Previous studies have demonstrated that mammalian FMR1 has RNA-binding activity that is conferred by the two KH domains and the RGG box. In an RNA homopolymer binding assay, in vitro-translated, 35S-labeled hFMR1 protein shows strong binding to poly(G), weaker but significant binding to poly(U), and no detectable binding to poly(C) and poly(A). Under the same binding conditions, the same RNA homopolymer binding profile was observed for Fmr1. The conservation of the RNA-binding activity between Fmr1 and hFMR1 lends further support to the conclusion that Fmr1 is functionally related to the vertebrate FMR1/FXR proteins. To assess whether the KH domains of Fmr1 confer its RNA-binding capability, point mutations predicted to inactivate the function of either KH domain were engineered into the Drosophila Fmr1 cDNA by site-directed mutagenesis. A codon for a highly conserved isoleucine residue within each of the KH domains was mutated to a codon for asparagine (I244N or I307N). These KH domain mutations are analogous to a mutation identified in the FMR1 protein of a fragile X patient, and such analogous mutations in human FMR1 have been shown to impair RNA binding in vitro. Furthermore, solution structure data of these KH domains predict that these substitutions will disrupt an alpha-helix structure within the KH domain. RNA homopolymer binding assays with these mutant forms of Fmr1 show that either mutation impairs the ability of Fmr1 to bind poly(U) in vitro as is observed for the analogous hFMR1 mutants (Wan, 2000).
Another biochemical feature of hFMR1 and hFXR proteins is their capacity to form heteromers with other FMR1/FXR proteins. To determine if Fmr1 has a similar capacity, in vitro binding experiments were performed to test the interaction of purified recombinant GST-Fmr1 with Fmr1 and with hFMR1 and hFXR produced by in vitro transcription and translation. GST-Fmr1 binds with the highest avidity to hFMR1, less well to hFXR2, and very weakly to hFXR1 and to itself. In reciprocal experiments in which hFMR1, hFXR1, and hFXR2 were immobilized as GST fusion proteins, the same profiles of relative binding avidity were observed. The order of preference of binding of Fmr1 suggests that each of the FMR1/FXR proteins has a characteristic selectivity of protein-protein (Wan, 2000).
FMRP is a negative translational regulator (Laggerbauer, 2001; Li, 2001; Schaeffer, 2001). What potential targets for Fmr1 could explain its regulation of synaptic structure and transmission in the eye and NMJ? Futsch, the Drosophila homolog of the mammalian microtubule-associated protein MAP1B, regulates the microtubule cytoskeleton to mediate dendritic, axonal, and synaptic growth. Hypomorphic futsch mutants display a distinctive NMJ morphology phenotype similar to dfxrNOE (neuronal overexpression of Fmr1), i.e., fewer and larger synaptic boutons. Moreover, microtubules play an essential role in the transport and subsequent regulation of synaptic vesicle dynamics underlying neurotransmission. Based on these lines of reasoning, immunoprecipitation and Western analyses were performed to determine whether Futsch expression is regulated by Fmr1 (Zhang, 2001).
First, tests were carried out for a physical interaction between Fmr1 protein and futsch mRNA. Immunoprecipitation analyses were performed using a monoclonal anti-Fmr1 to identify mRNAs that associate with the protein: (1) as a positive control, RT-PCR shows that the protein complex immunoprecipitated by anti-Fmr1 contains Fmr1 mRNA, in agreement with previous findings with mammalian FMRP (Ashley, 1993a; Brown, 1998); (2) futsch mRNA is also present in the immunoprecipitated protein complex. As a negative control, it was shown that anti-Fmr1 immunoprecipitation does not pull down alpha-tubulin mRNA, which is highly expressed in brain. Subsequent sequencing of the RT-PCR products confirms the association of Fmr1 and futsch mRNA. These results demonstrate that Fmr1 protein specifically binds futsch mRNA and may regulate Futsch expression at a translational level (Zhang, 2001).
Quantitative Western analyses have shown that Futsch expression in the nervous system is inversely correlated with Fmr1 expression. Initially, observed alterations were observed in the distribution and intensity of Futsch immunoreactivity in the nervous systems of Fmr1 mutants. To quantify these changes in Futsch expression, Western analyses was performed on adult head extracts from Fmr1 mutants and dfxrNOE lines. Futsch protein levels are significantly decreased to an average of 78.2% ± 2.5% of control levels in dfxrNOE. In Fmr1 null mutants, in contrast, Futsch is altered in the opposite direction, displaying an increase to 208% ± 32.4% of control levels. These results demonstrate that Fmr1 negatively regulates the expression of Futsch in the nervous system. The negative regulation of Futsch by Fmr1, together with the binding of Fmr1 with futsch mRNA as well as reports of a role of FMRP in translational regulation, suggests that Fmr1 acts as a negative translational regulator of Futsch (Zhang, 2001).
Futsch regulates microtubules at the Drosophila NMJ (Roos, 2000). Therefore, it was hypothesized that Fmr1-dependent Futsch regulation might mediate the control of synaptic structure and function, explaining the synaptic dysfunction observed in Fmr1 mutants. Since Fmr1 null mutants elevate Futsch expression, this model predicts that Futsch overexpression should mimic Fmr1 synaptic phenotypes. Similarly, Fmr1 overexpression decreases Futsch expression, so futsch hypomorph mutants should display dfxrNOE phenotypes. Finally, double mutants of Fmr1;futsch would be predicted to rescue Fmr1 synaptic structure and function phenotypes. These predictions were tested in futsch mutants, futsch transgenic NOE lines, and Fmr1;futsch double mutants by assaying synaptic structure and neurotransmission at both the larval NMJ and adult eye (Zhang, 2001).
Consistent with the hypothesis, futschNOE causes an NMJ overgrowth phenotype similar to Fmr1 null mutants, with increased synaptic area, branching, and bouton number. Also consistent with the hypothesis, hypomorphic futsch mutants display reduced NMJ growth and enlarged synaptic boutons, similar to dfxrNOE. Therefore, a Fmr1;futsch double mutant (Fmr1 null allele 50M; futsch hypomorph allele N94) and NMJ structure were assayed. The double mutant forms a structurally normal NMJ indistinguishable from wild-type with regard to synaptic branching and bouton number. These results demonstrate that upregulation of Futsch is sufficient to explain the synaptic structural defects caused by Fmr1 loss-of-function (Zhang, 2001).
Functional assays of neurotransmission in the eye and NMJ were performed with futsch hypomorphs and NOE lines to determine whether the regulation fits predictions from the model. Consistent with the hypothesis, both futsch hypomorphs and futschNOE elevate neurotransmission at the NMJ. Also consistent with the hypothesis, both futsch hypomorphs and futschNOE significantly reduce photoreceptor neurotransmission. It is particularly striking that either loss or overexpression of both Fmr1 and futsch have identical effects on synaptic transmission, i.e., elevated at the NMJ and suppressed in the eye (Zhang, 2001).
These observations suggest that precise regulation of Futsch levels by Fmr1 is required to properly maintain transmission in central and peripheral synapses. If the defects observed in Fmr1 mutants are due to the upregulation of Futsch levels, then one should be able to bring the levels of Futsch down to compensate by combining Fmr1 nulls with futsch hypomorphs in the same genome. Double mutants of Fmr1;futsch were generated and synaptic transmission at the NMJ and in the eye was assayed. A remarkable suppression of synaptic defects observed in Fmr1 mutants was seen. In the NMJ, the double mutant reduces transmission to levels indistinguishable from controls, whereas in the eye, the double mutant increases neurotransmission to wild-type levels. This genetic suppression provides convincing evidence that Fmr1 regulates synaptic mechanisms entirely through its regulation of Futsch. Taken together, this study strongly supports the hypothesis that Fmr1 acts as a translational repressor of Futsch to regulate microtubule dynamics and thereby control synaptic structure and function (Zhang, 2001).
RNA interference (RNAi) is a flexible gene silencing mechanism that responds to double-stranded RNA by suppressing homologous genes. This study reports the characterization of RNAi effector complexes (RISCs) that contain small interfering RNAs and microRNAs (miRNAs). Two putative RNA-binding proteins, the Drosophila homolog of the mammalian fragile X mental retardation protein (FMRP), Fmr1, and VIG (Vasa intronic gene), are identified through their association with RISC. FMRP, the product of the human fragile X locus, regulates the expression of numerous mRNAs via an unknown mechanism. The possibility that Fmr1, and potentially FMRP, use, at least in part, an RNAi-related mechanism for target recognition suggests a potentially important link between RNAi and human disease (Caudy, 2002).
Biochemical purification of the RNAi effector nuclease, RISC, revealed the Drosophila Argonaute-2 (Ago-2) protein as a core component of this complex (Hammond. 2001). In an effort to identify additional RISC components, large-scale biochemical purification was performed following both RISC activity and Ago-2 protein by Western blotting. After five purification steps, a number of additional proteins also consistently co-purified. Among those were two RNA-binding proteins, VIG and Fmr1. Each protein was identified in multiple independent preparations of purified RISC (Caudy, 2002).
The VIG protein is encoded from within an intron of the Vasa gene. This protein has no recognizable protein domains, other than an RGG box, a motif that is known to bind RNA. VIG is an evolutionarily conserved protein, with homologs in C. elegans, Arabidopsis, mammals, and Schizosaccharomyces pombe. Little is known regarding the function of this protein family. However, a human homolog of VIG, PAI-RBP-1, was originally identified as a protein with affinity for an AU-rich element in the 3'-UTR of the plasminogen activator inhibitor (PAI) RNA, which regulates its stability (Caudy, 2002).
