene region inserted in the genome, suggesting a potential piRNA-dependent mechanism that may mediate transgene silencing as Bel function is inhibited (Lo, 2016).

Transgene silencing refers to the activity of various host defense responses that ordinarily act on natural foreign or parasitic sequences such as transposable elements (TEs), viroids, RNA and DNA viruses, and bacterial DNA. Since transgenes or their transcripts can resemble these cellular invaders in a number of ways, they naturally become the targets of host protective reactions. There are at least two distinct host defense systems responsible for silencing transgenes. One performs its effect via de novo DNA methylation at the genome level. The second defense system operates post-transcriptionally to silence transgenes, which involves sequence-specific RNA degradation in the cytoplasm. Therefore, transgene silencing involves complex cell immune systems including epigenetic and RNA silencing mechanisms. Although many factors involved in transgene silencing have been identified, and several mechanisms have been proposed, there remains much to understand regarding this vital aspect of the cell immune system (Lo, 2016).

Drosophila oogenesis, which involves the generation of the female gamete (oocyte), nurse cells, and follicle cells, is an excellent system for the study of TE and transgene silencing. The egg chamber, the developmental unit of oogenesis, contains the germline cells (one oocyte and 15 nurse cells) and a layer of surrounding somatically derived epithelial follicle cells. Both the germline cells and follicle cells can produce small RNAs to silence TE expression. The nuage, a perinuclear structure within Drosophila nurse cells, is an RNA-rich organelle unique to the germline. The nuage is required for the processing and localization of germline mRNAs and for the biogenesis of PIWI-interacting RNAs (piRNAs), a class of small non-coding RNAs that function as the cell immune system for silencing TEs. In D. melanogaster, most primary piRNAs are produced from discrete pericentromeric and telomeric heterochromatic loci (called piRNA clusters) containing damaged repeated TE sequences. In fly germline cells, an additional step of piRNA biogenesis, the 'ping-pong cycle' mechanism, is employed to generate the secondary piRNAs. Multiple factors localized in the nuage of germline cells have been discovered to be essential for secondary piRNA biogenesis, including Aub, AGO3, Spindle-E (Spn-E), and Vasa. In follicle cells, piRNAs are only produced from piRNA clusters (e.g., flamenco ) via PIWI and other related nuclear factors, and there is no secondary piRNA biogenesis involved. Intriguingly, besides piRNA clusters, euchromatic transposon insertion sites have been identified as another origin to produce piRNAs and endo-siRNAs. This mechanism provides another layer of defense to suppress TE activity and can also serve as a way to affect expression of coding genes and microRNA (miRNA) genes adjacent to inserted TEs (Lo, 2016).

Vasa and Spn-E belong to a family of DEAD-box proteins defined by multiple distinct conserved motifs including the D-E-A-D (Asp-Glu-Ala-Asp) motif. Among the identified DEAD-box proteins, one subfamily is highly conserved from yeast to human, which includes orthologs in yeast (DED1), Drosophila (Belle (Bel)), Xenopus (An3), mice (PL10), and humans (DDX3). These DEAD-box subfamily proteins possess the ATP-dependent RNA helicase activity to unwind double-stranded RNA and remodel RNA-protein interactions. Yeast DED1 is a multifunctional protein that functions to regulate multiple stages of RNA processing and translation. DED1 has also been shown to play a specific role in cell-cycle control. DDX3, the human homolog of DED1, is known to be involved in modulating multiple biological processes, including antiviral innate immunity, mitotic chromosome segregation in somatic cells, the suppression of spermatogenesis, G1-S transition of the cell cycle, epithelial-mesenchymal transition (EMT), a bona fide component of the RNAi pathway, TNF-related apoptosis, and WNT signaling (Lo, 2016 and references therein).

Vasa, a paralog of Bel, is required for the formation and function of nuage to suppress TE expression by being involved in the production of piRNAs. Most recently, Xiol (2014) reported that Vasa is a key component in the piRNA amplifier complex in the nuage and serves as a protein platform to recruit PIWI proteins, the Tudor protein Qin/Kumo and antisense piRNA guides in an ATP-dependent manner for the ping-pong-loop amplification of secondary piRNAs. Bel colocalizes with Vasa in the nuage and at the oocyte posterior during oogenesis, and is required for female fertility (Johnstone, 2005). Recent findings have shown that loss of bel delays activation of Notch signaling in follicle cells, which in turn leads to delayed cell differentiation and defects in the switch from the mitotic cycle to the endocycle (Poulton, 2011). However, unlike Vasa, the specific roles of Bel in the nuage of germline cells, and whether it is involved in piRNA biogenesis, remain unknown (Lo, 2016).

From previous studies, it was unexpectedly found that the Gal4-driven expression of a UASp-Bel:GFP transgene was silenced in bel mutant germline cells. This silencing effect was not specific to bel-based transgenes because 13 out of 22 different transgenic lines tested could be silenced in either germline or somatic bel mutant cells, or both. Subsequently the RNA helicase Spn-E, the epigenetic regulator Modifier of mdg4 [Mod(mdg4)] and the miRNA biogenesis enzyme Dcr-1 was identified as crucial factors for this bel-related transgene silencing. Their abrogation could either partially or completely rescue the transgene-silencing phenotype induced by loss of bel. Importantly, small RNA deep sequencing analysis suggests that a piRNA-mediated mechanism is potentially involved in Bel-inactivation-induced transgene silencing. Together, these studies genetically link the function of Bel to Spn-E, Mod(mdg4), and Dcr-1, and suggest that transgene silencing induced by Bel inactivation may involve RNA processing, piRNA, miRNA, and epigenetic mechanisms (Lo, 2016).

This article reports that loss-of-bel function triggers transgene silencing, which occurs through reduction in transgene RNA levels. Furthermore, genetic studies indicate that this transgene silencing effect induced by bel abrogation requires the RNA helicase Spn-E, the insulator modulator Mod(mdg4) and/or the miRNA biosynthesis enzyme Dcr-1. Based on the functional roles of these three molecules, the data suggest that this transgene silencing effect may involve RNA processing, chromatin remodeling and/or miRNA biogenesis. This transgene silencing event occurring under various bel mutant backgrounds implies that Bel may regulate these three molecular mechanisms to sustain transgene expression in the normal physiological condition. Intriguingly, these studies also identified additional complexity in the relationship between Bel and Spn-E because the mislocalization of nuage components in spn-E mutants requires Bel. Therefore, these findings, taken together, provide new insight into Bel function and expand the molecular interaction network radiating from Bel (Lo, 2016).

