InteractiveFly: GeneBrief

RNA-binding Fox protein 1: Biological Overview | References

Gene name - RNA-binding Fox protein 1

Synonyms - Ataxin-2 binding protein 1

Cytological map position - 67E4-67E5

Function - RNA-binding protein

Keywords - targets pumilio mRNA for destabilization and translational silencing, thereby promoting germ cell development, oogenesis - regulated by memory suppressor microRNA miR-980 - homolog of an autism-susceptibility gene

Symbol - Rbfox1

FlyBase ID: FBgn0052062

Genetic map position - chr3L:10,481,412-10,593,403

Classification - RRM: RNA recognition motif

Cellular location - nuclear and cytoplasmic

NCBI link: EntrezGene
Rbfox1 orthologs: Biolitmine
Recent literature
Nazario-Toole, A. E., Robalino, J., Okrah, K., Corrada-Bravo, H., Mount, S. M. and Wu, L. P. (2018). The splicing factor RNA-binding fox protein 1 mediates the cellular immune response in Drosophila melanogaster. J Immunol [Epub ahead of print]. PubMed ID: 29997126
The uptake and destruction of bacteria by phagocytic cells is an essential defense mechanism in metazoans. To identify novel genes involved in the phagocytosis of Staphylococcus aureus, a major human pathogen, this study assessed the phagocytic capacity of adult blood cells (hemocytes) of the fruit fly, Drosophila melanogaster, by testing several lines of the Drosophila Genetic Reference Panel. Natural genetic variation in the gene RNA-binding Fox protein 1 (Rbfox1) correlated with low phagocytic capacity in hemocytes, pointing to Rbfox1 as a candidate regulator of phagocytosis. Loss of Rbfox1 resulted in increased expression of the Ig superfamily member Down syndrome adhesion molecule 4 (Dscam4). Silencing of Dscam4 in Rbfox1-depleted blood cells rescued the fly's cellular immune response to S. aureus, indicating that downregulation of Dscam4 by Rbfox1 is critical for S. aureus phagocytosis in Drosophila. This study is the first to demonstrate a link between Rbfox1, Dscam4, and host defense against S. aureus.
McCarthy, A., Sarkar, K., Martin, E. T., Upadhyay, M., Jang, S., Williams, N. D., Forni, P. E., Buszczak, M. and Rangan, P. (2022). Msl3 promotes germline stem cell differentiation in female Drosophila. Development 149(1). PubMed ID: 34878097
Gamete formation from germline stem cells (GSCs) is essential for sexual reproduction. However, the regulation of GSC differentiation is incompletely understood. Set2, which deposits H3K36me3 modifications, is required for GSC differentiation during Drosophila oogenesis. The H3K36me3 reader Male-specific lethal 3 (Msl3) and histone acetyltransferase complex Ada2a-containing (ATAC) cooperate with Set2 to regulate GSC differentiation in female Drosophila. Msl3, acting independently of the rest of the male-specific lethal complex, promotes transcription of genes, including a germline-enriched ribosomal protein S19 paralog RpS19b. RpS19b upregulation is required for translation of RNA-binding Fox protein 1 (Rbfox1), a known meiotic cell cycle entry factor. Thus, Msl3 regulates GSC differentiation by modulating translation of a key factor that promotes transition to an oocyte fate.
Rose, M., Domsch, K., Bartle-Schultheis, J., Reim, I. and Schaub, C. (2022). Twist regulates Yorkie activity to guide lineage reprogramming of syncytial alary muscles. Cell Rep 38(4): 110295. PubMed ID: 35081347
Genesis of syncytial muscles is typically considered as a paradigm for an irreversible developmental process. Notably, transdifferentiation of syncytial muscles is naturally occurring during Drosophila development. The ventral longitudinal heart-associated musculature (VLM) arises by a unique mechanism that revokes differentiation states of so-called alary muscles and comprises at least two distinct steps: syncytial muscle cell fragmentation into single myoblasts and successive reprogramming into founder cells that orchestrate de novo fiber formation of the VLM lineage. This study provides evidence that the mesodermal master regulator twist plays a key role during this reprogramming process. Acting downstream of Drosophila Tbx1 (Org-1), Twist is regulating the activity of the Hippo pathway effector Yorkie and is required for the initiation of syncytial muscle dedifferentiation and fragmentation. Subsequently, fibroblast growth factor receptor (FGFR)-Ras-mitogen-activated protein kinase (MAPK) signaling in resulting mononucleated myoblasts maintains Twist expression, thereby stabilizing nuclear Yorkie activity and inducing their lineage switch into founder cells of the VLM./a> that is critical for regulating myosin activity, leads to structural defects. It was further shown that Rbfox1 directly binds the 3'-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type-specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type-specific splicing and expression dynamics of identity genes and structural proteins.
Nikonova, E., Mukherjee, A., Kamble, K., Barz, C., Nongthomba, U. and Spletter, M. L. (2022). Rbfox1 is required for myofibril development and maintaining fiber type-specific isoform expression in Drosophila muscles. Life Sci Alliance 5(4). PubMed ID: 34996845
Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. This study reports that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type-specific gene and splice isoform expression, notably loss of an indirect flight muscle-specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. It was further shown that Rbfox1 directly binds the 3'-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type-specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type-specific splicing and expression dynamics of identity genes and structural proteins.