A human ortholog of Fmr1, FMRP, is encoded from a locus on the X chromosome that is epigenetically silenced in Fragile X syndrome. FMRP is expressed not only in neuronal cells but also in numerous other tissues, and Fragile X patients display additional phenotypes, including macroorchidism. The human genome also encodes two additional FMRP homologs, FXR1 and FXR2. Fragile X family members each contain two copies of a KH domain (hnRNP K homology) and an RGG box. All three of these domains have been proposed to bind RNA (Caudy, 2002).
To test the association of VIG and Fmr1 with RISCs, epitope-tagged proteins were expressed in Drosophila S2 cells. Notably, both Vig and Fmr1 co-fractionate with Ago-2 and RISC activity in an ~500-kD complex, as predicted if all three were components of RISC. Both RISC and Fmr1 pellet in a high-speed centrifugation and can be extracted from the P100 pellet by treatment with 0.4 M KOAc. Essentially, all expressed Fmr1 co-fractionates with RISC through subsequent ion exchange columns. In accord with this hypothesis, immunoprecipitation of Fmr1 results in co-precipitation of Ago-2 protein. To test whether the interaction is bridged by binding separately to a common mRNA, immunoprecipitations were treated with RNAse A. Interaction between Ago-2 and Fmr1 is resistant to treatment of the immunoprecipitates with RNAse A up to concentrations of 10 mg/mL. Similarly, Ago-2 was also recovered in immunoprecipitations of epitope-tagged VIG, and VIG and Fmr1 were present in mutual immunoprecipitations (Caudy, 2002).
The presence of VIG and Fmr1 in RISCs would predict that these proteins would also interact with siRNAs, another established component of RISCs. To test this possibility, Drosophila S2 cells were co-transfected with 32P-labeled dsRNA triggers of ~500 nt in length and either tagged VIG or Fmr1. In each case, immunoprecipitation selectively recovered ~21 nt siRNAs, even though these are a minor component in analyses of total RNA extracted from cells transfected with labeled silencing triggers (Caudy, 2002).
To examine the requirement for Fmr1 and VIG in RNAi, RNAi was used to reduce the expression of each of these gene products in S2 cells. This approach has been used previously to test the dependency of RNAi on Dicer and Argonaute family members in Drosophila cells, mammalian cells, and C. elegans and as a means to identify components of the RNAi machinery in worms. Pretreatment of cells with dsRNAs covering the coding sequences of either Fmr1 or VIG partially impair silencing. In the former case, expression returned to 50% of the unsuppressed control, whereas in the latter case, luciferase was de-repressed to ~70% of the control. The partial effect of suppressing Fmr1 can be interpreted in several ways. First, Fmr1 could be an accessory factor for RISCs, affecting efficiency of the complex but not being absolutely required for RNAi. Second, Fmr1 could be essential to only a subset of RISCs, whereas other (perhaps KH domain) proteins replace Fmr1 in alternative versions of RISCs. Third, Fmr1 might not be required for RNAi per se; however, loss of Fmr1 could affect the long-term stability of some RISC complexes. Another Argonaute family member expressed in S2 cells, Ago-1, appears to have little effect on the efficiency of RNAi in S2 cells. Ago-1 can be biochemically separated from active RISCs. This study by no means excludes a role for Ago-1 in RNAi since previous genetic studies have indicated that Ago-1 is required for efficient RNAi during embryogenesis (Caudy, 2002).
The possibility that a sequence-specific nuclease activity might co-immunoprecipitate with either of these components was tested. Immunoprecipitates of Fmr1 substantially degraded the cognate but not the noncognate substrate. In the case of Fmr1, >80% of the homologous substrate was degraded in 1 h of incubation (Caudy, 2002).
The majority of Fragle X mutations occur in the 5'-UTR of the gene and represent a trinucleotide repeat expansion that is thought to act by targeting FMR1 for epigenetic silencing. However, one point mutation in the FMRP protein itself has also been linked to the disease (I304N). This hypomorphic mutation in the human protein causes defects in ribosome association and RNA binding. An analogous mutation was constructed in the second KH domain of the Drosophila protein, I307N, and it was asked whether this mutation affects association with the RNAi machinery. Biochemical fractionation showed a significant alteration in the ability of the mutant Fmr1 to associate with RISC and Ago-2, with ~30% of the mutant protein shifting from the ribosome pellet (where Ago-2 localizes) to a soluble cytoplasmic form. This fraction is completely devoid of Ago-2, and Ago-2 cannot be found in association with cytoplasmic Fmr1 (Caudy, 2002).
The specific biochemical role of the human protein FMRP is not known; however, numerous studies suggest that it regulates the expression of a large number of genes at the level of protein synthesis. Recent studies of Dicer function and of Argonaute family members in C. elegans have revealed that endogenously encoded small hairpin RNAs (generically, miRNAs) regulate the expression of protein coding genes via pathways related to RNAi. The presence of Fmr1 in RISCs suggests the hypothesis that FMRP or FMRP family members may regulate gene expression via a RISC-like complex (Caudy, 2002).
Little is known of the effector complexes that contain miRNAs. The current study indicates that siRNAs and miRNAs are distinguished to some extent and preferentially, although not exclusively, assembled into separate, functional effector RNPs. A recent report has shown association of miRNAs in human cells with a 15S complex containing Gemin3, Gemin4, and eIF2c, a member of the Argonaute protein family. Gemin3 is a DEAD-box family RNA helicase, and a helicase has previously been predicted as an essential activator of siRNA-containing complexes in Drosophila. Also, in C. elegans two DExH/D box helicases, one of which is Dicer, associate with Rde-4 and are required for RNAi. Gemin4 lacks both recognizable protein motifs and an identifiable homolog in the Drosophila genome. Thus, the relationship between the previously characterized miRNP complex and RISC remains unclear; this led to further characterization of the association between miRNAs and RISCs (Caudy, 2002).
A significant portion of two miRNAs, mir2b and mir13a, in Drosophila S2 cells, are found in association with polyribosomes, as would be predicted for small RNAs that regulate protein synthesis. Furthermore, a functional connection between siRNAs and ribosome association has been made in trypanosomes. miRNAs can be extracted from a high-speed pellet from S2 cell extracts (which contains polysomes) using buffers containing elevated salt concentrations. Extracted material was fractionated by size-exclusion chromatography, and the behavior of miRNAs and siRNAs was compared with that of known RISC subunits. The majority of siRNAs co-fractionate with Ago-2 and with VIG and Fmr1. In contrast, the majority of miRNAs fractionate in an ~250-kD complex, although ~20% of miRNAs are present in fractions containing RISC. However, the possibility cannot be ruled out that this represents a tail of the 250-kD peak, given the nonquantitative nature of Western blotting. Considering the established role of Fmr1 in the regulation of endogenous protein-coding genes, the possibility that miRNAs might be present in Fmr1-containing complexes was examined. Immunoprecipitates of Fmr1 were prepared from column fractions using appropriate affinity resins. Notably, mir2b was detected in Fmr1 immunoprecipitates from transfected cells. Similarly, VIG could be used to immunoprecipitate mir2B directly from extracts (Caudy, 2002).
The forgoing data suggest that miRNAs associate with RISCs that also contain Fmr1 and VIG. This is consistent with recent data suggesting that miRNAs can direct cleavage by RISC, given appropriate homologous substrates. However, the observation of an ~250-kD peak containing the majority of miRNAs raised questions regarding the composition of these smaller complexes. Another Argonaute family member, Ago-1, showed co-fractionation of this protein with the major miRNA peak both on sizing columns and subsequently on ion exchange columns. To test the association of Ago-1 with miRNAs, immunoprecipitation experiments were performed with an epitope-tagged Ago-1 protein. Both mir2b and mir13a could be detected in association with Ago-1. Furthermore, Ago-1 immunoprecipitates also contained siRNAs. Reciprocal immunoprecipitations and Western blots indicate that Fmr1 and Vig do not detectably associate with Ago-1 (Caudy, 2002).
The forgoing data raises the possibility that siRNAs and miRNAs assemble into separate, functional effector RNPs. Recently, it has been shown that Ago-1 is required for efficient interference with endogenous genes in Drosophila embryos. Notably, Ago-1 complexes in S2 cells are similar in size to the active effector complexes, RISC*, that have previously been detected in embryo lysates. The failure of this study to detect substantial RISC activity in Ago-1 complexes and the failure to detect a substantial dependence on Ago-1 for RNAi in S2 cells could reflect developmental differences in the utilization of Ago family members or experimental differences in the assays used to detect RISCs, namely measuring loss of substrate (in this case) versus endonucleolytic cleavage as in the other studies (Caudy, 2002).
The identification of the Drosophila homolog of the fragile X protein as a RISC subunit suggests that disruptions in RNAi-related pathways may contribute to human disease. The fragile X protein has previously been implicated in the regulation of gene expression at the level of protein synthesis. In Drosophila, a bona fide Fmr1 regulatory target has been identified as Futsch, a Map1B homolog. Indeed, Fmr1 and Futsch are epistatic with respect to the neuronal defects of Fmr1-mutant flies, although they are not epistatic with respect to circadian defects. Many miRNAs in Drosophila show complementarity to common sequence motifs that negatively regulate gene expression at the posttranscriptional level. These K boxes, Brd boxes, and GY boxes are present in numerous mRNAs. Notably, a K box and a GY box are found in the 3'-UTR of Futsch. However, a sequenced miRNA has not been definitively linked to suppression of Futch expression. In humans, FMRP has been isolated as a component of large cytoplasmic mRNA complexes and, through this association, has been implicated in the regulation of numerous mRNAs. None of the several proteins known to bind FMRP has been implicated in RNAi. However, there are many possible resolutions to this seeming contradiction. First, FMRP could function through several completely independent mechanisms. Second, FMRP could be an accessory factor for (rather than an integral component of) RISCs, using RISCs for identifying FMRP's cognate targets that may be regulated via mechanisms that involve its interaction with other proteins. This would be consistent with the partial effects of Fmr1 depletions on RNAi efficiency in S2 cells (Caudy, 2002).