These studies show that loss of bel gave rise to transgene silencing via decreased transgene RNA levels. This phenomenon could be attributable to either transcriptional suppression or increased RNA degradation. Some support for an RNA degradation/targeting mechanism comes from the finding that Spn-E is required for transgene silencing induced by loss of bel. In germline nurse cells, Spn-E, which is located in the cytoplasmic nuage, is crucial for properly maintaining the subcellular localization of piRNA-related protein factors in the nuage, the ping-pong reaction of piRNA biogenesis, and silencing of TEs. Spn-E is also required for the proper localization of RNA transcripts (e.g., Bicoid and Oskar) during oogenesis, which might be related to its role in organizing a cytoskeletal framework. Therefore, it is plausible that the Spn-E-dependent RNA processing activity and/or Spn-E-generated piRNAs mediate transgene RNA degradation. In addition, it is possible that piRNAs can also elicit transcriptional silencing of transgenes based on their nuclear epigenetic role in TE silencing. Another striking finding from these studies shows that Bel is involved in disrupting the subcellular localization of nuage components when Spn-E is abrogated. In contrast, loss of Bel alone had no impact on the localization of piRNA-related nuage protein components. These findings suggest that there could be a competitive relationship between Bel and Spn-E, where these two molecules negatively regulate each other. This hypothesis is also supported by observations that Spn-E is required for transgene silencing when Bel is abrogated. According to these findings, a model is envisioned that Bel may function as a negative regulator for ping-pong-cycle-mediated piRNA biogenesis via disrupting the nuage localization of piRNA-related proteins as Spn-E function is abrogated, whereas Spn-E may be aberrantly activated by loss of Bel, in turn leading to transgene silencing. Whether piRNAs generated from the Spn-E-mediated ping-pong cycle are involved in transgene silencing is currently unknown. However, a recent study has shown that piRNAs can be generated directly from the transposon-derived transgene insertion area located in the euchromatic genome, which have been proposed to be involved in transgene silencing. This finding links piRNAs to transgene silencing. Preliminary small RNA deep sequencing analysis showed that the viable trans-heterozygous bel74407/neo30 mutant ovaries, which manifest a partial transgene silencing phenotype, displayed no significant defect in overall piRNA biogenesis from piRNA clusters and the ping-pong cycle, indicating that unlike Vasa, Bel is not involved in regulating piRNA generation. This finding is consistent with the result that homozygous bel mutants had no defect in nuage protein localization, which is different from other piRNA-related nuage proteins whose defects can significantly disturb the localization of other nuage protein components. Nevertheless, deep sequencing analysis also identified de novo piRNAs generated in bel74407/neo30 mutant ovaries (but not in wild-type ovaries) that could be mapped to the integrated P-element-derived transgene sequence area (P[LacW]). This result is in line with previous findings and indicates that the de novo generation of piRNAs from the inserted transgene region in the genome occurs under the bel mutant background. Given that Bel is a paralog of Vasa and the current genetic findings also suggest a competitive relationship between Bel and Spn-E, loss of Bel may disrupt its normal regulation of some small RNA-related helicases and co-factors, which in turn aberrantly activates the small RNA pathway(s). Therefore, it is possible that loss of Bel may promote de novo piRNA biogenesis from the transgene insertion sites by freeing these small RNA regulators and provoking the activation of their related small RNA pathway(s), which is one possible mechanism leading to transgene silencing. Since it was not possible create viable mutant progeny bearing mutations at both bel and spn-E gene loci, it is still uncertain whether Spn-E is involved in regulating de novo piRNA biogenesis from transgene insertion sites. Although it was not possible to elucidate the detailed mechanism due to technical hurdles, exploring the regulatory roles of Bel and Spn-E in this new type of piRNA biogenesis will be key, interesting research for understanding molecular mechanisms underlying transgene silencing (Lo, 2016).

Another unexpected finding is that the spn-E mutant rescue of transgene silencing associated with loss of bel also occurs in somatic follicle cells. This indicates that in addition to its well-known function in germline cells, Spn-E may have a somatic function. As Spn-E is not implicated in somatic piRNA biogenesis (e.g., flamenco), it is uncertain how Spn-E mediates somatic transgene silencing and whether piRNAs participate in this event. Therefore, it will be important in the future to unravel whether the mechanism by which Spn-E facilitates transgene silencing in bel mutant somatic cells is the same as that in germline cells (Lo, 2016).

The common feature for transgenes silenced by Bel abrogation is their P-element-based integration into genomic DNA. The observation that some P-element-based transgenes were not silenced in bel mutant cells raises an interesting question about what factors can determine whether a transgene can be silenced or not. From genetic analysis of a series of transgenes, it was observed that six examined transgenes (ci-LacZ, dMyc-LacZ, dom-LacZ, C306-Gal4, ptc-Gal4, Tj-Gal4), which were generated by the insertion of two different P-element-derived vector sequences (P[LacW] and P[GawB]), were silenced under the bel mutant background. In contrast, two transgenes (histone-GFP, histone-RFP) generated by the insertion of P[His2Av]-derived vector sequences were not silenced by Bel inactivation. Although these data are not a conclusive result, they imply that the inserted transgene sequence itself, not the insertion location in the genome, may be a critical determinant for Bel-dependent transgene silencing since this silencing phenotype seems to be transgene-specific and a change in the transgene insertion location in the genome has no influence on whether this transgene can be silenced or not when Bel function is inhibited. It is possible that the transgene sequence determines a local chromosomal conformation and whether the transgene can be silenced under the bel mutant background is determined by whether its chromosomal structure can be recognized by epigenetic regulators involved in transgene silencing. Although further investigations are still needed to verify this hypothesis due to limited cases in this study, it raises the possibility that epigenetic regulation at the chromatin level may be involved in Bel-dependent transgene silencing. Indeed, besides Spn-E, the genetic study identified Mod(mdg4) as another crucial factor required for transgene silencing in both germline and somatic cells when Bel is abrogated. The mod(mdg4) gene encodes multiple nuclear factors through trans-splicing and this protein family is functionally involved in the modification of the properties of insulators, which are genomic elements that regulate gene expression. Mod(mdg4) proteins can function as chromatin modulators engaged in the organization of highly ordered chromatin domains. The involvement of Mod(mdg4) in transgene silencing suggests that nuclear epigenetic events are also crucial for induction of transgene silencing when Bel is inactivated. However, the role of Mod(mdg4) in transgene silencing might also be indirect, such as through its regulation of other genes that could contribute to silencing. Nevertheless, the findings suggest that the co-ordination between nuclear and cytoplasmic events mediated by Mod(mdg4) and Spn-E, respectively, is mandatory for induction of transgene silencing when Bel is functionally inhibited (Lo, 2016).

The miRNA biogenesis enzyme Dcr-1 is the third factor identified from these studies that is crucial for transgene silencing in the absence of Bel. Interestingly, the block in transgene silencing in this case (double mutant for bel and Dcr-1) only occurred in germline cells, but not in somatic cells. This discovery raises the possibility that there are additional miRNA-targeted proteins present in germline cells, but not in somatic cells, and they can interact with factors essential for germline transgene silencing. If this is a case, aberrantly elevated levels of these miRNA-targeted proteins in Dcr-1 mutant germline cells might interfere with transgene silencing. Besides this possible indirect role, another possibility is that the Dcr-1-dependent miRNA pathway may play a direct role in transgene silencing in germline cells as Bel is abrogated. The miRNA pathway has been shown to be implicated in transgene silencing in Drosophila S2 cells. Although the silencing mechanism is unclear, this finding raises a possible direct role of the Dcr-1-dependent miRNA pathway in bel-mutant transgene silencing. A study from Zhou has shown that Bel proteins were cofractionated with the miRNA-dependent RNA-induced silencing complexes (miRISCs) and co-immunoprecipitated with Ago1, the protein component of miRISCs (Zhou, 2008). This finding suggests a compelling possibility that Bel may be directly involved in the miRNA pathway to regulate miRISC-dependent RNA silencing and Bel inactivation may result in the aberrant functionality of miRISC and its related RNA silencing. Since miRNAs can target mRNAs via their short seed sequences, a possibility which cannot be ruled out is that some miRNAs may directly target transgene RNAs to regulate their levels. Future investigation is needed to reveal which possibility is more relevant (Lo, 2016).

In conclusion, these findings provide novel insights into the regulatory role of Bel in the expression of transgenes in Drosophila and its functional linkage to crucial factors implicated in RNA processing, chromatin remodeling and miRNA biogenesis. These findings further advance understanding of the complex cellular functions of Bel. These studies of the role for Bel in transgene expression may have important, future implications for understanding the regulation of expression of newly invaded or transposed TEs and virus-retrotransposon DNA chimeras generated from viral infection as they may share the similar scenario as transgene integration (Lo, 2016).