RNA-binding Fox (Rbfox) proteins have well-established roles in regulating alternative splicing, but specific Rbfox isoforms lack nuclear localization signals and accumulate in the cytoplasm. The potential splicing-independent functions of these proteins remain unknown. This study demonstrates that cytoplasmic Drosophila Rbfox1, called such to conform to mammalian homologs, but given the FlyBase designation Ataxin-2 binding protein 1 (A2bp1), regulates germ cell development and represses the translation of mRNAs containing (U)GCAUG elements within their 3'UTRs. During germline cyst differentiation, Rbfox1 targets pumilio mRNA for destabilization and translational silencing, thereby promoting germ cell development. Misexpression of pumilio results in the formation of germline tumors, which contain cysts that break down and dedifferentiate back to single, mitotically active cells. Together, these results reveal that cytoplasmic Rbfox family members regulate the translation of specific target mRNAs. In the Drosophila ovary, this activity provides a genetic barrier that prevents germ cells from reverting back to an earlier developmental state. The finding that Rbfox proteins regulate mRNA translation has implications for Rbfox-related diseases (Carreira-Rosario, 2016).

RNA-binding proteins play an integral role in mRNA metabolism, splicing, transport, and translation. An increasing number of studies link mutations in genes encoding RNA-binding proteins with a variety of diseases, highlighting the importance of these proteins with regard to human health. Rbfox proteins represent one such family and contain a highly conserved, centrally located RNA recognition motif (RRM) flanked by intrinsically disordered regions (Auweter, 2006, Jin, 2003 and Ponthier, 2006). Mammals have three Rbfox paralogs: RBFOX1 (A2BP1), RBFOX2 (RBM9) , and RBFOX3 (NeuN) . Nuclear isoforms of these genes regulate alternative splicing by directly binding to intronic (U)GCAUG elements, resulting in the exclusion or inclusion of downstream or upstream exons, respectively. In mice, disruption of Rbfox1 in neurons leads to neuronal hyperactivity, while loss of Rbfox2 results in cerebellum development defects ( Gehman, 2011 and Gehman, 2012). Rbfox1 and Rbfox2 have been implicated in a number of diseases including cancer, diabetes, and neurological disorders such as autism, mental retardation, and epilepsy. In all these examples, the observed phenotypes have been attributed to perturbations in normal mRNA splicing patterns (Carreira-Rosario, 2016).

Specific isoforms of Rbfox genes localize to the cytoplasm of cells in a variety of tissues across species. While the molecular functions of these isoforms remain poorly understood, both nuclear and cytoplasmic isoforms appear to act as tumor suppressors in the context of glioblastomas. Loss of cytoplasmic Rbfox1 has also been associated with colorectal cancer, and abnormal cytoplasmic inclusions of Rbfox1 are often observed in spinocerebellar ataxia type II patients. Recent studies have also shown that Rbfox proteins bind to many different 3'UTRs in the mammalian brain. These observations suggest that Rbfox proteins carry out additional functions beyond their established roles in splicing (Carreira-Rosario, 2016 and references therein).

The Drosophila genome contains a single Rbfox homolog called A2bp1, referred to as Rbfox1 to remain consistent with nomenclature across species. Mutations in the Drosophila Rbfox1 result in germline tumor formation (Tastan, 2010). This study shows that cytoplasmic Rbfox1 is necessary for Drosophila germline development and regulates the stability and translation of specific mRNAs by binding to (U)GCAUG elements contained within their 3'UTR sequences. It was further shown that within the germline Rbfox1 targets pumilio to promote differentiation. Thus, this study reveals a splicing-independent function of Rbfox proteins, the disruption of which may contribute to RBFOX-linked diseases (Carreira-Rosario, 2016).

Rbfox family members from different species localize to either the nucleus or the cytoplasm. While isoforms that localize to the nucleus play a clear role in regulating alternative splicing (Gehman, 2011, Hamada, 2013 and Lee, 2009), the function of cytoplasmic isoforms has remained less clear. Previous results showed that loss of Rbfox1 in Drosophila resulted in a block of germ cell differentiation (Tastan, 2010). This study sought to determine the extent to which disruption of either nuclear or cytoplasmic Rbfox1 isoforms contributed to this phenotype. Transgenic rescue, isoform-specific RNAi knockdown and isoform-specific knockout experiments provide strong evidence that two cytoplasmic Rbfox1 isoforms specifically promote germ cell differentiation during the early stages of germline cyst development. The Drosophila genome does not encode for another redundant Rbfox family member. Thus the Drosophila ovary represents a unique platform on which to explore the function of cytoplasmic Rbfox family members in an in vivo setting (Carreira-Rosario, 2016).