The precise mechanisms through which FMRP is delivered to its regulatory targets is unclear. Selection of random RNA oligonucleotides has provided evidence that FMRP binds G-quartet structures that lie adjacent to short RNA helices. This interaction required only the RGG motif and did not involve the KH domain. Thus, it was proposed that FMRP recognizes and regulates G-quartet-containing mRNAs. This study, however, proposes an alternative mechanism of regulation by fragile X protein. The results are consistent with FMRP1 being targeted to its substrates as part of RNAi complexes, which are guided by miRNAs. Recognition of G-quartets might be an important determinant of further specificity or might be critical for translational regulation. It is further proposed that FMRP might be one of many distinct protein subunits that join RISCs, depending on the tissue, subcellular localization, and the developmental stage. In this way, RISCs could function as a flexible platform on which might be constructed a variety of regulatory machines that alter gene expression at the level of mRNA metabolism, at the level of protein synthesis, and at the level of genome structure (Caudy, 2002).
In Drosophila, Fmr1 binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. A Fmr1-associated complex has been isolated that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The Fmr1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. Dmp68 is also required for efficient RNAi. Fmr1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that Fmr1 is part of the RNAi-related apparatus. These findings suggest a model in which the RNAi and Frm1-mediated translational control pathways intersect in Drosophila. The findings also raise the possibility that defects in an RNAi-related machinery may cause human disease (Ishizuka, 2002).
The identification of AGO2 as a Fmr1-interacting protein is particularly striking. AGO2 is a member of the Argonaute gene family and is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNAi in Drosophila. Therefore, the finding that Fmr1 forms a complex in vivo with AGO2 suggests that Fmr1 may function in RNAi. To test this, RNAi was used to suppress the endogenous proteins, much as had been done previously to establish a role for AGO2 in RNAi. Suppression of ribosomal proteins L5 and L11 with specific dsRNAs made S2 cells so sick that their roles in RNAi could not be assessed. However, treatment of S2 cells with dsRNAs homologous to AGO2, Fmr1, or Dmp68 markedly reduces the levels of these proteins. The ability of these cells to carry out RNAi was tested by transfection with enhanced green fluorescent protein (EGFP) expression plasmid in combination with an EGFP dsRNA. Suppression of AGO2 expression correlates with a pronounced reduction in the ability of cells to silence the reporter EGFP by RNAi. Interestingly, RNAi targeting Dmp68 results in inhibition of RNAi in S2 cells. These results suggest that the DEAD-box helicase Dmp68 not only interacts with Fmr1 but is also required for efficient RNAi in S2 cells. Dmp68 is a Drosophila ortholog of human p68, which has been demonstrated to unwind short but not long dsRNAs in an ATP-dependent manner. It is concluded that at least two of the Fmr1-interacting proteins, AGO2 and Dmp68, are required for RNAi in cultured Drosophila S2 cells. In contrast, depletion of Fmr1 did not appear to affect the EGFP silencing. Therefore, although Fmr1 interacts strongly with AGO2 and Dmp68 in vivo, Fmr1 does not appear to be essential for efficient RNAi (Ishizuka, 2002).
Recent work in numerous organisms has shown that RNAi shares features with a developmental gene regulatory mechanism that involves miRNAs. These small RNAs (siRNAs and miRNAs) are thought to be incorporated into silencing complexes that mediate mRNA destruction during RNAi and translational control during development, respectively. Therefore, it is suggested that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation. AGO2 and Dmp68 are essential for RNAi in Drosophila. However, Fmr1 appears to be a translation repressor. Because Fmr1 interacts with AGO2 and Dmp68 in vivo, it was of interest to examine whether Fmr1 is also present in an AGO2- and/or Dmp68-associated complex. To do this, TAP-tagged AGO2 (AGO2-TAP) or Dmp68 (Dmp68-TAP) were expressed in S2 cells. Cytoplasmic lysate of the cells expressing AGO2-TAP or Dmp68-TAP was prepared and subjected to TAP purifications. In reciprocal assays, endogenous Fmr1 and AGO2 were found to associate with each other. In addition, endogenous AGO2 was copurified with AGO2-TAP. Endogenous Fmr1 and AGO2 were also found to be present in the Dmp68-associated complex. Because AGO2 can be coimmunoprecipitated with Dicer, which initiates RNAi by processing dsRNA silencing triggers into siRNAs, and also processing miRNA precursors into mature miRNAs, the possibility was considered that Fmr1 might also interact physically with Dicer. Indeed, endogenous Dicer can be copurified not only with AGO2-TAP but also with Fmr1-TAP, and it was also shown that Fmr1 remains associated with AGO2 after RNAi induction. It is well established that siRNAs associate with AGO2 during RNAi in S2 cells. Therefore, these results indicate that Fmr1 may be a part of RISC. Finally, analogous to the human AGO2 ortholog (eIF2C2)-associated complex that contains a DEAD-box type RNA helicase and miRNAs, it was of interest to test whether miRNAs are also found in AGO2- and/or Fmr1-associated complexes. RNA molecules copurified with AGO2-TAP or Fmr1-TAP were recovered, dissolved on a 15% polyacrylamide denaturing gel, and subjected to Northern blot analysis. A known miRNA, miR-2b, in Drosophila S2 cells could be detected both in the AGO2- and Fmr1-associated complexes. Together, these data show that Fmr1 is present in a complex with components of RNAi and miRNAs in cultured Drosophila S2 cells (Ishizuka, 2002).
Fmr1 is thought to have important roles in the translation of some specific mRNAs such as futsch mRNA. Although it is unclear how Fmr1 regulates translation of such mRNAs, these findings may hold some clues. Because Fmr1 interacts with ribosomal L5/5S rRNA and L11, all of which are located at the top of the 60S ribosomal subunit, it is likely that this interaction brings Fmr1 to the 60S ribosomal subunit. Therefore, the association of Fmr1 with the 60S ribosomal subunit through direct interactions with ribosomal L5/5S rRNA and L11 may inhibit translation by preventing the assembly of initiation complexes or by giving rise to structural change of the ribosomes, which, in turn, influences a step(s) after translation initiation. Alternatively, because 5S rRNA is the only known RNA species that binds ribosomal proteins, including L5, and forms a 5S RNP before it is incorporated into the ribosomes, Fmr1 may interact with the cytoplasmic nonribosome-associated 5S RNP, which, in turn, influences the formation of the mature 60S ribosomal subunit. It is interesting to note in this context that only about half of the 5S rRNA molecules in mammalian cells are associated with the 60S ribosomal subunit and that although 5S rRNA enhances ribosomal activity, it is not absolutely essential for it (Ishizuka, 2002).
Fmr1 is present in a complex isolated from S2 cells, which also contains AGO2 and Dicer. AGO2 and Dicer are essential components of RNAi. The interaction between Fmr1 and AGO2 remains constant before and after RNAi induction, suggesting that Fmr1 is part of RISC during RNAi. However, there is no evidence to support the notion that RISC formation is induced by treatment of S2 cells with dsRNA. As one of the functions of the RNAi apparatus is to silence transposons and repetitive sequences residing naturally in the Drosophila genome, these cells are therefore likely to be full of pre-formed RISC complexes, irrespective of dsRNA treatment. Therefore, it is possible that Fmr1 is part of the pre-formed RISC complexes and remains to be part of the active RISC after ATP-dependent siRNA unwinding (Ishizuka, 2002).
The involvement of another Fmr1-interacting protein, Dmp68, in RNAi further suggests the close association of Fmr1 with RNAi. The p68 RNA helicase was first identified by cross-reaction with a monoclonal antibody that was originally raised against SV40 large T antigen two decades ago. The helicase plays important roles in cell proliferation and organ maturation and belongs to a large family of highly evolutionarily conserved proteins, the so-called DEAD-box family of putative ATPases and helicases. Recent studies have revealed that several RNA helicases, including mut6, SDE3, mut14 , drh-1, and spindle-E are required for RNAi and related posttranscriptional gene silencing (PTGS) pathways. Dmp68 is similar to, but clearly not an ortholog of these proteins. Therefore, Dmp68 is a novel component of RNAi. Because ATP-dependent unwinding of the siRNA duplex remodels the RISC to generate an active RISC in the RNAi pathway, Dmp68 may mediate the unwinding process. It is also conceivable that Dmp68 may be involved in downstream events such as target RNA recognition, as an RNA chaperone or an RNPase (Ishizuka, 2002).