RNA helicase Belle (DDX3) is essential for male germline stem cell maintenance and division in Drosophila

This study showed that RNA helicase Belle (DDX3) was required intrinsically for mitotic progression and survival of germline stem cells (GSCs) and spermatogonial cells in the Drosophila melanogaster testes. Deficiency of Belle in the male germline resulted in a strong germ cell loss phenotype. Early germ cells are lost through cell death, whereas somatic hub and cyst cell populations are maintained. The observed phenotype is related to that of the human Sertoli Cell-Only Syndrome caused by the loss of DBY (DDX3) expression in the human testes and results in a complete lack of germ cells with preservation of somatic Sertoli cells. This study found the hallmarks of mitotic G2 delay in early germ cells of the larval testes of bel mutants. Both mitotic cyclins, A and B, are markedly reduced in the gonads of bel mutants. Transcription levels of cycB and cycA decrease significantly in the testes of hypomorph bel mutants. Overexpression of Cyclin B in the germline partially rescues germ cell survival, mitotic progression and fertility in the bel-RNAi knockdown testes. Taken together, these results suggest that a role of Belle in GSC maintenance and regulation of early germ cell divisions is associated with the expression control of mitotic cyclins (Kotov, 2016).

This study shows that RNA helicase Belle (DDX3) is required cell-autonomously for the survival and divisions of GSCs in Drosophila testes. In bel6/neo30 mutants as well as in the case of germline-specific RNAi belKD rapid elimination of germ cells via apoptosis occurred. But what events could trigger apoptosis? To address this issue, larval testes were analyzed. Testes of bel6/neo30 mutant larvae still contained all populations of early germ cells. This observation indicates that primordial germ cells (PGCs) correctly migrate into embryonic gonads during mid to late embryogenesis. In the mutant larval testes the wild-type hub and GSCs adjacent to the hub were clearly detected. It is known that the mechanism of capturing GSCs and CySCs to the hub involves a high level of adhesion molecule E-Cad on the hub/stem cells interface. In testes with STAT depletion the expression of E-Cad is severely disrupted accounting for the defects in hub-GSC adhesion and for the subsequent loss of GSCs. However, it was determined that STAT expression (in CySCs) and consequently upstream Upd signaling from the hub were not disrupted in the bel6/neo30 testes. Although the amount of Belle was strongly reduced in the bel6/neo30 testes, no reduction of E-Cad level was observed in CySCs. On the contrary, high ectopic expression of E-Cad was detected on the surface of CySCs surrounding the hub. The influence of Belle on the adherens junction formation in GSCs cannot be directly estimated. However, due to adhesion failures a loss of GSCs via premature differentiation could be expected followed by normal development of newly formed germline cysts. In contrast, this study detected a reduced germ cell content and morphological abnormalities of early germ cells including their giant nuclear and cellular sizes. It is assumed that these germ cells could not enter mitosis and are delayed in the G2 phase. Failure to enter mitosis after G2 delay appears to induce germ cell apoptosis in the bel testes, as previously has been shown for the how testes (Kotov, 2016).

It is known that Drosophila mitotic cyclins, Cyclin A, Cyclin B and Cyclin B3, each form complexes with Cdc2, and they appear to function synergistically to provide a progression throughout mitosis. Sufficient levels of mitotic cyclins must be accumulated at the end of G2 to ensure the onset of mitosis. To date, cell cycle regulation of GSCs and their daughter gonial cells is still poorly understood. It is known that PGCs suppress mitotic activity during their migration to embryonic gonads due to translational repression of maternal cycB mRNA via its 3'UTR by Pumilio-Nanos complex and other unidentified factors. Pumilio and Nanos are also known to be expressed in GSCs of gonads of adult flies and are found to be essential for GSC maintenance. However, factors overriding the repressive Pumilio-Nanos-dependent signal and providing expression of zygotic Cyclin B protein during normal testis development are currently unknown (Kotov, 2016).

It has been shown that Cyclin B and Cyclin A, but not Cyclin B3, are required in the gonad for the maintenance of early germ cells. A mutational depletion of Cyclin B leads to a complete missing of germ cells in the adult testes and their significant loss in the ovaries. However, the requirements for Cyclin A expression for the survival of early germ cells are currently obscure. It is known that overexpression of Cyclin A or expression of its nondegradable form leads to a rapid loss of GSCs in the ovaries (Kotov, 2016).

This study found that the previously published cycB testis phenotype mimicked that in the case of bel6/neo30 mutants and germline-specific RNAi belKD. It was determined that both of the mitotic cyclins, but not Cyclin E and Cdc2, were significantly decreased in the belEY08943/neo30mutant testes. Furthermore, a considerable decrease was found of cycA and cycB mRNA levels. These results suggest a specific contribution of Belle to the transcriptional regulation of mitotic cyclins in the germline. It was also revealed that the constitutive level of Cyclin B expression in control testes was significantly lower than in control ovaries. Assuming that Belle regulates mitotic cyclins in a similar way in the germline of both sexes, it is believed that deficiency of Belle has a more severe effect on spermatogenesis, due to a sharply reduced level of Cyclin B protein below a threshold, whereas its dose in the bel6/neo30 ovaries is still sufficient to allow mitosis to occur. In support of this hypothesis a partial rescue was achieved of the RNAi belKD testis phenotype by transgenic germline-specific expression of Cyclin B, but not by Cyclin A overexpression (Kotov, 2016).

The reduction of cycB transcription in belEY08943/neo30 testes places cycB downstream of bel. In rescue experiments a third copy of cycB was added to the system employing the germinal nos-Gal4 driver in combination with UAS-bel RNAi hairpin. It is assumed that the reduction of Belle in RNAi belKD testes would negatively affect the expression of both endogenous and transgenic cycB. In accordance with this assumption only a partial restoration of the Cyclin B protein level and only partial rescue of spermatogenesis was achieved. The data indicate that at least one crucial requirement for Belle in early germ cells is relevant to Cyclin B level maintenance for ensuring germ cell mitosis (Kotov, 2016).

To date, evidences of participation of DDX3 proteins in cell cycle control both at the level of transcription and translations have been obtained. DDX3 specifically cooperates with transcription factor Sp1 to positively regulate the transcription of p21waf gene. A temperature-sensitive mutation of ddx3 gene in golden hamster cell culture at nonpermissive temperature leads to G1 arrest, which is accompanied by a decline of cycA mRNA and rather suggests the transcriptional level of regulation for cycA. It has been shown that in human HeLa cells DDX3 interacts with the GC-rich, highly structured 5'UTR of Cyclin E1 mRNA and regulates its translation initiation and a knockdown of DDX3 delays the entry to the S phase. DED1, a Schizosaccharomyces pombe homolog of DDX3, is involved in the translational control of B-type cyclin mRNAs (Cig2 and Cdc13), which have extended and expectedly highly structured 5'UTRs. It is known that only a single cyclin-dependent kinase and two B-type cyclins regulate both the S phase and mitosis in yeasts. Indeed, temperature-sensitive mutations of ded1 gene inhibit B-type cyclin translation and arrest cell cycle at both S phase and G2/M transition, whereas both cig2 and cdc13 mRNA levels remain unchanged (Kotov, 2016 and references therein).

This study has presented experimental evidence that Belle has specific and essential functions in the male germline associated with proper transcriptional regulation of mitotic cyclin expression. The testis phenotype observed in Drosophila is similar to the SCOS phenotype in human testes, indicating a conserved function of DDX3 in spermatogenesis. Understanding the molecular basis for DBY (DDX3) functions in mammalian germ cell maintenance has proven to be challenging. The functions and regulation of A-type and B-type cyclins in mammalian spermatogenesis are not clearly understood. In this case, a study in the Drosophila model provides a useful insight into the mechanism of GSC maintenance in the male germline. The current findings support a mechanism according to which the determination of the fate of male GSCs is closely connected with the control of mitosis via the regulation of mitotic cyclin levels (Kotov, 2016).