Further experiments showed that cytoplasmic Rbfox1 regulates gene expression through a 3'UTR-dependent mechanism. The defining RRM domain of the Rbfox protein family is highly conserved across species. In vitro and in vivo experiments presented here indicate that Drosophila Rbfox1 physically associates with RNAs that contain GCAUG elements, similar to mammalian Rbfox proteins. Recent studies using RNA-crosslinking immunoprecipitation approaches have shown that mammalian Rbfox1, Rbfox2, and Rbfox3 all physically interact with 3'UTR sequences that contain GCAUG sites or other similar elements (Weyn-Vanhentenryck, 2014). The current experiments show that the presence of GCAUG sites within mRNA 3'UTRs results in modest decreases in mRNA stability, and more much dramatic decreases in protein expression. These observations suggest that Drosophila Rbfox1 acts to repress the translation of specific target mRNAs (Carreira-Rosario, 2016).

An increasing number of GCAUG sites within 3'UTRs appeared to have an additive effect on target gene expression in the context of germ cells. The presence of one site had little or no effect, at least in the context of the reporters that were used, while the presence of two or three sites resulted in a clear repression of protein expression in Rbfox1-expressing cells. While the repression of 3'UTR GCAUG reporters occurred in both the germline and within specific neurons, it remains possible that cytoplasmic Rbfox family members may regulate gene expression in a different manner in different contexts. For example, a newly published study shows that mammalian Rbfox proteins can promote the stability and translation of a target gene in cell culture (Lee, 2016). The functional significance of this regulation remains to be tested in vivo. Regardless, these findings, together with results presented here, indicate that the ability of cytoplasmic Rbfox family members to regulate protein expression has been conserved across species. The direction and degree of cytoplasmic Rbfox-dependent gene regulation may depend on different cell-specific proteins or on the presence of other 3'UTR regulatory elements within a given target transcript. The discovery of this function has significant implications for understanding of how Rbfox family members regulate normal development, as well as the disorders linked with disruption of Rbfox genes such as epilepsy, autism, and cancer (Carreira-Rosario, 2016).

The search for functionally relevant endogenous mRNA targets of Drosophila Rbfox1 led to the finding that Rbfox1 represses Pumilio protein expression during early germline cyst differentiation. Previous studies noted the presence of Pumilio protein in GSCs, cystoblasts, and two-cell cysts, but the mechanisms responsible for the stage-specific decrease of Pumilio expression in four-, eight-, and 16-cell cysts, and the functional significance of this expression pattern, have remained unknown. This study shows that Pumilio expression decreases as Rbfox1 expression increases. Examining the 3'UTR sequence of pumilio revealed the presence of four GCAUG sites, two of which showed extensive sequence conservation across many Drosophila species. Strikingly, Rbfox1, Rbfox2, and Rbfox3 also physically associate with Pumilio1 and Pumilio2 mRNA in the mouse nervous system (Carreira-Rosario, 2016).

Further analysis showed that Pumilio expression in the germline is regulated through a 3'UTR-dependent mechanism. A wild-type pumilio 3'UTR reporter exhibited an expression pattern similar to the endogenous protein, displaying decreased expression in the presence of Rbfox1. Mutating each of the four GCAUG elements within the pumilio 3'UTR sequence resulted in a striking expansion of reporter expression into the four-, eight-, and 16-cell cyst stages, suggesting that Rbfox1 negatively regulates Pumilio expression. qRT-PCR analysis of synchronously differentiating germ cells showed that endogenous pumilio mRNA levels increased in the absence of Rbfox1. These findings are in contrast to data obtained comparing the 3x Rbfox1 sensor to the mutant reporter, and suggest that Rbfox1 may influence the stability of specific target mRNAs in different contexts. Nonetheless, the degree to which Pumilio protein expression increases in the absence of Rbfox1 in these experiments is consistent with the model that Rbfox1 also regulates the expression of Pumilio, at least in part, at the level of translation. Other translational regulators, such as Bruno, also appear to influence mRNA stability (Carreira-Rosario, 2016).

The repression of Pumilio expression by Rbfox1 helps to promote germ cell differentiation. Loss of Rbfox1 results in germline tumor formation and an expansion of Pumilio expression. Strikingly, knockdown of pumilio strongly suppresses the Rbfox1 tumorous phenotype, leading to the formation of egg chambers with polyploid nuclei. While cytoplasmic Rbfox1 likely regulates the expression of other genes, the strength of this genetic interaction indicates that pumilio represents a major functional target of Rbfox1 in regard to germ cell differentiation. Mis-expression of a pumilio transgene, lacking the endogenous 3'UTR, in an otherwise wild-type background, phenocopies Rbfox1 mutants. These data indicate that germ cells must repress Pumilio expression before they can proceed into the next stage of development. Pumilio homologs are essential genes for germ cell maintenance across species. Given the conservation of Rbfox1-binding sites within the 3'UTR of pumilio mammalian homologs, repression of pumilio by Rbfox may represent a conserved mechanism that promotes germline differentiation (Carreira-Rosario, 2016).