The connection that has been established between Fmr1, components of RNAi, miRNAs, and the general translation machinery is of considerable significance because they provide intriguing clues and possible connections to the function of Fmr1 and the pathways with which it may intersect. Recent work in numerous organisms has shown that RNAi shares features with a developmental gene regulatory mechanism that involves miRNAs. For example, both the foreign dsRNAs that trigger RNAi and the endogenous miRNA precursors that function in development are processed into small RNAs by Dicer. Members of the Argonaute gene family are also involved in both the siRNA and miRNA pathways. In C. elegans, Dicer, the dsRNA-binding protein RDE-4, and a conserved DExH-box RNA helicase (DRH-1) are in a complex with RDE-1, an AGO2 ortholog. Furthermore, the human AGO2 ortholog, eIF2C2, is in a complex, the miRNP, that contains the DEAD-box RNA helicase Gem3. Therefore, Argonaute proteins appear to be in a complex that contains an RNA helicase(s), Dicer and small guide RNAs, and function in a variety of homology-dependent mechanisms that involve base-pairing between small guide RNAs and target mRNAs. The findings that Fmr1 interacts with AGO2, Dmp68, Dicer, miRNAs, and the general translation machinery, provide a means to link RNAi enzymes to translational control pathways, and are also consistent with the fact that the RISC nuclease fractionates with ribosomes (Ishizuka, 2002).
It appears that Fmr1 is important for translational control, possibly mediated by RNAi-related components and miRNAs. Although recent studies have identified a list of mRNAs that are potential FMRP targets, these results further suggest a model in which FMRP may not directly bind its mRNA targets but rather it is targeted to its mRNA substrates as part of RNAi-related apparatus, which are guided by miRNAs. How then might FMRP regulate translation of its mRNA targets? In the case of lin-4, a prototype of miRNAs, its mRNA targets (lin-14 and lin-28) are translationally repressed yet remain associated with polyribosomes, suggesting a block at a step after translation initiation. FMRP may form an miRNP complex on its mRNA targets and the association of this complex with ribosomal L5/5S rRNA and L11 may inhibit translation at one or more postinitiation steps, including elongation, termination, or the release of functional protein as discussed above. Finally, it is proposed that fragile X syndrome may be the result of protein synthesis abnormality caused by a defect(s) in an RNAi-related apparatus (Ishizuka, 2002).
Neuronal plasticity requires actin cytoskeleton remodeling and local protein translation in response to extracellular signals. Rho GTPase pathways control actin reorganization, while the fragile X mental retardation protein (FMRP) regulates the synthesis of specific proteins. Mutations affecting either pathway produce neuronal connectivity defects in model organisms and mental retardation in humans. CYFIP, the fly ortholog of vertebrate FMRP interactors CYFIP1 and CYFIP2, is specifically expressed in the nervous system. CYFIP mutations affect axons and synapses, much like mutations in Drosophila Fmr1 and in Drosophila Rho GTPase Rac1. CYFIP interacts biochemically and genetically with Fmr1 and Rac1. Finally, CYFIP acts as a Rac1 effector that antagonizes Fmr1 function, providing a bridge between signal-dependent cytoskeleton remodeling and translation (Schenck, 2003).
Fragile X syndrome is caused by loss-of-function mutations in the
fragile X mental retardation 1 (FMR1) gene. How FMR1 affects
the function of the central and peripheral nervous systems is still
unclear. FMR1 is an RNA binding protein that associates with a small
percentage of total mRNAs in vivo. It remains largely unknown what
proteins encoded by mRNAs in the FMR1-messenger ribonuclear protein
(mRNP) complex are most relevant to the affected physiological
processes. Loss-of-function mutations in the Drosophila fragile
X-related (dfmr1) gene, which is highly homologous to the
human fmr1 gene, decrease the duration and percentage of time
that crawling larvae spend on linear locomotion. Overexpression of
DFMR1 in multiple dendritic (MD) sensory neurons increases the time
percentage and duration of linear locomotion; this phenotype is
similar to that caused by reduced expression of the MD neuron
subtype-specific degenerin/epithelial sodium channel (DEG/ENaC)
family protein Pickpocket1 (PPK1). Genetic analyses indicate that
PPK1 is a key component downstream of DFMR1 in controlling the
crawling behavior of Drosophila larvae. DFMR1 and ppk1 mRNA
are present in the same mRNP complex in vivo and can directly bind to
each other in vitro. DFMR1 downregulates the level of ppk1
mRNA in vivo, and this regulatory process also involves Argonaute2
(Ago2), a key component in the RNA interference pathway. These
studies identify ppk1 mRNA as a physiologically relevant in
vivo target of DFMR1. The finding that the level of ppk1 mRNA
is regulated by DFMR1 and Ago2 reveals a genetic pathway that
controls sensory input-modulated locomotion behavior (Xu, 2004).
To study subtle behavioral defects caused by dfmr1
mutations, the crawling behavior of wild-type and mutant larvae were
examined at the wandering stage with DIAS software in an environment
devoid of cues for chemotaxis and phototaxis; because larvae were
placed on a flat surface, as verified by a level, there were
presumably no cues for geotaxis. The larval crawling behavior is
relatively simple and stereotyped and can be separated into two
phases: linear locomotion and nonlocomotive turning. As shown by
analysis of the centroid paths of crawling larvae, wild-type and
dfmr14 mutant larvae have different crawling
patterns. Analysis at higher magnification showed that the wild-type
larvae had longer linear paths and made fewer turns than
dfmr14 mutants (Xu, 2004).
To quantify differences between wild-type and dfmr14 mutant larvae, the direction change was determined, defined as the absolute value of the difference in direction from one frame to the next. Computer analysis of crawling routes indicated that, during 1.5 min of recording (150 data points), a representative dfmr14 mutant larva had 17 data points with direction changes larger than 60°, and a representative wild-type larva had only eight such points (Xu, 2004).
Because the number of direction changes varied substantially among larvae of a given genotype, a large number of larvae were recorded and analyzed to quantify the difference at the population level. The average direction change for a larva is the mean value of all the data points. More dfmr14/dfmr14 mutants than wild-type larvae exhibited an average direction change greater than 20°. dfmr14/dmr14 mutant larvae showed a greater average direction change than wild-type larvae. No significant differences were seen between dfmr14/+ heterozygous larvae and wild-type larvae. To confirm that the abnormal crawling behavior was caused by dfmr1 mutations, mutant larvae were examined that had a combination of different dfmr1 loss-of-function alleles that were independently generated from the genetic screen. Larvae with different dfmr1 alleles all exhibited similar crawling behaviors. In addition, the alteration in direction change could be rescued by introducing a genomic DNA fragment containing the wild-type dfmr1 gene, indicating that dfmr1 was indeed responsible for the observed phenotype in mutant larvae (Xu, 2004).
To further characterize the alterations in the crawling pattern of dfmr1 mutant larvae, 'linear locomotion' was defined as the time period during which at least five consecutive frames showed a direction change that was smaller than 20°. Wild-type larvae spent a greater percentage of time on linear locomotion than dfmr14 mutant larvae. Mutant larvae with different dfmr1 alleles showed a phenotype similar to that of dfmr14 mutant larvae. In addition, the average duration of linear locomotion of dfmr14 mutant larvae was shorter than that of wild-type larvae. Both alterations were rescued by the introduction of a genomic DNA fragment containing the wild-type dfmr1 gene. These findings indicate that dfmr1 mutations significantly alter the crawling pattern of wandering larvae (Xu, 2004).
DFMR1 has been shown to play a role in the proper development of higher-order fine dendritic processes of MD sensory neurons in the PNS of Drosophila larvae. Most MD neurons elaborate extensive dendritic arbors just underneath the epidermis in each segment. To test whether MD neurons modulate crawling behavior, DFMR1 was expressed in MD neurons under the control of Gal4 109(2)80, which drives target gene expression in all MD neurons and less than 100 central neurons. This manipulation decreased the average direction change and increased the time percentage and duration of linear locomotion; together, these create a phenotype opposite that of dfmr1 mutant larvae. Furthermore, expression of normal DFMR1 protein by the same Gal4 driver in a dfmr1 mutant background rescued the defects in crawling behavior. These results are consistent with the notion that changes in DFMR1 activity in MD neurons affect the crawling behavior of Drosophila larvae (Xu, 2004).
To uncover the molecular mechanism underlying DFMR1 function, the association between DFMR1 and several mRNAs that encode known channel molecules was examined. A monoclonal antibody against DFMR1 was used to immunoprecipitate DFMR1-mRNP complexes and reverse transcription-polymerase chain reaction (RT-PCR) was used to demonstrate the presence or absence of a particular mRNA in the complexes. The mRNA encoding PPK1, an MD neuron subtype-specific member of the DEG/ENaC family, was associated with DFMR1 in vivo. Lysates from dfmr14 mutant larvae served as negative controls. No association was detected in the same immunoprecipitation experiment between DFMR1 and the mRNAs encoding Hyperkinetic (HK) and Shaker, both of which have been implicated in larval crawling behavior. To further investigate the association between DFMR1 and ppk1 mRNA, GST-DFMR1 fusion protein was purified and it was found that ppk1 mRNA could bind in vitro directly to GST-DFMR1 but not GST alone. To demonstrate the binding specificity, a competition binding experiment was performed. The affinity of DFMR1 for ppk1 mRNA was at least one order of magnitude higher than that for a control mRNA. These findings demonstrate that DFMR1 is associated with ppk1 mRNA in vivo and that they can directly bind to each other at least in vitro (Xu, 2004).