Drosophila DDX3/Belle exerts its function outside of the Wnt/Wingless signaling pathway

The helicases human DDX3 and Drosophila Belle (Bel) are part of a well-defined subfamily of the DEAD-box helicases. Individual subfamily-members perform a myriad of functions in nuclear and cytosolic RNA metabolism. It has also been reported that DDX3X is involved in cell signaling, including IFN-α and IFN-β inducing pathways upon viral infection as well as in Wnt signaling. This study used a collection of EMS-induced bel alleles recovered from a Wingless (Wg) suppressor screen to analyze the role of the Drosophila homolog of DDX3 in Wg/Wnt signaling. These EMS alleles, as well as a P-element induced null allele and RNAi-mediated knock down of bel, all suppressed the phenotype of ectopic Wg signaling in the eye. However, they did not affect the expression of known Wg target genes like senseless, Distalless or wingful/Notum. Ectopic Wg signaling in eye imaginal discs induces apoptosis by increasing grim expression. Mutations in bel revert grim expression to wild-type levels. Together, these results indicate that Bel does not function as a core component in the Drosophila Wg pathway, and that mutations affecting its helicase function suppress the effects of ectopic Wg signaling downstream of the canonical pathway (Jenny, 2016).

The findings indicate that Bel plays a role in the suppression of the phenotype caused by ectopic Wg signaling in Drosophila eye imaginal discs. EMS derived alleles, a functional null allele (P-element induced) and bel knock down by RNAi, were all able to suppress the sev-wg phenotype. The bel alleles also showed an eye phenotype in a neutral background: these eyes are smaller and mis-structured, suggesting that Bel also plays a role in normal eye development (Jenny, 2016).

The role was tested of bel in endogenous Wg signaling in eye and wing progenitor tissues. In wing discs, the protein expression of two known Wg target genes, Sens and Dll, was unaffected in homozygous mutant bel clones or in compartments where bel was knocked down. Measuring expression levels of the Wg target gene wf (wingful or Notum) by qRT PCR and RNAseq in eye imaginal discs confirmed that Wg target gene expression does not depend on Bel. However, the sev-wg induced gene grim showed reduced transcript levels in bel mutants. Mutations in pygo, a bona-fide Wg pathway component, reduced both Wg target genes (wf and fz3) and grim expression. These findings suggest that Bel's role lies downstream of the canonical Wg signaling cascade and upstream of grim expression. The data is in contradiction with the recent report that the mammalian Bel homolog DDX3 is involved in the upstream Wnt signal cascade, acting as a regulatory subunit of CK1-ε, to promote the phosphorylation of Dsh. This study attributed the Wnt/Wg signaling function to DDX3's C-terminus (amino acids 456-662) and suggested it was independent of the helicase activity. The alleles obtained from the current EMS screen reveal a different picture. All mutations precisely map to conserved DEAD-box helicase motifs. This striking bias in localization of randomly induced mutations suggests that in Drosophila the helicase function plays a key role. Additionally, the current analysis with the Bel specific antibody indicates that the mis-sense alleles are expressed at normal levels and thus should not affect a potential scaffold function of Bel (Jenny, 2016).

The finding that Bel does not have a role in Wg signaling is not entirely unexpected in light of the mechanism proposed by Cruciat et al., who suggest that the relevant interaction partner is CK1-ε. In Drosophila, the role of CK1-ε in Wg signaling is uncertain. CK1-ε is implicated in non-canonical Wg signaling. When overexpressed in cultured Drosophila cells disc overgrown (dco, encoding the Drosophila CK1-ε homolog) may affect canonical signaling. However, in vivo one study suggests that sens expression is reduced in dco clones, another study with the null-allele dcoLE88 failed to find evidence for a role for Dco in the Wg pathway. Further confusion is added by the finding that Bel was ascribed a negative role in Wg transduction based on the results of a genome-wide RNAi screen in Drosophila S2 cells (Jenny, 2016).

Regardless of the role of DCO, the current results show that in Drosophila Bel does not act as a core Wnt signaling component, however it is involved in the suppression of cell death induced by ectopic Wg signaling, downstream of the canonical pathway (Jenny, 2016).

Translational control by the DEAD Box RNA helicase belle regulates ecdysone-triggered transcriptional cascades

Steroid hormones act, through their respective nuclear receptors, to regulate target gene expression. Despite their critical role in development, physiology, and disease, however, it is still unclear how these systemic cues are refined into tissue-specific responses. This study identified a mutation in the evolutionarily conserved DEAD box RNA helicase belle/DDX3 that disrupts a subset of responses to the steroid hormone ecdysone during Drosophila melanogaster metamorphosis. belle directly regulates translation of E74A, an ets transcription factor and critical component of the ecdysone-induced transcriptional cascade. Although E74A mRNA accumulates to abnormally high levels in belle mutant tissues, no E74A protein is detectable, resulting in misregulation of E74A-dependent ecdysone response genes. The accumulation of E74A mRNA in belle mutant salivary glands is a result of auto-regulation, fulfilling a prediction made by Ashburner nearly 40 years ago. In this model, Ashburner postulates that, in addition to regulating secondary response genes, protein products of primary response genes like E74A also inhibit their own ecdysone-induced transcription. Moreover, although ecdysone-triggered transcription of E74A appears to be ubiquitous during metamorphosis, belle-dependent translation of E74A mRNA is spatially restricted. These results demonstrate that translational control plays a critical, and previously unknown, role in refining transcriptional responses to the steroid hormone ecdysone (Ihry, 2012).

The microRNA pathway regulates the temporal pattern of Notch signaling in Drosophila follicle cells

Multicellular development requires the correct spatial and temporal regulation of cell division and differentiation. These processes are frequently coordinated by the activities of various signaling pathways such as Notch signaling. From a screen for modifiers of Notch signaling in Drosophila the RNA helicase Belle, a recently described component of the RNA interference pathway (Ulvila, 2006; Zhou, 2008), was identified as an important regulator of the timing of Notch activity in follicle cells. Loss of Belle delays activation of Notch signaling, which results in delayed follicle cell differentiation and defects in the cell cycle. Because mutations in well-characterized microRNA components phenocopied the Notch defects observed in belle mutants, Belle might be functioning in the microRNA pathway in follicle cells. The effect of loss of microRNAs on Notch signaling occurs upstream of Notch cleavage, as expression of the constitutively active intracellular domain of Notch in microRNA-defective cells restored proper activation of Notch. Furthermore, evidence is presented that the Notch ligand Delta is an important target of microRNA regulation in follicle cells and regulates the timing of Notch activation through cis inhibition of Notch. This study has uncovered a complex regulatory process in which the microRNA pathway promotes Notch activation by repressing Delta-mediated inhibition of Notch in follicle cells (Poulton, 2011).

The strict regulation of important cellular processes, such as the temporal activity of signaling pathways like Notch, is an essential point of control in guiding the development of multicellular organisms. Cells have therefore evolved a complex array of mechanisms to regulate signaling pathways. miRNA regulation of gene expression has rapidly emerged as one of the most important of these regulatory mechanisms. This study has shown that the correct timing of Notch activity in follicle cells requires the miRNA pathway and the newly identified RNAi component Bel. The data suggest that one important target of miRNA-based regulation of Notch signaling in follicle cells is Delta, in which Delta acts as a repressor of Notch activity (Poulton, 2011).

These findings that two core components of miRNA production are required to properly initiate the mitotic-to-endocycle switch in follicle cells by promoting Notch signaling describe a novel mechanism by which the miRNA pathway regulates this key developmental event. Interestingly, the miRNA pathway appears to control the overall timing of Notch activity, as disruption of the miRNA pathway results in a delay of Notch activation and inactivation in follicle cells. Previous work has shown that certain miRNAs, known as heterochronic miRNAs, regulate the timing of important developmental processes on a wide biological scale, from changes in cell cycle to the transition from juvenile to adult. This research identifies a new example of heterochrony mediated by miRNAs, in which cell cycle switches and differentiation are shifted in time as a result of delayed Notch signaling activity (Poulton, 2011).