Strikingly, morphological and molecular markers suggest that overexpression of Pumilio results in the dedifferentiation of germ cells. Rbfox1 mutants exhibit a similar phenotype. Pumilio overexpressing ovaries contain large tumors with multicellular cysts throughout their germaria. As these cysts continue to age and move toward the posterior of germaria, they begin to break down, as marked by the fragmentation of fusomes and ring canals. Similar observations have been made in both the Drosophila ovary and testis when germline cysts are experimentally prompted to undergo dedifferentiation. Furthermore, these germ cells re-acquire the expression of cytoplasmic Sxl, which typically marks GSCs, cystoblasts and two-cell cysts. Single cells derived from cyst breakdown remain mitotically active. These data indicate that germ cells must actively shut down gene expression programs that foster self-renewal and early differentiation before they can advance to the next stage of development. Failure to do so results in the reversion of the cells back to an earlier developmental state. It is anticipated that loss of Rbfox1, and the corresponding mis-expression of Rbfox1 target genes, may have similar effects in different tissues and in different species (Carreira-Rosario, 2016).

miR-980 is a memory suppressor microRNA that regulates the autism-susceptibility gene A2bp1

MicroRNAs have been associated with many different biological functions, but little is known about their roles in conditioned behavior. This study demonstrates that Drosophila miR-980 is a memory suppressor gene functioning in multiple regions of the adult brain. Memory acquisition and stability were both increased by miR-980 inhibition. Whole cell recordings and functional imaging experiments indicated that miR-980 regulates neuronal excitability. This study identified the autism susceptibility gene, A2bp1, as an mRNA target for miR-980. A2bp1 levels varied inversely with miR-980 expression; memory performance was directly related to A2bp1 levels. In addition, A2bp1 knockdown reversed the memory gains produced by miR-980 inhibition, consistent with A2bp1 being a downstream target of miR-980 responsible for the memory phenotypes. These results indicate that miR-980 represses A2bp1 expression to tune the excitable state of neurons, and the overall state of excitability translates to memory impairment or improvement (Guven-Ozkan, 2016).

MicroRNAs (miRNAs) are small (21–23 nt), non-coding RNAs that repress gene expression to regulate cellular development and physiology. A short seed sequence (6–8 nt) located at the 5' end of miRNAs binds to complementary sequences in the 3'-UTR of target mRNAs torepress mRNA expression by blocking translation and/or promoting degradation of the mRNA target). Thus, miRNAs offer a relatively rapid, analog, and cell-type-specific control mechanism for the epigenetic expression of genomic information in both time and space (Guven-Ozkan, 2016).

One aspect of miRNA function that remains understudied concerns the roles for these molecules in learning and memory, a primary adaptive function of the CNS. Prior studies revealed that broad insults to the miRNA processing pathway impairs memory formation in both Drosophila and the mouse. Although eukaryotic genomes encode hundreds of distinct miRNAs and they are generally expressed at high levels in the CNS, only a handful of specific miRNAs have been studied and implicated in memory formation through roles in neuronal maturation, connectivity, and synaptic plasticity (Guven-Ozkan, 2016).

To identify the miRNAs that participate in the biology of memory formation, a large scale, comprehensive screen was conducted using a transgenic approach to systematically inhibit 134 different miRNAs, using a 'microRNA sponge' technique (Ebert, 2007). The influences of 134 miRNAs were surveyed for effects on intermediate term (ITM, i.e., at 3 hr after conditioning), olfactory aversive memory. From this screen, several new miRNAs were identified that function to inhibit or promote memory formation at this time point. MiR-980, when inhibited, was shown to enhance memory formation. Thus, MiR-980, a member of the miR-22 family of vertebrate miRNAs, was classified as having a memory suppressor function (Guven-Ozkan, 2016).

This study characterize the memory suppressing function of miR-980. Among the mRNA targets for miR-980, it was demonstrated that the autism-susceptibility gene, Ataxin2 binding protein 1 (A2bp1, also known as Rbfox-1, Fox-1) is a primary target responsible for miR-980-directed memory suppression. A2bp1 is a known RNA binding protein involved in alternative splicing of a network of critical neuronal genes during development and in adults (Lee, 2009; Fogel, 2012) and in addition to autism (ASD), is associated with intellectual disability and epilepsy. Opposite to the role for miR-980, A2bp1 as a memory-promoting gene. Combined data advance understanding of the miR-22 family of miRNAs, showing that in Drosophila the magnitude of memory formation is a direct function of miR-980 abundance and of its primary mRNA target for this function, A2bp1 (Guven-Ozkan, 2016).