The level of ppk1 mRNA is regulated by DFMR1 in MD sensory neurons. dfmr1 mutant larvae have been shown to have slightly more higher-order dendritic branches of sensory neurons than wild-type larvae. It seems that ppk1 mRNA and dendrite development are independently regulated by DFMR1, because overexpression of Rac1 in MD neurons increased dendritic branching without affecting the level of ppk1 mRNA and locomotion behavior. Recent studies demonstrate that DFMR1 associates with microRNAs and proteins involved in the RNAi pathway. Although it was reported that DFMR1 could affect the efficiency of RNAi in vitro, it was found that DFMR1, unlike Ago2, is not required for efficient RNAi-mediated degradation of ppk1 mRNA in vivo. Deletion mutations were generated in ago2 and it was found that Ago2 also regulates the ppk1 mRNA level in a manner similar to DFMR1. Genetic-interaction studies further support the notion that the two molecules work in the same genetic pathway to control ppk1 mRNA level. A GST-Ago2 fusion protein was purified and it was found that Ago2 itself does not bind to ppk1 mRNA. These findings suggest a model in which DFMR1 binds to ppk1 mRNA and recruits Ago2 and presumably other components to regulate the level of ppk1 mRNA, which in turn modulates larval locomotion behavior. Interestingly, overexpression of PPK1 by itself does not affect locomotion behavior, possibly due to the fact that PPK1 is only a subunit of a functional channel. Therefore, other channel subunits or downstream components may be coordinately regulated by DFMR1. The association of DFMR1 and Ago1 has also been reported (Jin, 2004). Argonaute family proteins are involved not only in RNAi pathways but also in microRNA-mediated gene regulation. These studies provide strong evidence that sensory-channel molecules can be regulated by components associated with microRNA pathways (Xu, 2004).
Although DFMR1 is known to be an RNA binding protein, the RNAs that associate with it in vivo have not been systematically identified. Recent studies suggest that a small percentage of mRNAs are associated with FMR1-mRNA complexes in mouse brain and cell lines. Since DFMR1 and human FMR1 share a high degree of sequence homology and are likely to function similarly, DFMR1 might also regulate multiple mRNAs in Drosophila. It remains a major challenge to identify the key mRNA targets that mediate DFMR1's effect on a particular physiological pathway. In this study, by using both biochemical and genetic approaches, it has been shown that regulation of ppk1 mRNA by DFMR1 contributes to MD sensory neuron-modulated larval crawling behavior (Xu, 2004).
Because DEG/ENaC superfamily proteins are highly conserved through evolution, it would be of great interest to determine which DEG/ENaC channel molecules in mammals are also regulated by FMR1. Further understanding of the functional alterations in the sensory pathway caused by DFMR1/FMR1 mutations may provide deeper insights into the mechanisms underlying fragile X syndrome in humans (Xu, 2004).
Fragile X syndrome, the most common form of inherited mental retardation, is caused by loss of function for the Fragile X Mental Retardation 1 gene (FMR1). FMR1 protein (FMRP) has specific mRNA targets and is thought to be involved in their transport to subsynaptic sites as well as translation regulation. A saturating genetic screen of the Drosophila autosomal genome was used to identify functional partners of dFmr1. Nineteen mutations were recovered in the tumor suppressor lethal (2) giant larvae (dlgl) gene and 90 mutations at other loci. dlgl encodes a cytoskeletal protein involved in cellular polarity and cytoplasmic transport and is regulated by the PAR complex through phosphorylation. Direct evidence is provided for a Fmrp/Lgl/mRNA complex, which functions in neural development in flies and is developmentally regulated in mice. The data suggest that Lgl may regulate Fmrp/mRNA sorting, transport, and anchoring via the PAR complex (Zarnescu, 2005).
This study reports the identification of Lgl as a functional partner of the Fragile X protein, Fmrp. Lgl forms a large macromolecular complex with Fmrp, which is developmentally regulated and modulates the architecture of the neuromuscular junction in the fly. At the cellular level, Lgl and Fmrp are temporally and spatially coexpressed during development and colocalize in granules in the soma and the developing neurites of mouse cultured cells. Fractionation experiments show that Fmrp and Lgl comigrate with Golgi membrane-associated complexes. Furthermore, the Fmrp/Lgl complex contains a subset of mRNAs and interacts physically and genetically with the PAR complex, an essential component of the cellular polarization pathway. These results suggest that Lgl functions with Fmrp to regulate a subset of target mRNAs during synaptic development and/or function. It is proposed that Lgl may regulate Fmrp/mRNA containing RNPs by (1) sorting at the Golgi, (2) transport in neurites, and (3) anchoring at specific membrane domains, such as subsynaptic sites. The neurite transport function may involve molecular motors such as myosin II and kinesin, previously shown to associate with Lgl and Fmrp, respectively. The anchoring mechanism may involve the PAR complex, which has a demonstrated role in defining membrane domains and has recently been shown to generate asymmetry in the C. elegans embryo by stabilizing RNPs at the posterior pole (Zarnescu, 2005).
The data demonstrate that Fmrp and Lgl form a functional complex in living neurons, and this is conserved in flies and mice. In the mouse brain, mFmrp associates with mLgl preferentially at a time of increased synaptogenesis, demonstrating a developmentally regulated interaction between Lgl and Fmrp. The data suggest that dLgl acts to regulate a subset of dFmr1-associated mRNAs with some encoding circadian regulated molecules (CG3348 and CG9681) and some encoding secreted or transmembrane proteins (CG6136, CG4101, CG9681) among others. dLgl also associates with mRNA independent of dFmr1; thus, it is formally possible that dLgl interacts with other RNA binding proteins, which remain to be determined (Zarnescu, 2005).
Fmrp has been implicated in the translational regulation of specific target mRNAs, perhaps via the RNAi pathway. Also, it has been proposed that Fmrp is involved in the transport and localization of mRNAs: cellular fractionation and immunolocalization data revealed the association of Fmrp-containing complexes with molecular motors such as kinesin and myosin V. Lgl functions in cellular polarity via regulating myosin motor activity and/or vesicle transport and has been shown to regulate polarized delivery by sorting at the Golgi. Taken together, these concepts suggest that Lgl may act as a scaffold for Fmrp granules, possibly at the Golgi, and perhaps aids in carrying specific mRNA targets to sites of locally controlled translation (such as subsynaptic sites) (Zarnescu, 2005).
It is also possible that Lgl anchors Fmrp at specific membrane domains such as synapses, perhaps via the PAR complex. Lgl function is regulated by the PAR complex, specifically via phosphorylation by aPKC-zeta and by direct binding to PAR6. The genetic interaction data suggest that PAR6 and Baz antagonize dFmr1 function, which is in accordance with previously published work showing that the PAR complex inhibits dlgl and with data that demonstrate that dlgl functions cooperatively with dFmr1. aPKC-zeta was shown to antagonize most dLgl functions with the exception of its role in regulating neuroblast apical size and the data are be consistent with such reports. Loss of function for aPKC-zeta suppresses gain of function sev:dFmr1 as well as the loss-of-function phenotype of dFmr1 at the NMJs. Taken together, these data suggest a dynamic relationship between the various members of the complex. One possible interpretation of the results is that aPKC/PAR can act on Fmrp directly or via Lgl depending on the developmental and/or cellular context. Furthermore, this is consistent with sucrose fractionation experiments, which suggest the existence of at least two complexes comprising dFmr1/PAR proteins and dFmr1/dLgl/PAR proteins (Zarnescu, 2005).
The PAR complex not only functions in cell polarity, but also at the synapse, where it is believed to function in synaptic tagging. Synaptic tags have been proposed to transiently mark a synapse after activation in a way that will translate the local events into persistent functional changes (such as long-term depression), processes in which Fmrp is also thought to act. Thus, the Fmrp/Lgl/PAR complex may act in synaptic plasticity linking synaptic input to the remodeling of the cytoskeleton and mediating required translational changes (Zarnescu, 2005).
Translational regulation of maternal mRNAs in distinct temporal and spatial
patterns underlies many key decisions in developing eggs and embryos. In
Drosophila, Orb is responsible for mediating the translational activation of
mRNAs localized within the developing oocyte. Orb is a germline-specific RNA
binding protein and is one of the founding members of the CPEB family of
translational regulators. Orb associates with the Drosophila
Fragile X Mental Retardation (dFMR1) protein as part of a ribonucleoprotein
complex that controls the localized translation of mRNAs in developing egg
chambers. One of the key orb regulatory targets is orb mRNA, and this
autoregulatory activity is critical for ensuring that Orb protein is expressed
at high levels in the oocyte. dFMR1 functions as a negative
regulator in the orb autoregulatory circuit, downregulating orb mRNA
translation (Costam, 2005).
To identify factors that could function in regulating Orb activity and/or
localization proteins that are physically associated with Orb in vivo were sought. For this purpose ovarian extracts were immunoprecipitated with Orb antibodies. Besides Orb, the immunoprecipitates contained a complex set of proteins that were absent in the control immunoprecipitates. Attention was focused on four larger proteins because they are present in nearly the same yield as Orb (as judged by
staining) and are well resolved from other polypetides. The association of these four proteins with Orb is RNase resistant; they are observed not only when an RNase inhibitor is present, but also when the immunoprecipitates are treated with RNase or when the immunoprecipitation is done in the presence of RNase (Costam, 2005).
The four proteins were identified by mass spectroscopy. The largest 150 kd species corresponds to Lingerer. Lingerer is expressed at high levels in the nervous system, imaginal discs, and gonads. It is required for viability and has been implicated in sexual behavior. The 120 kd species is CG18811-PA. Nothing is known about its function in flies; however, related proteins called Caprins have been identified in vertebrates including humans and are thought to function in cell proliferation. The species migrating slightly more slowly than Orb is Rasputin (Rin). Rin is the fly homolog of the RasGAP SH3 binding protein (G3BP),
and it is thought to regulate RNA metabolism in response to Ras signaling.