Bel is a DEAD-box RNA helicase that was recently identified in two Drosophila cell culture screens as necessary for effective siRNA knockdown (Ulvila, 2006; Zhou, 2008). Precisely how Bel functions in this process is unknown, although data from the Zhou screen suggest that Bel acts downstream of siRNA production and loading. Interestingly, although Bel did not significantly disrupt miRNA-based assays in that screen, Bel was found to be in a complex with components of both the miRNA and siRNA pathways, and Bel immunoprecipitation pulled down both miRNAs and siRNAs, suggesting that Bel might be involved in both pathways. The similarities described between the bel mutant phenotype and the phenotypes of the miRNA pathway components Dicer (Dcr-1) and pasha imply that Bel might function in the miRNA pathway. Attempts were made to test the role of Bel in the miRNA pathway more directly using the GFP-tagged Delta 3'UTR sensor line, the expression of which is regulated by miRNA activity, but the results of these experiments were inconclusive. Although Bel appears to function in the siRNA pathway, this study found that the siRNA pathway is not involved in regulating Notch in follicle cells. A few reports have also identified several phenotypes associated with disruption of bel that indicate that Bel functions in the G1/S transition in the eye disc by affecting Hedgehog signaling and Dacapo expression (Ambrus, 2010; Ambrus, 2007), as well as identifying a role for Bel with the zinc-finger protein Zn72D in regulating the splicing and translation of maleless transcripts (Worringer, 2009). It will be interesting to determine whether the function of Bel in these other important cellular processes is also related to a role in RNAi pathways (Poulton, 2011).

Notch can be both activated and inhibited by its ligands. In oogenesis, it is known that Delta from the germline cells functions in trans to activate Notch in the surrounding follicle cells. This study found that Delta expressed in the follicle cells operates in its repressive capacity to prevent premature activation of Notch. Because Delta is actually upregulated in the germline by stage 5/6, well before the expression of Notch target genes at stage 7, and in light of the data on the inhibitory role of follicle cell Delta, it is likely that the presence of Delta from the germline alone is not what determines the precise timing of Notch activity. Instead, a model is favored in which the timing of Notch activity is determined by a titration of trans-activating germline Delta relative to the cis-inhibitory effects of follicle cell Delta. Therefore, loss of follicle cell Delta, as in the Delta mutant clone experiments, allows earlier activation of Notch by the lower levels of Delta presented by the germline before stage 7, as well as higher levels of Notch activity relative to wild-type cells in mid-oogenesis. This antagonistic relationship between germline and follicle cell Delta suggests that there must be a precise balance between these two populations of Delta that determines exactly when Notch is activated during oogenesis; analysis of the miRNA pathway suggests that miRNAs might help to fine-tune this balance (Poulton, 2011).

The conclusion that Delta is a relevant target of miRNA-based control of Notch activity in follicle cells is supported by the following observations. First, expression of NICD is sufficient to restore proper activation of Notch in the Dcr-1 mutant, indicating that the relevant miRNA target functions upstream in the Notch pathway (prior to ligand-induced Notch cleavage). Because ligand-based inhibition by Delta acts upstream of Notch cleavage, Delta is a logical candidate of miRNA regulation. Second, Delta,Dcr-1 double-mutant analysis strongly suggests that Delta is an important target of miRNAs. Specifically, in Dcr-1 single-mutant clones, Notch signaling is delayed, yet removal of Delta along with loss of Dcr-1 leads to premature activation of Notch, as seen in Delta single-mutant clones. This indicates that the inhibitory effects on Notch signaling caused by loss of miRNAs requires the presence of Delta. However, the possibility cannot be ruled out that the activating effects of loss of Delta on Notch might be stronger than the inhibitory effects of loss of miRNAs on repressing Notch activity through some other miRNA target. Third, Delta is an apparent direct target of the miRNA pathway, as indicated by experiments demonstrating that follicle cell clones of Dcr-1 and pasha result in increased Delta protein and increased expression of a Delta 3'UTR sensor. Together, the ectopic expression of Delta protein and of the Delta 3'UTR sensor in the Dcr-1,pasha clones, in conjunction with the Delta,Dcr-1 double-mutant analysis, strongly suggest that the miRNA pathway regulates Notch activity by repressing Delta protein levels (Poulton, 2011).

Cis inhibition of Notch has also been described for Ser, raising the possibility that Ser might be functioning in follicle cells in a similar capacity to that which was discovered for Delta. However, Ser mutant follicle cell clones possess no defects in Notch activity markers. To determine whether Ser is repressed by the miRNA pathway in follicle cells, Ser protein levels were examined in follicle cells double mutant for Dcr-1 and pasha, and no changes were observed in Ser expression, which in the wild type was essentially undetectable. It is concluded that Ser does not play a role in regulating Notch activity in follicle cells (Poulton, 2011).

More than two dozen miRNAs are predicted to target Delta mRNA. Owing largely to a lack of readily available mutants to conduct a thorough loss-of-function screen for the miRNA(s) involved, it remains unknown which miRNAs are important in governing the timing of Notch signaling in follicle cells. Both loss of function and overexpression of miR-1, which has been previously demonstrated to regulate Delta in Drosophila heart development, were tested; however, neither produced any phenotype consistent with the described Notch defects. As the genetic tools available to investigate the roles of specific miRNAs improve, and the ability to predict which miRNAs target certain transcripts also improves, it should be possible to identify the relevant miRNAs involved in this process (Poulton, 2011).

These findings describe a complex system by which developing egg chambers regulate the timing of several key events, including cell cycle programs and differentiation. Mechanistically, it was found that the miRNA pathway controls the temporal pattern of Notch activity, apparently by limiting Delta protein levels in follicle cells, in which Delta exerts an inhibitory effect on Notch. The data support a model in which the timing of Notch activation is determined not just by the expression of germline Delta, but also by a multi-layered regulatory system in which follicle cell Delta prevents premature Notch activation, while miRNAs serve to counter this inhibitory effect by limiting Delta expression. Such a model of miRNA function in follicle cells fits well with the developing theme that miRNAs commonly serve to fine-tune developmental processes by subtle regulation of key regulators. It will be interesting to determine whether miRNAs also regulate Notch signaling in other tissues of the fly through a similar mechanism of ligand-mediated inhibition of Notch, and it will be particularly exciting to investigate whether this regulatory network is utilized in other animals (Poulton, 2011).

Patterning of the Drosophila oocyte by a sequential translation repression program involving the d4EHP and Belle translational repressors

During Drosophila development, translational control plays a crucial role in regulating gene expression, and is particularly important during pre-patterning of the maturing oocyte. A critical step in translation initiation is the binding of the eukaryotic translation initiation factor 4E (eIF4E) to the mRNA cap structure, which ultimately leads to recruitment of the ribosome. d4EHP is a translational repressor that prevents translation initiation by out-competing eIF4E on the cap structure for a subset of mRNAs. However, only two examples of mRNAs subject to d4EHP translation repression in Drosophila are known. This study shows that the belle (bel) mRNA is translationally repressed by the d4EHP protein in the Drosophila ovary. Consistent with this regulation, d4EHP overexpression in the ovary phenocopies the bel mutant. Evidence is provided that the Bel protein binds to eIF4E and may itself function as a translation repressor protein, with bruno as a potential target for Bel repression in the oocyte. Bruno is known to repress the mRNA of the key oocyte axis determinant oskar (osk) during oogenesis, and this study found that an increase in the level of Bruno protein in bel mutant ovaries is associated with a reduction in Osk protein. Overall, these data suggest that a translational regulatory network exists in which consecutive translational repression events act to correctly pattern the Drosophila oocyte (Yarunin, 2011).