A behaviorally based 'miRNA sponge screen' was conducted to systematically identify the miRNAs involved in Drosophila olfactory aversive learning and memory (Busto, 2015). The results offer five major advances in knowledge about the function of this class of regulatory molecules: (1) miR-980 functions to suppress memory formation by acting in multiple types of neurons within the olfactory nervous system; (2) miR-980 works as a suppressor of acquisition and memory stability; (3) miR-980 suppresses the excitability of excitatory neurons; (4) the memory suppressor functions of miR-980 are mediated largely by the inhibition of the autism-susceptibility gene, A2bp1; and (5) A2bp1, itself, is a memory-promoting gene (Guven-Ozkan, 2016).

One surprising observation made in this study was that inhibition of miR-980 in multiple neurons within the olfactory nervous system enhances memory performance, as was anticipated, finding a single cellular focus for its effects. Initially, it was difficult to understand how a single microRNA could modify behavioral memory when altered in one of many different types of neurons. This was reconciled by showing that excitability of projection neurons is enhanced with inhibited miR-980 function, offering the explanation that increased signaling, in general, within the olfactory nervous system enhances behavioral memory. This model provides a general explanation for the effects of miR-980 that function in multiple classes of excitable neurons (Guven-Ozkan, 2016).

It is proposed that the role of miR-980 in excitability accounts for the increased acquisition when the miRNA is inhibited. An increase in excitable state may simply enhance the signaling through different neuron types within the olfactory nervous system as the organism integrates sensory information into memory. A corollary of this idea is that normal acquisition is a composite effect of multiple neurons within the circuit conveying the sensory information being learned. Although it is possible that increased acquisition also accounts for the increased memory performance observed when immediate performance scores were normalized, an alternative possibility is that miR-980 may have distinct roles in acquisition and memory stability. For instance, although the increased acquisition is attributed to increased neuronal excitability, the increased memory after acquisition may be due to altered regulation of molecules involved in synaptic transmission (Guven-Ozkan, 2016).

miR-980 belongs to the miR-22 family of miRNAs found in mammals. Within the nervous system, the miR-22 family has been reported to participate in neuroprotection, neurodegeneration, neuroinflammation, neurodevelopment. Thus, although this family appears to have multiple roles in the nervous system and disease, the current studies identify members of this family as specifically involved in the suppression of memory formation. Given the functional association between miR-980 and A2bp1 shown here, it is also tempting to speculate that the miR-980/miR-22 family of miRNAs might be associated with autism spectrum disorders. No evidence for this possibility has yet been reported, but the expression of miR-22 is reduced in attention deficit hyperactivity disorder (ADHD) and is genetically associated with panic disorder and anxiety in humans. Thus, there are neuropsychiatric links to miR-22 , which could potentially be through a role in excitability. Moreover, miR-22 represses the tumor suppressor gene PTEN in transformed human bronchial epithelial cells, and PTEN is known to be involved in Cowden syndrome and ASD in humans (Guven-Ozkan, 2016).

Behavioral, molecular, cellular, and genetic data together argue that A2bp1 is a primary target of miR-980 for memory suppression. First, A2bp1 is broadly expressed in the fly brain, consistent with a broad nervous system requirement for miR-980. Second, there are three miR-980 binding sites in A2bp1 3' UTR making it a strong candidate mRNA target for miR-980 regulation. Third, an in vitro mRNA binding experiment was performed using biotinylated mature miR-980 as bait, and eight times more A2bp1 mRNA was successfully captured using wild-type miR-980 versus a form mutated for the seed region. Fourth, A2bp1 shows the precise abundance/behavior relationship predicted as a direct target of miR-980. Overexpression of A2bp1 increases memory; miR-980 suppression increases memory. A2bp1 knockdown impairs memory; miR-980 overexpression impairs memory. Fifth, A2bp1 protein abundance varies as expected by manipulation of miR-980 levels. Overexpression of miR-980 decreases A2bp1 protein abundance and miR-980 suppression increases A2bp1 protein abundance. Finally, reducing A2bp1 levels using RNAi in miR-980-inhibited flies reversed the memory improvement. This finding is consistent with the model that A2bp1 is genetically downstream of miR-980 and a major mediator of the phenotype. However, the possibilities cannot be excluded that there may be additional miR-980 targets that participate in memory suppression and miR-980 regulation of A2bp1 could be indirect. A simple model for miR-980/A2bp1 interactions and function seem to be at odds with an observation made about A2bp1 using mammalian models. In the mouse, neuronal-specific knockout of A2bp1 increases excitability in the dentate gyrus, a result opposite of that predicted by the current model. This difference might reflect species or cell type differences, the complexity of the gene with its dozens of isoforms, or the multiple layers of regulation on A2bp1 expression. Bioinformatics analyses predict multiple miRNAs as binding to the A2bp1 3' UTR and regulating its expression. Thus, its basal or regulated expression level due to changes in physiological state could be a composite of (Guven-Ozkan, 2016).