Finally, the smallest protein, which is the subject of this study, corresponds
to the Drosophila Fragile X Mental Retardation protein (dFMRP) (Costam, 2005).
Though dFMR1 is a KH domain RNA binding protein, it is associated with
Orb in an RNase resistant complex. Although this could indicate that Orb and
dFMR1 complex with each other through (direct or indirect) protein-protein
interactions, an inability to produce soluble recombinant Orb has precluded
testing for direct interactions in vitro. In addition, it should be noted that
the fact that the Orb-dFMR1 complex in ovarian extracts is RNase resistant does
not exclude the possibility that Orb and dFMR1 coassemble into mRNPs because
the two proteins recognize sequences in the same RNA species. That there must
be some type of target specificity in Orb-dFMR1 complex assembly is suggested
by both Western and confocal analysis. Though dFMR1 appears to be present in
Orb immunoprecipitations in near molar yield, Western blots indicate that only
a subfraction of the total dFMR1 in ovaries is in an immunoprecipitable complex
with Orb. One reason for this is that all of the somatic cells in the ovary
have dFMR1, whereas they do not contain Orb. However, even within the germline
not all of the dFMR1 is associated with Orb. For example, in the germarium
dFMR1 is found in stem cells and the mitotic cysts, whereas Orb is not
readily detected until the 16-cell cyst is formed. Even after the 16-cell cysts
are formed and begin to develop, a significant fraction of the germline dFMR1
is localized in nurse cells, and with the exception of perinuclear particles,
this protein does not seem to be associated with Orb. In contrast, much
of the dFMR1 protein present in the oocyte appears to be specifically
associated with Orb. Moreover, it is in the oocyte where Orb activity is known
to be required. In previtellogenic chambers, dFMR1 and Orb colocalize around
the oocyte nucleus, concentrating most heavily at the posterior pole. After the
onset of vitellogenesis, Orb and dFMR1 colocalize along most of the oocyte
cortex except at the very posterior pole where there is a relatively low level
of dFMR1 and a high level of Orb (Costam, 2005).
An important question is whether the
Orb-dFMR1 association in the oocyte (and in the perinuclear particles in the
nurse cells) has regulatory consequences. Several lines of evidence argue that
it does. (1) dfmr1 interacts genetically with orb. orb
is weakly haploinsufficient and a significant fraction of the eggs laid by
females heterozygous for strong loss of function mutations such as
orb343 have D-V polarity defects indicative of a failure in
the grk-EGFR signaling pathway. This haploinsufficiency is enhanced by
transgenes, like HD19G, that express orb 3′ UTR sequences
fused to heterologous LacZ protein coding sequence. The HD19G
mRNAs compete with the endogenous orb mRNA for Orb protein binding and
further compromise the orb positive autoregulatory circuit.
D-V polarity defects of HD19G orb343/+ females can be
suppressed by reducing the dose of dfmr1 in half, whereas they can be
enhanced by adding an extra dfmr1 gene. Genetic interactions between
dfmr1 and orb are also seen in the absence of the HD19G
transgene. Moreover, in this case the D-V polarity defects seen at 18°C
for females heterozygous for orb343 or for another allele,
orbdec, can be almost completely suppressed by eliminating
dfmr1 altogether. (2) As would be expected if dfmr1 exerts
its effects on D-V polarity by modulating the orb autoregulatory
circuit, it was found that dfmr1 negatively regulates Orb protein expression.
Consistent with the suppression of D-V polarity defects, Orb accumulation in
HD19G orb343/+ females can be increased by reducing the dose
of dfmr1. Conversely, as would be expected from the finding that excess
dFMR1 enhances the frequency of D-V polarity defects, increasing the dose of
dfmr1 decreases Orb expression. (3) The effects of dfmr1 on
Orb accumulation are not restricted to circumstances in which orb
autoregulation is partially compromised. It is also seen in females that are
wild-type for orb. In this case, eliminating dfmr1 leads to the
overexpression of Orb protein. (4) orb interacts genetically with
dfmr1. Two independent orb mutations dominantly suppress the
excess germ cell phenotype seen in egg chambers from dfmr1 mutant
females. The fact that this phenotype can be suppressed by reducing the
orb gene dose would suggest that it arises, either directly or
indirectly, from the overproduction of Orb protein. (5) While
the inhibitory effects of dfmr1 on Orb
expression cannot be
unambiguously attributed to the particular subfraction of the dFMR1 protein that is complexed
specifically with Orb, the data indicate that dfmr1 activity is required
in the germline in order to regulate Orb expression (Costam, 2005).
Based on the pattern of Orb accumulation when dfmr1 activity is reduced, it would appear that
dFMR1 is initially required to repress the translation of orb mRNAs
while they are in transit from the nurse cells to the oocyte. If dFMR1 action
is direct in the nurse cells, it presumably associates with orb message
soon after it is synthesized in the nurse cell nuclei and acts to represses
translation. In this respect, it may be of interest that dFMR1-Orb
particles are observed around the edge of the nurse cell nuclei. These perinuclear particles
resemble the sponge bodies that are thought to be involved in assembling newly
synthesized mRNAs into translationally dormant mRNPs so that they can be
transported from the nurse cells to the oocyte. Although dFMR1 clearly
functions to block Orb expression in nurse cells, the fact that the amount of
Orb in nurse cells in the absence of dfmr1 activity is still much lower
than it is in the oocyte argues that there must be other factors besides dFMR1
that inhibit the translation of orb mRNA while it is in transit. As
observed in the nurse cells, Orb accumulation is upregulated in
dfmr13 oocytes. Orb expression is also upregulated in the
oocytes of HD19G orb343/+ egg chambers when dfmr1
activity is reduced, whereas it is repressed when dfmr1 activity is
increased. The effects of dfmr1 on Orb expression in the oocyte suggest
that it functions to attenuate the orb positive autoregulatory feedback
loop; however, the fact that Orb levels do not become excessive in the mutant
oocytes indicates that there must be other mechanisms to prevent Orb over
accumulation (Costam, 2005).
Unlike the nurse cells, most of the dFMR1 in the oocyte
colocalizes with Orb. Consequently, it would be reasonable to suppose that
dFMR1 modulates the orb autoregulatory circuit in the oocyte through its
association with Orb complexes that contain orb mRNA. As noted above,
while Orb-dFMR1 complexes in ovary extracts were found ot be RNase
resistant, it is suspected that complex assembly may depend upon the presence of
recognition sequences for each protein in the mRNA. In the case of orb
mRNA, previous studies indicate that Orb interacts with several sequences in
the 3′ UTR in vivo and in extracts. While the 3′ UTR does not
have sequences that match the dFMR1 consensus, there are three recognition
motifs in the 5′ UTR, at the beginning of the protein coding sequence. Thus,
a plausible idea is that these 5′ and 3′ recognition motifs serve
to recruit dFMR1 and Orb into the orb mRNA RNPs. That the dFMR1
recognition motifs may, in fact, be important for dFMR1 regulation is suggested
by the behavior of the HD19G mRNA, which contains all of the known Orb
target sequences in the orb 3′ UTR but does not have the dFMR1
motifs. The HD19G mRNA mimics the endogenous orb mRNA with
respect to its dependence on orb activity for both localization within
the oocyte and translational activation. However, HD19G mRNA responds
differently from the endogenous orb message to changes in dfmr1
activity (Costam, 2005).
Seemingly similar specificities are evident in the requirements for
orb and dfmr1 activity in regulating fs(1)K10 and
osk mRNA translation. Both of these mRNAs have Orb target sequences in
their 3′ UTRs, whereas only fs(1)K10 has potential dFMR1
recognition motifs. Whereas both fs(1)K10 and osk depend upon
orb activity for proper localization and translation, the
expression of Fs(1)K10 is upregulated in the absence of dfmr1 activity,
while there is no apparent effect on Osk. Because Orb is overexpressed in
dfmr1 mutants, one explanation for the upregulation of Fs(1)K10 is that
it is an indirect consequence of excess orb activity. However, since Osk
levels are unaltered, the idea is favored that dfmr1 functions to inhibit
orb-mediated activation of fs(1)K10 mRNA translation but does not
have a role in osk regulation. In this respect, it may be significant
that Orb specifically promotes the translation of osk mRNA localized at
the posterior pole in vitellogenic stage egg chambers. The posterior pole
differs from elsewhere along the cortex in that there seems to be much less
dFMR1 associated with Orb. Presumably other factors, such as Bruno and another
KH domain RNA protein, Bicaudal-C, would function in repressing osk
translation and counteracting orb activation. Much like dFMR1, these
proteins coimmunoprecipitate with Orb and are necessary for proper repression
of osk translation (Costam, 2005).
An intriguing question is whether the connection between Orb and dFMR1 in fly ovaries is relevant to the neurological phenotypes induced by inactivation of the fragile X gene in humans and mice. It is interesting in this regard that recent studies have implicated both CPEBs and FMRPs as key players not only in CNS development but also in learning and memory. Moreover, much as is observed in the fly germline, CPEBs and FMRPs appear to function antagonistically in regulating the localized translation of specific target mRNAs. If the activity and/or expression of mammalian CPEBs is negatively regulated by FMRPs, as is the case for Orb in flies, then it seems possible that the loss of FMRP activity may lead to excess CPEB activity in the nervous system and perturb the proper spatial or temporal regulation of translation (Costam, 2005).