DEAD-box RNA helicase Belle/DDX3 and the RNA interference pathway promote mitotic chromosome segregation

During mitosis, faithful inheritance of genetic material is achieved by chromosome segregation, as mediated by the condensin I and II complexes. Failed chromosome segregation can result in neoplasm formation, infertility, and birth defects. Recently, the germ-line-specific DEAD-box RNA helicase Vasa was demonstrated to promote mitotic chromosome segregation in Drosophila by facilitating robust chromosomal localization of Barren (Barr), a condensin I component. This mitotic function of Vasa is mediated by Aubergine and Spindle-E, which are two germ-line components of the Piwi-interacting RNA pathway. Faithful segregation of chromosomes should be executed both in germ-line and somatic cells. However, whether a similar mechanism also functions in promoting chromosome segregation in somatic cells has not been elucidated. This study presents evidence that belle (vasa paralog) and the RNA interference pathway regulate chromosome segregation in Drosophila somatic cells. During mitosis, belle promotes robust Barr chromosomal localization and chromosome segregation. Belle's localization to condensing chromosomes depends on dicer-2 and argonaute2. Coimmunoprecipitation experiments indicated that Belle interacts with Barr and Argonaute2 and is enriched at endogenous siRNA (endo-siRNA)-generating loci. These results suggest that Belle functions in promoting chromosome segregation in Drosophila somatic cells via the endo-siRNA pathway. DDX3 (human homolog of belle) and DICER function in promoting chromosome segregation and hCAP-H (human homolog of Barr) localization in HeLa cells, indicating a conserved function for those proteins in human cells. These results suggest that the RNA helicase Belle/DDX3 and the RNA interference pathway perform a common role in regulating chromosome segregation in Drosophila and human somatic cells (Pek, 2011).

Although Vasa and Belle have been implicated in the piRNA and endo-siRNA pathways, respectively, it is not known whether DDX3 is also involved in the RNAi pathway. The fact that DDX3 is involved in viral RNA sensing offers the possibility that DDX3 may be a component of the RNAi pathway in humans. Furthermore, the DCR-dependent localization of DDX3, both during interphase and prophase, suggests that DDX3 may function downstream of DCR. Further investigation into the nature of the genomic loci and RNAi pathway components that associate with DDX3 and the nature of the noncoding RNAs involved in this process will provide greater insight into its molecular mechanism in human cells (Pek, 2011).

This study has indicated that the robust chromosomal localization of Barr/hCAP-H is regulated by the Vasa/Belle/DDX3 class of DEAD-box RNA helicases in both germ-line and somatic Drosophila cells and human somatic cells. This finding suggests the possibility of a common pathway that regulates chromosome segregation by the Vasa/Belle/DDX3 class of RNA helicases. Although chromosome segregation appears to be regulated by RNAi machinery, the necessary small RNA pathway components vary notably between the germ-line and somatic cells. The piRNA pathway components are required in the germ-line cells, whereas the endo-siRNA pathway components function as their somatic counterparts. This finding suggests that various cell types can use the existing small RNAs and RNAi factors to achieve a common goal of robust Barr/hCAP-H localization. This study also provides a framework for future studies investigating the molecular mechanism of the cooperation between the Vasa/Belle/DDX3 RNA helicases and the RNAi factors to ensure proper chromosome segregation (Pek, 2011).

Mutation of the DEAD-box helicase belle downregulates the cyclin-dependent kinase inhibitor Dacapo

The retinoblastoma protein (pRB) negatively regulates cell proliferation by limiting the activity of the family of E2F transcription factors. In Drosophila, mutation of the DEAD-box helicase belle (bel) relieves an E2F/pRB induced G(1) cell cycle arrest; however, the mechanism of this rescue is unknown. This study shows that the level of the cyclin-dependent kinase inhibitor Dacapo (Dap), homolog of mammalian p21/p27, is strongly reduced both in bel mutant cells in vivo and in tissue culture cells depleted of Bel by RNA interference. Interestingly, the loss of bel also partially alleviates an ectopically induced G(1) cell cycle arrest. Additionally, Bel was shown to undergo nucleocytoplasmic shuttling. Thus, inactivation of bel renders cells less sensitive to several anti-proliferative signals inducing G(1) arrest (Ambrus, 2010).

The zinc finger protein Zn72D and DEAD box helicase Belle interact and control maleless mRNA and protein levels

The Male Specific Lethal (MSL) complex is enriched on the single X chromosome in male Drosophila cells and functions to upregulate X-linked gene expression and equalize X-linked gene dosage with XX females. The zinc finger protein Zn72D is required for productive splicing of the maleless (mle) transcript, which encodes an essential subunit of the MSL complex. In the absence of Zn72D, MLE levels are decreased, and as a result, the MSL complex no longer localizes to the X chromosome and dosage compensation is disrupted. To understand the molecular basis of Zn72D function, proteins were identified that interact with Zn72D. Among several proteins that associate with Zn72D, the DEAD box helicase Belle (Bel) was found. Simultaneous knockdown of Zn72D and bel restored MSL complex localization to the X chromosome and dosage compensation. MLE protein was restored to 70% of wild-type levels, although the level of productively spliced mle transcript was still four-fold lower than in wild-type cells. The increase in production of MLE protein relative to the amount of correctly spliced mle mRNA could not be attributed to an alteration in MLE stability. These data indicate that Zn72D and Bel work together to control mle splicing and protein levels. Thus Zn72D and Bel may be factors that coordinate splicing and translational regulation (Worringer, 2009).

Why is it important to regulate MLE protein levels? MLE localizes to all chromosomes and throughout the nucleus when overexpressed. Incorrect MLE expression is detrimental to the development of the fly, because heat shock over-expression of transgenic MLE protein results in male and female lethality. It is possible that translational repression by Zn72D and Bel is one mechanism by which levels of MLE protein are tightly controlled. Expression of a transgenic mle cDNA in S2 cells resulted in overproduction of MLE protein; however, inclusion of the first two introns in the same transgene reduced the amount of MLE protein produced from the transgene. This suggests that perhaps recruitment of Zn72D to the mle transcript has the effect of not only productively splicing the transcript but also targeting it for translational regulation (Worringer, 2009).

Like Zn72D, its human homologue ZFR is also found mainly in the nucleus, with a subset in the cytoplasm. Cytoplasmic ZFR colocalizes in neuronal granules with Staufen2, a protein involved in mRNA transport and localization. ZFR interacts with and is required for cytoplasmic localization of the Staufen262 isoform. As neuronal granules are involved in translational regulation and localization of mRNAs, these data suggest that ZFR may have a role regulating cytoplasmic localization of mRNAs. If Zn72D has a similar function in flies, it has the potential to regulate gene expression at two steps. Zn72D may first promote productive splicing of mRNAs and then later affect their cytoplasmic localization, which in turn may impact translation (Worringer, 2009).

This study has identified several proteins that interact with Zn72D, including the DEAD box helicase Bel. Co-knockdown of both bel and Zn72D restores the MSL complex localization to the X chromosome and dosage compensation of X-linked genes that was lost in the absence of Zn72D. In addition, it was found that co-knockdown of Zn72D and bel resulted in restoration of MLE protein levels to 70% of wild-type levels, despite a four-fold reduction in properly spliced mle mRNA. These data implicate Zn72D and bel as being factors that target spliced mRNAs for localized, regulated translation in the cytoplasm (Worringer, 2009).