A2bp1 is associated with autism and epilepsy in human patients functioning presumably by regulating alternative splicing during both development and in adults). It has been proposed that changes in gene-splicing alter the relative abundance of protein isoforms, which remodels protein networks and increases the risk for autism. Consistent with this thought, transcriptome analyses from ASD brains identified A2bp1 as one hub gene that is dysregulated in patients with autism. A2bp1 was originally identified through its interaction with Ataxin-2. Pn-specific knockdown of Ataxin-2 impairs long-term olfactory habituation-associated structural and functional plasticity by regulating the miRNA pathway. Future studies will shed light on whether memory phenotypes of A2bp1 are dependent on Ataxin-2. It is intriguing that the current studies show that adult stage-specific increases in A2bp1 abundance improve aversive olfactory memory, independent of any developmental function for the protein, and human ASD is a spectrum brain disorder that is associated with poor to extraordinarily robust learning and memory capacities. It is speculated that the different protein interaction networks that form due to varying levels of A2bp1 function account for the range of intellectual abilities observed in ASD. Drosophila may prove to be a much speedier and simpler system to dissect the specific effect of A2bp1 abundance on the emergence of protein interaction networks and their influence on cognitive abilities (Guven-Ozkan, 2016).

Drosophila ataxin 2-binding protein 1 marks an intermediate step in the molecular differentiation of female germline cysts

In the Drosophila ovary, extrinsic signaling from the niche and intrinsic translational control machinery regulate the balance between germline stem cell maintenance and the differentiation of their daughters. However, the molecules that promote the continued stepwise development of ovarian germ cells after their exit from the niche remain largely unknown. This study reports that the early development of germline cysts depends on the Drosophila homolog of the human ataxin 2-binding protein 1 (A2BP1/Rbfox1) gene. Drosophila A2BP1 protein expression is first observed in the cytoplasm of 4-, 8- and 16-cell cysts, bridging the expression of the early differentiation factor Bam with late markers such as Orb, Rbp9 and Bruno encoded by arrest. The expression of A2BP1 is lost in bam, sans-fille (snf) and mei-P26 mutants, but is still present in other mutants such as rbp9 and arrest. A2BP1 alleles of varying strength produce mutant phenotypes that include germline counting defects and cystic tumors. Phenotypic analysis reveals that strong A2BP1 alleles disrupt the transition from mitosis to meiosis. These mutant cells continue to express high levels of mitotic cyclins and fail to express markers of terminal differentiation. Biochemical analysis reveals that A2BP1 isoforms bind to each other and associate with Bruno, a known translational repressor protein. These data show that A2BP1 promotes the molecular differentiation of ovarian germline cysts (Tastan, 2010).

A2BP1 is expressed in a novel pattern during early cyst development and mutations in A2BP1 disrupt early oogenesis, resulting in the formation of cystic tumors. These findings suggest that A2BP1 helps regulate changes in gene expression programs during the intermediate steps of germline cyst development (Tastan, 2010).

Past studies have sought to characterize the mechanisms that control bam expression in germline stem cells and cystoblasts. These efforts led to the understanding that dpp signaling from the cap cells initiates a phosphorylation cascade that results in the transcriptional repression of bam in stem cells through a well-defined element within its promoter. Once a stem cell daughter leaves the niche, this repression subsides resulting in active bam transcription. The expression of Bam continues up until the eight-cell cyst stage whereupon it is again repressed. Given these findings and the lack of two-, four- and eight-cell cyst specific markers, the prevailing view has been that all Bam-expressing cysts are roughly equivalent on a molecular level. Subsequently, fusome branching has served as a widely used marker to track the progress of cyst differentiation. However, the expression of A2BP1 now shows that the number of mitotic divisions undertaken by a cyst does not necessarily reflect the underlying molecular state of these cells. In addition to undergoing successive rounds of incomplete mitotic divisions, cystoblasts, and two, four- and eight-cell cysts also exhibit distinct changes in their gene expression programs. For example, the cystoblast expresses both cytoplasmic Sxl protein and Bam. In two-cell cysts, Sxl expression begins to recede while Bam levels increase. In four-cell cysts, cytoplasmic Sxl protein is no longer detectable, Bam expression continues and A2BP1 protein expression is induced. In eight-cell cysts, Bam expression begins to decrease while A2BP1 expression continues to increase. Finally, in 16-cell cysts, Bam is absent, A2BP1 is present and the expression of other proteins such as Nanos, Orb, Rpb9 and Bruno is upregulated. These markers probably reflect much broader changes in gene expression during cyst development (Tastan, 2010).