During the cleavage stage of animal embryogenesis, cell numbers increase dramatically without growth, and a shift from maternal to zygotic genetic control occurs called the midblastula transition. Although these processes are fundamental to animal development, the molecular mechanisms controlling them are poorly understood. This study demonstrates that Drosophila fragile X mental retardation protein (dFMRP) is required for cleavage furrow formation and functions within dynamic cytoplasmic ribonucleoprotein (RNP) bodies during the midblastula transition. dFMRP is observed to colocalize with the cytoplasmic RNP body components Maternal expression at 31B (ME31B) and Trailer Hitch (TRAL) in a punctate pattern throughout the cytoplasm of cleavage-stage embryos. Complementary biochemistry demonstrates that dFMRP does not associate with polyribosomes, consistent with their reported exclusion from many cytoplasmic RNP bodies. By using a conditional mutation in small bristles (sbr), which encodes an mRNA nuclear export factor, to disrupt the normal cytoplasmic accumulation of zygotic transcripts at the midblastula transition, the formation of giant dFMRP/TRAL-associated structures was observed, suggesting that dFMRP and TRAL dynamically regulate RNA metabolism at the midblastula transition. Furthermore, dFMRP associates with endogenous tral mRNA and is required for normal TRAL protein expression and localization, revealing it as a previously undescribed target of dFMRP control. It was also shown genetically that tral itself is required for cleavage furrow formation. Together, these data suggest that in cleavage-stage Drosophila embryos, dFMRP affects protein expression by controlling the availability and/or competency of specific transcripts to be translated (Monzo, 2006).
The data suggest that in cleavage-stage Drosophila embryos, dFMRP affects translational initiation of specific mRNA molecules within cytoplasmic RNP bodies by controlling their availability and/or modulating their competency to be translated. dFMRP does not measurably associate with polyribosomes under a wide range of conditions in cleavage-stage Drosophila extracts, similar to results obtained for Drosophila S2 cells, but in contrast to reports in other systems. Instead, dFMRP colocalize and cosediment was observed with TRAL and ME31B, known components of translationally quiescent cytoplasmic RNP bodies. Although dAGO2 cosediments with polyribosomes in cleavage-stage embryo extracts and could directly suppress translational elongation or termination, a similar role for dFMRP is unlikely. In fact, there is no indication that endogenous dFMRP directly interacts with dAGO2 in cleavage-stage Drosophila embryos, in contrast to their observed association in Drosophila S2 cell extracts. This discrepancy could result from a fundamental difference in RNA metabolism between S2 cells and cleavage-stage embryos undergoing the MBT (Monzo, 2006).
tral mRNA represents a previously undescribed in vivo target of dFMRP regulation. Although there is no direct evidence that dFMRP and TRAL form a stable complex in cleavage-stage embryos in vitro, dFMRP activity is clearly required for normal TRAL protein expression in vivo. Mislocalization of TRAL protein but not ME31B in both fmr1- and sbrts148 mutant embryos suggests that a specific functional relationship exists between dFMRP and TRAL. In fmr1- embryos, TRAL protein levels also are reduced 2-fold, indicating that TRAL does not simply get redistributed into abnormal structures, its rate of synthesis and/or degradation must also be affected. The co-IP of tral mRNA with dFMRP from WT embryo extracts demonstrates that dFMRP and tral mRNA form a stable RNP complex and suggests that dFMRP is involved in tral mRNA metabolism. Although it has not yet been determined whether dFMRP directly binds tral mRNA, this analysis of the tral mRNA sequence, by using the fast RNA motif/pattern searcher RNABOB identified a single G-quartet stem-loop structure within the tral 3' UTR, a motif that FMRP can bind with high affinity. Regardless of whether dFMRP binds tral mRNA directly, dFMRP could control the assembly of a translationally competent tral mRNP complex and/or its localized delivery for translation. The transient association of tral mRNA with cytoplasmic RNP bodies in a translationally quiescent state might be required for dFMRP to promote the assembly of a translationally competent tral mRNP. Alternatively, the restricted translation of tral mRNA, controlled by dFMRP-dependent localized release from cytoplasmic RNP bodies, might promote the normal assembly of a functional TRAL RNP complex. In either case, lower steady-state TRAL protein levels resulting from decreased synthesis and/or increased degradation in fmr1- embryos could be related to abnormal TRAL RNP complex assembly, observed as large structures by IF. Interestingly, the higher steady-state level of tral mRNA observed in fmr1- embryo extracts is reminiscent of the increased levels of another dFMRP target, pickpocket mRNA, observed in fmr1- embryo extracts and may reflect a common feature of dFMRP mRNA processing (Monzo, 2006).
In conclusion, it is believed that a system of cytoplasmic RNP bodies exists in cleavage-stage embryos that associates with maternal and zygotic mRNAs to mediate their degradation or processing for subsequent release for translation during the MBT. A large proportion of these cytoplasmic RNP bodies contain dFMRP. It is likely that the cleavage furrow formation defect observed in fmr1- mutants is the result of disrupting TRAL function. Indeed, tral- embryos have a cellularization phenotype that resembles that of fmr1- embryos. A similar requirement has also been found for the Caenorhabditis elegans homolog of tral, car-1, in cleavage furrow formation. However, as with Fragile X syndrome, it is possible that the altered expression of many targets is responsible for the full fmr1- cellularization phenotype (Monzo, 2006).
The expression of the RNA-binding factor Fragile X mental retardation protein (FMRP) is disrupted in the most common inherited form of cognitive deficiency in humans. FMRP controls neuronal morphogenesis by mediating the translational regulation and localization of a large number of mRNA targets, and these functions are closely associated with transport of FMRP complexes within neurites by microtubule-based motors. However, the mechanisms that link FMRP to motors and regulate its transport are poorly understood. This study shows that FMRP is complexed with Bicaudal-D (BicD) through a domain in the latter protein that mediates linkage of cargoes with the minus-end-directed motor dynein. In Drosophila the motility and, surprisingly, levels of FMRP protein are dramatically reduced in BicD mutant neurons, leading to a paucity of FMRP within processes. Functional evidence is provided that BicD and FMRP cooperate to control dendritic morphogenesis in the larval nervous system. These findings open new perspectives for understanding localized mRNA functions in neurons (Bianco, 2010).
BicD proteins (BicD in Drosophila and BicD1 and BicD2 in
mammals) play roles in the transport of a subset of cargoes
by the minus-end-directed microtubule motor dynein. The N-terminal two-thirds of BicD interact with dynein and its accessory complex dynactin, and the C-terminal third (the C-terminal domain [CTD]) mediates mutually exclusive association
with different cargoes. The best-characterized roles of BicD proteins are in the bidirectional transport of Golgi vesicles and a subset of asymmetrically localized Drosophila mRNAs, which are mediated by binding of the CTD to the
membrane-associated G protein Rab6 and the RNA-binding
protein Egalitarian (Egl), respectively. The interactions
of the BicD CTD with both proteins are inhibited by the
K730M substitution in the Drosophila BicD protein, which
is a null mutation in vivo. K730M does not, however, inhibit
binding of the BicD CTD to other copies of BicD, indicating
that it specifically effects association of BicD with motor cargoes (Bianco, 2010).
In an attempt to elucidate the basis of linkage of other cargoes
to dynein, a GST pull-down from fly embryonic
extracts was performed with the Drosophila BicD CTD (amino acids 536-782) and an equivalent K730M mutant protein as a specificity
control. Mass spectrometry revealed that a protein of 80-85
kDa reproducibly recruited only by the wild-type CTD was Drosophila FMRP (27 unique peptides), and this was confirmed by western blotting. Endogenous
BicD and FMRP were specifically coimmunoprecipitated
from Drosophila embryonic extracts. Unlike known Egl-interacting proteins, FMRP was not coimmunoprecipitated with a GFP-tagged Egl protein. This finding,
together with the observation that binding of both Egl and
FMRP to BicD is impaired by the K730M mutation, suggests that BicD:FMRP complexes are largely, or completely, distinct from BicD:Egl complexes (Bianco, 2010).
The ability to detect FMRP in CTD pull-downs and BicD
immunoprecipitations from extracts was abolished by treatment
with RNase. In contrast, the complex of Egl with BicD was not sensitive to RNase treatment. Thus, the stable association of BicD and FMRP
in extracts is dependent on RNA. Nonetheless, a weak interaction of the BicD CTD with a subfragment of FMRP (aa 220-618) was found in yeast two-hybrid assays. This interaction was specific, as shown by the fact that it was disrupted by the K730M mutation within the BicD CTD. These findings raise the possibility of a direct contact of BicD and FMRP in vivo that is stabilized by the association of FMRP
with RNA targets and possibly other RNA-associated proteins (Bianco, 2010).
These results suggest that BicD could be a functional
interactor of FMRP in vivo. Subsequent studies therefore focused on neurons, where FMRP plays a prominent role. As previously observed, endogenous FMRP is
enriched in puncta within the cell body and neurites of
Drosophila primary neurons cultured from larval brains. Endogenous BicD was also found in puncta in these cells, but these were much more frequent than those
containing FMRP. Although there was overlap of a subset of FMRP puncta with BicD puncta, the widespread distribution of BicD precluded
a meaningful interpretation about the extent of complex
formation of BicD and FMRP in fixed primary neurons (Bianco, 2010).