Comparative analysis of argonaute-dependent small RNA pathways in Drosophila

The specificity of RNAi pathways is determined by several classes of small RNAs, which include siRNAs, piRNAs, endo-siRNAs, and microRNAs (miRNAs). These small RNAs are invariably incorporated into large Argonaute (Ago)-containing effector complexes known as RNA-induced silencing complexes (RISCs), which they guide to silencing targets. Both genetic and biochemical strategies have yielded conserved molecular components of small RNA biogenesis and effector machineries. However, given the complexity of these pathways, there are likely to be additional components and regulators that remain to be uncovered. A comparative and comprehensive RNAi screen was undertaken to identify genes that impact three major Ago-dependent small RNA pathways that operate in Drosophila S2 cells. Subsets of candidates were identified that act positively or negatively in siRNA, endo-siRNA, and miRNA pathways. These studies indicate that many components are shared among all three Argonaute-dependent silencing pathways, though each is also impacted by discrete sets of genes (Zhou, 2008).

belle (bel) emerged from this screen and from other studies as an RNAi pathway candidate (Ulvila, 2006). It encodes a DEAD-box RNA helicase, which is required for viability and in the germ line (Johnstone, 2005). In order to assess RNAi efficiency in flies, transgenics were used carrying an inverted repeat of the white (w) gene under the control of the eye-specific GMR promoter (GMR-wIR). These flies display a pale eye color due to strong suppression of white. Because bel is essential, an eye-specific mitotic recombination system was used to generate mosaics in which the impact of a bel allele (bel⁶) on w silencing could be examined in clones. Eye cells homozygous for a Dcr-2 mutation, Dcr-2f^sL811X, display a dark red color, indicating loss of silencing. Eyes predominantly homozygous for bel⁶ are rough and small, suggesting that bel is required for cell viability. Importantly, patches of cells with increased pigmentation are observed in homozygous bel⁶ clones. hsc70-4 mutant eyes were examined, which are also small and rough, and pigmentation was found to be unaffected, suggesting that the bel⁶ phenotype is specific. These observations suggest that bel⁶ mutant clones are defective in RNAi (Zhou, 2008).

Next, whether Bel associates with RNAi components was examined. Cytoplasmic extract was prepared from S2 cells and fractionated by gel filtration. Individual fractions were immunoblotted using antibodies against Bel and components of RISC, including VIG, FMR, Ago2, or Ago1. The majority of Bel cofractionates with Ago1, whereas a smaller fraction coelutes with Ago2, FMR, and VIG, components of the siRISC. FLAG-tagged Bel was immunoprecipitated from S2 cells and robust coprecipitation of Ago2, FMR, or VIG was found. RNase treatment reduced interaction with Ago2 and VIG, but not as substantially with FMR (Zhou, 2008).

To test interactions of Bel with small RNAs, FLAG-tagged Bel or known components of the RISC were expressed together with an artificial siRNA (CXCR4) generated from a perfectly complementary hairpin expression construct. As expected, robust CXCR4 signals could be detected in the Ago2 or VIG complexes. Importantly, CXCR4 was also present in the Bel immunoprecipitate, as was esi-2.1, though they are probably bound directly to another protein in the complex. It is concluded that Bel likely acts directly as part of the RNAi machinery, as it resides in a complex that also contains both protein and RNA components of RISC. Interestingly, more CXCR4 siRNA was present in the Ago1 immunoprecipitate than was detected in the Ago2 sample. While this could be attributed to some intrinsic characteristics of the CXCR4 siRNA mimetic, it is also possible that the coupling between miRNA processing and loading steps accounts for this observation (Zhou, 2008).

Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells

Double-stranded RNA (dsRNA) fragments are readily internalized and processed by Drosophila S2 cells, making these cells a widely used tool for the analysis of gene function by gene silencing through RNA interference (RNAi). The underlying mechanisms are insufficiently understood. To identify components of the RNAi pathway in S2 cells, this study developed a screen based on rescue from RNAi-induced lethality. Argonaute 2, a core component of the RNAi machinery, and three gene products previously unknown to be involved in RNAi in Drosophila were identified: DEAD-box RNA helicase Belle, 26 S proteasome regulatory subunit 8 (Pros45), and clathrin heavy chain, a component of the endocytic machinery. Blocking endocytosis in S2 cells impaired RNAi, suggesting that dsRNA fragments are internalized by receptor-mediated endocytosis. Indeed, using a candidate gene approach, two Drosophila scavenger receptors, SR-CI and Eater, were identified that together accounted for more than 90% of the dsRNA uptake into S2 cells. When expressed in mammalian cells, SR-CI was sufficient to mediate internalization of dsRNA fragments. These data provide insight into the mechanism of dsRNA internalization by Drosophila cells. These results have implications for dsRNA delivery into mammalian cells (Ulvila, 2006).

Belle is a Drosophila DEAD-box protein required for viability and in the germ line

DEAD-box proteins are ATP-dependent RNA helicases that function in various stages of RNA processing and in RNP remodeling. This study reports identification and characterization of the Drosophila protein Belle (Bel), which belongs to a highly conserved subfamily of DEAD-box proteins including yeast Ded1p, Xenopus An3, mouse PL10, human DDX3/DBX, and human DBY. Mutations in DBY are a frequent cause of male infertility in humans. Bel can substitute in vivo for Ded1p, an essential yeast translation factor, suggesting a requirement for Bel in translation initiation. Consistent with an essential cellular function, strong loss of function mutations in bel are recessive lethal with a larval growth defect phenotype. Hypomorphic bel mutants are male-sterile. Bel is also closely related to the Drosophila DEAD-box protein Vasa (Vas), a germ line-specific translational regulator. Bel and Vas colocalize in nuage and at the oocyte posterior during oogenesis, and bel function is required for female fertility. However, unlike Vas, Bel is not specifically enriched in embryonic pole cells. It is concluded that the DEAD-box protein Bel has evolutionarily conserved roles in fertility and development (Johnstone, 2005).

Functions of Belle orthologs in other species

HIV-1 blocks the signaling adaptor MAVS to evade antiviral host defense after sensing of abortive HIV-1 RNA by the host helicase DDX3

The mechanisms by which human immunodeficiency virus 1 (HIV-1) avoids immune surveillance by dendritic cells (DCs), and thereby prevents protective adaptive immune responses, remain poorly understood. This study showed that HIV-1 actively arrested antiviral immune responses by DCs, which contributed to efficient HIV-1 replication in infected individuals. The RNA helicase DDX3 was identified as an HIV-1 sensor that bound abortive HIV-1 RNA after HIV-1 infection and induced DC maturation and type I interferon responses via the signaling adaptor MAVS. Notably, HIV-1 recognition by the C-type lectin receptor DC-SIGN activated the mitotic kinase PLK1, which suppressed signaling downstream of MAVS, thereby interfering with intrinsic host defense during HIV-1 infection. Finally, PLK1-mediated suppression of DDX3-MAVS signaling was shown to be a viral strategy that accelerated HIV-1 replication in infected individuals (Gringhuiis, 2017).

The DEAD-Box RNA helicase DDX3 interacts with NF-kappaB subunit p65 and suppresses p65-mediated transcription

RNA helicase family members exhibit diverse cellular functions, including in transcription, pre-mRNA processing, RNA decay, ribosome biogenesis, RNA export and translation. The RNA helicase DEAD-box family member DDX3 has been characterized as a tumour-associated factor and a transcriptional co-activator/regulator. This study demonstrated that DDX3 interacts with the nuclear factor (NF)-kappaB subunit p65 and suppresses NF-kappaB (p65/p50)-mediated transcriptional activity. The downregulation of DDX3 by RNA interference induces the upregulation of NF-kappaB (p65/p50)-mediated transcription. The regulation of NF-kappaB (p65/p50)-mediated transcriptional activity was further confirmed by the expression levels of its downstream cytokines, such as IL-6 and IL-8. Moreover, the binding of the ATP-dependent RNA helicase domain of DDX3 to the N-terminal Rel homology domain (RHD) of p65 is involved in the inhibition of NF-kappaB-regulated gene transcription. In summary, the results suggest that DDX3 functions to suppress the transcriptional activity of the NF-kappaB subunit p65 (Xiang, 2016).