In situ hybridization suggests that the regulation of A2BP1 expression occurs at the level of transcription. However, given the complexity of the A2BP1 locus, the possibility cannot be ruled out that alternative splicing and translational regulation also restrict A2BP1 expression. Examining A2BP1 protein expression in various mutant backgrounds has allowed further subdivision of cystic tumors. Consistent with previous phenotypic characterization, A2BP1 expression shows that mei-P26, snf, rbp9 and arrest tumors are arrested at different stages of cyst differentiation. Loss of mei-P26 and snf blocks the molecular differentiation of cysts prior to the induction of A2BP1 expression. The absence of A2BP1 expression within mei-P26 and snf mutant germline cysts indicates that mitotic divisions can continue in the absence of molecular differentiation. These findings suggest mei-P26, snf and, by inference, sxl help drive cyst development to a point defined by A2BP1. In turn, A2BP1 promotes cyst progression towards a terminally differentiated state marked by reduced levels of Bam and Sxl and increased levels of late markers such as Rbp9 and Orb (Tastan, 2010).

What is the developmental function of A2BP1? A number of mutations that result in germline tumors have been isolated. Some of these mutations disrupt genes involved in the regulation of sex-specific splicing and germline sexual identity. However, the issue of why defects in sexual identity result in tumorous phenotypes remains largely unresolved. Other mutations such as mei-P26 and arrest do not have a clear role in establishing sexual identity, suggesting that disruption of other molecular pathways can also block cyst differentiation. The current data, together with previous findings, suggests cysts must turn off earlier programs of gene expression to move to the next stage of differentiation. For example, Bam expression must be repressed in 16-cell cysts. In encore and A2BP1 mutants, the expansion of Bam expression results extra mitotic divisions and a subsequent delay in meiosis. Significantly, bam null mutations can suppress both encore and weak A2BP1 mutant phenotypes. Similarly, expansion of cytoplasmic Sxl and Cyclin A in arrest and A2BP1 mutants correlates with an inability to enter meiosis (Tastan, 2010).

A2BP1 contains a highly conserved RNA recognition motif. The mammalian homolog of A2BP1 was first identified based on its association with ataxin 2. This study found little evidence that A2BP1 physically or genetically interacts with Drosophila Ataxin 2 in S2 cell extracts or during early cyst development. However, this conclusion is based on negative data and the findings that A2BP1 functions during nervous system development (Koizumi, 2007) leaves open the possibility that these two proteins may interact in different contexts (Tastan, 2010).

These studies suggest a functional link between A2BP1 and Bruno. Interestingly, high levels of A2BP1 expression precede high levels of Bruno expression. However, there are detectable levels of Bruno within four- and eight-cell cysts. These low levels of Bruno expression may be sufficient to coordinate with A2BP1 to promote cyst development. A2BP1 may also have additional functions that do not require interaction with Bruno. Differences between A2BP1 and arrest mutant phenotypes may reflect these separate functions. However, it is important to note that the Drosophila genome encodes three highly related Bruno-like proteins (FlyBase). Genetic redundancy between these genes may mask other functions during early cyst development. Regardless, the observed genetic and physical interactions between A2BP1 and Bruno support a model in which these two proteins cooperate to regulate germline cyst differentiation (Tastan, 2010).

Mammalian A2BP1 binds to UGCAUG RNA elements within introns and regulates the alternative splicing of specific messages. Given the RRM domain in A2BP1 is 90% identical to its mammalian homologs, A2BP1 may also bind to similar elements. The original study that defined the A2BP1 RNA binding sites in vitro showed that the protein associated with GCAUG sites with a slight bias for UGCAUG and AGCAUG sequences. Further work showed A2BP1 bound preferentially to UGCAUG sites in vivo. The cytoplasmic localization of A2BP1 protein in four-, eight- and 16-cell cysts and its association with Bruno suggests it may participate in translational repression. Examination of annotated sequences (FlyBase) reveals that a small number of Drosophila transcripts contain multiple UGCAUG and AGCAUG sites within their 3′UTRs. For example, sxl-RH 3′UTR has five AGCAUG sites but none of the preferred UGCAUG sites. The functional significance of these elements remains uncertain. Interestingly, Bruno has also been implicated in alternative splicing. Therefore, identifying in vivo mRNA targets of A2BP1 will help clarify its molecular functions and further enhance understanding of the complex genetic hierarchies that control germline cyst development (Tastan, 2010).


Search PubMed for articles about Drosophila A2bp1

Auweter, S. D., Fasan, R., Reymond, L., Underwood, J. G., Black, D. L., Pitsch, S. and Allain, F. H. (2006). Molecular basis of RNA recognition by the human alternative splicing factor Fox-1. EMBO J 25: 163-173. PubMed ID: 16362037

Busto, G. U., Guven-Ozkan, T., Fulga, T. A., Van Vactor, D. and Davis, R. L. (2015). microRNAs That Promote or Inhibit Memory Formation in Drosophila melanogaster. Genetics 200: 569-580. PubMed ID: 26088433

Carreira-Rosario, A., Bhargava, V., Hillebrand, J., Kollipara, R. K., Ramaswami, M. and Buszczak, M. (2016). Repression of Pumilio protein expression by Rbfox1 promotes germ cell differentiation. Dev Cell 36: 562-571. PubMed ID: 26954550

Ebert, M. S., Neilson, J. R. and Sharp, P. A. (2007). MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods 4: 721-726. PubMed ID: 17694064

Fogel, B. L., Wexler, E., Wahnich, A., Friedrich, T., Vijayendran, C., Gao, F., Parikshak, N., Konopka, G. and Geschwind, D. H. (2012). RBFOX1 regulates both splicing and transcriptional networks in human neuronal development. Hum Mol Genet 21: 4171-4186. PubMed ID: 22730494

Gehman, L. T., Stoilov, P., Maguire, J., Damianov, A., Lin, C. H., Shiue, L., Ares, M., Jr., Mody, I. and Black, D. L. (2011). The splicing regulator Rbfox1 (A2BP1) controls neuronal excitation in the mammalian brain. Nat Genet 43: 706-711. PubMed ID: 21623373

Gehman, L. T., Meera, P., Stoilov, P., Shiue, L., O'Brien, J. E., Meisler, M. H., Ares, M., Jr., Otis, T. S. and Black, D. L. (2012). The splicing regulator Rbfox2 is required for both cerebellar development and mature motor function. Genes Dev 26: 445-460. PubMed ID: 22357600

Guven-Ozkan, T., Busto, G. U., Schutte, S. S., Cervantes-Sandoval, I., O'Dowd, D. K. and Davis, R. L. (2016). miR-980 is a memory suppressor microRNA that regulates the autism-susceptibility gene A2bp1. Cell Rep 14: 1698-1709. PubMed ID: 26876166

Hamada, N., Ito, H., Iwamoto, I., Mizuno, M., Morishita, R., Inaguma, Y., Kawamoto, S., Tabata, H. and Nagata, K. (2013). Biochemical and morphological characterization of A2BP1 in neuronal tissue. J Neurosci Res 91: 1303-1311. PubMed ID: 23918472

Jin, Y., Suzuki, H., Maegawa, S., Endo, H., Sugano, S., Hashimoto, K., Yasuda, K. and Inoue, K. (2003). A vertebrate RNA-binding protein Fox-1 regulates tissue-specific splicing via the pentanucleotide GCAUG. EMBO J 22: 905-912. PubMed ID: 12574126

Koizumi, K., Higashida, H., Yoo, S., Islam, M. S., Ivanov, A. I., Guo, V., Pozzi, P., Yu, S. H., Rovescalli, A. C., Tang, D. and Nirenberg, M. (2007). RNA interference screen to identify genes required for Drosophila embryonic nervous system development. Proc Natl Acad Sci U S A 104: 5626-5631. PubMed ID: 17376868

Lee, J. A., Tang, Z. Z. and Black, D. L. (2009). An inducible change in Fox-1/A2BP1 splicing modulates the alternative splicing of downstream neuronal target exons. Genes Dev 23: 2284-2293. PubMed ID: 19762510

Lee, J. A., Damianov, A., Lin, C. H., Fontes, M., Parikshak, N. N., Anderson, E. S., Geschwind, D. H., Black, D. L. and Martin, K. C. (2016). Cytoplasmic Rbfox1 Regulates the Expression of Synaptic and Autism-Related Genes. Neuron 89: 113-128. PubMed ID: 26687839

Ponthier, J. L., Schluepen, C., Chen, W., Lersch, R. A., Gee, S. L., Hou, V. C., Lo, A. J., Short, S. A., Chasis, J. A., Winkelmann, J. C. and Conboy, J. G. (2006). Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16. J Biol Chem 281: 12468-12474. PubMed ID: 16537540

Tastan, O. Y., Maines, J. Z., Li, Y., McKearin, D. M. and Buszczak, M. (2010). Drosophila ataxin 2-binding protein 1 marks an intermediate step in the molecular differentiation of female germline cysts. Development 137: 3167-3176. PubMed ID: 20724451

Weyn-Vanhentenryck, S. M., Mele, A., Yan, Q., Sun, S., Farny, N., Zhang, Z., Xue, C., Herre, M., Silver, P. A., Zhang, M. Q., Krainer, A. R., Darnell, R. B. and Zhang, C. (2014). HITS-CLIP and integrative modeling define the Rbfox splicing-regulatory network linked to brain development and autism. Cell Rep 6: 1139-1152. PubMed ID: 24613350

Biological Overview

date revised: 12 June 2022

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