DDX3 modulates neurite development via translationally activating an RNA regulon involved in Rac1 activation

The RNA helicase DDX3 is a component of neuronal granules, and its gene mutations are linked to intellectual disability (ID). This study demonstrates that DDX3 depletion in neurons impairs neurite development by downregulating Rac1 level and activation. Moreover, DDX3 activates the translation of functionally coherent mRNAs involved in Rac1 activation including Rac1. Among the DDX3 regulon, Prkaca encodes the catalytic subunit of PKA, a potential activator of Rac1 in neurons. DDX3-modulated PKAcalpha and Rac1 expression tunes the strength of PKA-Rac1 signaling and thereby contributes to neurite outgrowth and dendritic spine formation. Inhibition of DDX3 activity or expression in neonatal mice impaired dendritic outgrowth and spine formation of hippocampal neurons, echoing neuronal deficits underling DDX3 mutation-associated ID. Finally, evidence is provided that DDX3 activates local protein synthesis through a 5' UTR-dependent mechanism in neurons. The novel DDX3 regulon may conduct a spatial and temporal control of Rac1 signaling to regulate neurite development (Chen, 2016).

Autoinhibitory interdomain interactions and subfamily-specific extensions redefine the catalytic core of the human DEAD-box protein DDX3

DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. This study defined the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. The crystal structure is described of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. This core fragment of DDX3 was used to test the function of two recurrent medulloblastoma variants of DDX3, and both were found to inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins (Floor, 2016).

Ezrin binds to DEAD-box RNA helicase DDX3 and regulates its function and protein level

Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. A small-molecule inhibitor, NSC305787, directly binds to ezrin and inhibits its function. This study used a proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. Direct interaction was validated between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. These data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5' untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, these findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane (Celik, 2015).

Search PubMed for articles about Drosophila Belle

Ambrus, A. M., Nicolay, B. N., Rasheva, V. I., Suckling, R. J. and Frolov, M. V. (2007). dE2F2-independent rescue of proliferation in cells lacking an activator dE2F1. Mol Cell Biol 27(24): 8561-8570. PubMed ID: 17923695

Ambrus, A. M. and Frolov M. V. (2010). Mutation of the DEAD-box helicase belle downregulates the cyclin-dependent kinase inhibitor Dacapo. Cell Cycle 9: 1016-1020. PubMed Citation: 20160476

Celik, H., Sajwan, K. P., Selvanathan, S. P., Marsh, B. J., Pai, A. V., Kont, Y. S., Han, J., Minas, T. Z., Rahim, S., Erkizan, H. V., Toretsky, J. A. and Uren, A. (2015). Ezrin binds to DEAD-box RNA helicase DDX3 and regulates its function and protein level. Mol Cell Biol 35(18): 3145-3162. PubMed ID: 26149384

Chen, H. H., Yu, H. I. and Tarn, W. Y. (2016). DDX3 modulates neurite development via translationally activating an RNA regulon involved in Rac1 activation. J Neurosci 36(38): 9792-9804. PubMed ID: 27656019

Floor, S. N., Condon, K. J., Sharma, D., Jankowsky, E. and Doudna, J. A. (2016). Autoinhibitory interdomain interactions and subfamily-specific extensions redefine the catalytic core of the human DEAD-box protein DDX3. J Biol Chem 291(5): 2412-2421. PubMed ID: 26598523

Gringhuis, S. I., Hertoghs, N., Kaptein, T. M., Zijlstra-Willems, E. M., Sarrami-Fooroshani, R., Sprokholt, J. K., van Teijlingen, N. H., Kootstra, N. A., Booiman, T., van Dort, K. A., Ribeiro, C. M., Drewniak, A. and Geijtenbeek, T. B. (2017). HIV-1 blocks the signaling adaptor MAVS to evade antiviral host defense after sensing of abortive HIV-1 RNA by the host helicase DDX3. Nat Immunol 18(2): 225-235. PubMed ID: 28024153

Ihry, R. J., Sapiro, A. L. and Bashirullah, A. (2012). Translational control by the DEAD Box RNA helicase belle regulates ecdysone-triggered transcriptional cascades. PLoS Genet 8(11): e1003085. PubMed ID: 23209440

Jenny, F. H. and Basler, K. (2016). Drosophila DDX3/Belle exerts its function outside of the Wnt/Wingless signaling pathway. PLoS One 11(12): e0166862. PubMed ID: 28030561

Johnstone, O., Deuring, R., Bock, R., Linder, P., Fuller, M. T. and Lasko, P. (2005). Belle is a Drosophila DEAD-box protein required for viability and in the germ line. Dev Biol 277(1): 92-101. PubMed ID: 15572142

Kotov, A. A., Olenkina, O. M., Kibanov, M. V. and Olenina, L. V. (2016). RNA helicase Belle (DDX3) is essential for male germline stem cell maintenance and division in Drosophila. Biochim Biophys Acta 1863: 1093-1105. PubMed ID: 26876306

Lo, P. K., Huang, Y. C., Poulton, J. S., Leake, N., Palmer, W. H., Vera, D., Xie, G., Klusza, S. and Deng, W. M. (2016). RNA helicase Belle/DDX3 regulates transgene expression in Drosophila. Dev Biol 412: 57-70. PubMed ID: 26900887

Pek, J. W. and Kai, T. (2011). DEAD-box RNA helicase Belle/DDX3 and the RNA interference pathway promote mitotic chromosome segregation. Proc Natl Acad Sci U S A 108(29): 12007-12012. PubMed ID: 21730191

Poulton, J. S., Huang, Y. C., Smith, L., Sun, J., Leake, N., Schleede, J., Stevens, L. M. and Deng, W. M. (2011). The microRNA pathway regulates the temporal pattern of Notch signaling in Drosophila follicle cells. Development 138(9): 1737-1745. PubMed ID: 21447549

Ulvila, J., Parikka, M., Kleino, A., Sormunen, R., Ezekowitz, R. A., Kocks, C. and Ramet, M. (2006). Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells. J Biol Chem 281(20): 14370-14375. PubMed ID: 16531407

Worringer, K. A., Chu, F. and Panning, B. (2008). The zinc finger protein Zn72D and DEAD box helicase Belle interact and control maleless mRNA and protein levels. BMC Mol. Biol. 10: 33. PubMed Citation: 19386123

Xiang, N., He, M., Ishaq, M., Gao, Y., Song, F., Guo, L., Ma, L., Sun, G., Liu, D., Guo, D. and Chen, Y. (2016). The DEAD-Box RNA Helicase DDX3 Interacts with NF-kappaB Subunit p65 and Suppresses p65-Mediated Transcription. PLoS One 11(10): e0164471. PubMed ID: 27736973

Xiol, J., Spinelli, P., Laussmann, M. A., Homolka, D., Yang, Z., Cora, E., Coute, Y., Conn, S., Kadlec, J., Sachidanandam, R., Kaksonen, M., Cusack, S., Ephrussi, A. and Pillai, R. S. (2014). RNA clamping by Vasa assembles a piRNA amplifier complex on transposon transcripts. Cell 157(7): 1698-1711. PubMed ID: 24910301

Yarunin, A., Harris, R. E., Ashe, M. P. and Ashe, H. L. (2011). Patterning of the Drosophila oocyte by a sequential translation repression program involving the d4EHP and Belle translational repressors. RNA Biol 8(5): 904-912. PubMed ID: 21788736

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date revised: 26 January 2017

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