Therefore neuronal cultures were established from brains of
transgenic larvae expressing FMRP::GFP and BicD::mCherry
and time-lapse microscopy was used to assay for cotransport of
puncta containing both proteins. These fluorescent fusion proteins retain function
and account for ~20% and 50% of the levels of total FMRP and BicD proteins, respectively, in transgenic larval brain extracts (Bianco, 2010).
Both BicD::mCherry and FMRP::GFP were widely distributed
in the cytoplasm of the primary neurons, but bidirectionally
transported FMRP::GFP puncta were found in all cells and
92.4% ± 3.2% of them were cotransported with a puncta of
BicD:mCherry. Thus, FMRP and BicD can be contained within the same motile transport complexes in neurons. The motility of FMRP::GFP in these experiments
will be described in more detail below. Only 77.2% ± 4.6%
of motile BicD::mCherry puncta were cotransported with
a puncta of FMRP::GFP (155 particles in 20 cells), indicating
that BicD may transport additional cargoes in these cells
and/or that a subset of BicD::mCherry complexes may contain
only nonfluorescent, endogenous FMRP (Bianco, 2010).
Next whether BicD has a functional role in FMRP:motor complexes in neurons was explored by assessing the subcellular localization of FMRP in third instar BicD mutant larvae. Because the high expression of FMRP expression in neighboring
nonneuronal cells obfuscates the distribution of the endogenous protein in thin neuronal processes, UAS-FMRP::GFP was expressed at low levels by using a panneuronal GAL4 driver. In neurons of zygotic BicD null mutant
larvae, which also lack detectable maternal BicD protein, the amount of FMRP::GFP within the neurites was greatly reduced compared to wild-type. Surprisingly, there was also a much weaker FMRP::GFP signal in the cell body of BicD mutant neurons relative to wild-type. Western blotting of third instar larval brain extracts confirmed a striking reduction in levels of both FMRP::GFP and endogenous
FMRP in the absence of BicD (Bianco, 2010).
Strong mutations in genes encoding the dynein and kinesin-1
motor proteins, which should inhibit microtubule-based FMRP
transport in Drosophila, did not alter the amount of FMRP. These findings, together with observations from interfering with dynactin function, suggest that
the reduction in FMRP protein levels in BicD mutants is caused
by a specific role of BicD rather than an indirect consequence
of inefficient FMRP transport (Bianco, 2010).
Levels of the Fmr1 mRNA, which encodes FMRP, were indistinguishable
in wild-type and BicD mutant brain extracts, as revealed by quantitative RT-PCR. Thus, the requirement for BicD in maintaining FMRP protein levels is
not associated with RNA decay or transcription. Further
evidence against a defect in Fmr1 transcription in BicD
mutants is provided by the strong reduction in the levels of
the GFP-tagged FMRP protein, which is transcribed under the control of yeast-derived UAS promoter elements. The FMRP::GFP transgene also lacks the untranslated
sequences from the Fmr1 gene, revealing that BicD’s regulation of FMRP protein
amount is mediated through the Fmr1 coding sequence. BicD may therefore influence FMRP protein stability through an unknown mechanism. However, it currently cannot be rule out that BicD regulates the translation of FMRP; at least in
mammals, the coding sequence of Fmr1 mRNA contains
a translational control element, which negatively regulates
protein production by binding FMRP. Distinguishing
between these and other possibilities will require long-term
studies. Interestingly, the underlying mechanism appears to
be restricted to certain cell types as shown by the fact that
FMRP levels in cultured Drosophila D-Mel cells (a derivative
of S2 cells) were not reduced by RNAi-mediated depletion of BicD (Bianco, 2010).
To investigate whether BicD also has a role in controlling
FMRP motility, the distribution of residual FMRP::GFP was examined in BicD mutant primary cultured larval neurons. There was a strong decrease in the proportion of FMRP::GFP particles that localized to neurites in BicD mutants compared
to wild-type, with FMRP particles also less likely
to reach the most distal regions of the mutant processes. The changes in FMRP distribution are unlikely to result from differences in cellular morphology or general effects on trafficking processes because the length and
complexity of neurites, as well as the distribution of mitochondria,
was comparable in BicD mutant and wild-type neurons (Bianco, 2010).
To test directly whether BicD is required for FMRP motility,
time-lapse imaging of FMRP::GFP particles was performed in
cultured larval neurons. FMRP particles in wild-type neurons were
usually stationary during several minutes of filming, but
some occasionally underwent periods of rapid, directed movement. Motile particles in the processes exhibited persistent motion both toward and away from the cell body, with some particles rapidly switching directions. There was
no overall bias in the length of directed, continuous movements
(run lengths) toward and away from the cell body, consistent with a completely mixed microtubule polarity in both neurites and the soma (Bianco, 2010).
Consistent with BicD’s well-characterized role in dynein/
dynactin-mediated transport, inhibition of dynactin function
by neuron-specific expression of a dominant-negative version
of the p150Glued subunit (DGlued) strongly reduced the
motility of FMRP puncta and their localization into neuronal processes. Despite the strong difference in efficiency of FMRP transport, the amounts of FMRP were indistinguishable between DGlued and wild-type extracts. This observation provides further evidence that the role of BicD in regulating FMRP protein levels is not due to a general effect of inhibiting transport (Bianco, 2010).
BicD is complexed with dynein and the plus end motor
kinesin-1 on at least some bidirectional cargoes, and
a kinesin-1 family member associates with FMRP complexes
and contributes to their transport in mammalian neurons.
In Drosophila primary neurons with a strong kinesin-1 heavy
chain mutant genotype (Khc17/27), there was a striking alteration
of FMRP appearance compared to wild-type cells, with
discrete particles not detectable above the diffuse cytoplasmic
signal. This observation, which is reminiscent of the reduced size of a kinesin-1 mRNP cargo in Drosophila oocytes, raises the possibility that both dynein
and kinesin-1 cooperate in FMRP transport in Drosophila neurons (Bianco, 2010).
BicD may have a direct role as a constituent of FMRP:motor
complexes. Alternatively, reduced levels of FMRP in BicD
mutants might have an indirect effect by reducing the probability
of FMRP encountering other transport factors. To
attempt to discriminate between these possibilities, advantage was taken of the observation that overexpression of BicD, even to a very large extent, does not alter the total amount of FMRP. This presumably reflects wild-type levels of BicD being nonlimiting for the function in controlling FMRP levels (Bianco, 2010).
2-fold overexpression of BicD (tagged with mCherry)
dramatically increased the run lengths and net displacements
of motile FMRP::GFP particles in cultured neurons, compared
to the wild-type. Run lengths in processes
were similar for movements both toward and away from the
cell body upon BicD overexpression. Nonetheless,
there was increased targeting of FMRP into distal
processes compared to wild-type. Once again, this presumably reflects the ability of long-distance, unbiased bidirectional transport to aid cargo spreading.
These results demonstrate that BicD is able to control motility
and subcellular localization of FMRP independently from the
role in regulating overall levels of the protein (Bianco, 2010).
The results of quantification of particle motility, together
with observations that (1) FMRP is recruited by means of
the domain of BicD involved in linking cargoes to dynein and (2) FMRP colocalizes in motile particles with BicD in vivo, provides strong
evidence that BicD plays a direct role in FMRP:motor
complexes. In the case of other cargoes studied, BicD is not
obligatory for their linkage to motor complexes but increases
their travel distances significantly. The residual directed transport of FMRP particles in BicD mutant neurons suggests that BicD may play a similar stimulatory role in this context. Other components of FMRP-containing transport
particles presumably also contribute to linkage with motor proteins (Bianco, 2010).
The functional significance of the BicD:FMRP
interaction in dendrites was explored by focusing on the role of FMRP in dendritic
morphogenesis. The well-characterized model
system for dendritic development in the Drosophila third instar
larva, the dorsal class IV dendritic arborization (da) neuron
ddaC, was explored (Bianco, 2010).
Dorsal ddaC neurons within zygotic BicD mutant larvae had
a much less extensively branched dendritic arbor than wildtype
cells. A similar inhibition of the dendritic branching program was observed in three different zygotic Fmr1 null mutant genotypes. Intermediate terminal branching
defects were also found in ddaC neurons heterozygous for
Fmr1D50M. This phenotype, which could be suppressed by the FMRP::GFP transgene, underscores the importance of correct
FMRP protein levels for neuronal morphogenesis (Bianco, 2010).
These results demonstrate that both BicD and FMRP are
required for efficient branching of the dendritic arbor in dorsal
ddaC neurons. Interestingly, FMRP negatively regulates
dendritic elaboration in mushroom body neurons in adult
brains. It has also previously been reported that mutating
Fmr1 increases branching of ventral da neurons, although
effects on specific classes of neurons within the cluster have
not been reported. The differential requirements for Fmr1 in controlling
the morphology of different neuronal cells is consistent
with previous findings. It has been showed that Fmr1 mutations cause overextended axons in LNv cells but a failure of axon extension in DC neurons. Cell type-specific effects of FMRP on neuronal morphogenesis may reflect differences in
the repertoire of its mRNA targets (Bianco, 2010).
BicD overexpression specifically in class IV da neurons
significantly increased the number of dendritic branches in
the distal regions of arbors in dorsal ddaC neurons compared
to wild-type. This result, together with the diminished branching in BicD mutant neurons, reveals a correlation between the amount of available BicD and the
degree of arborization of ddaC and that BicD can function
autonomously within neurons to control this process (Bianco, 2010).
Strikingly, the ability of overexpressed BicD to augment dendritic branching of ddaC appears to be due predominantly to its interaction with FMRP, as evidenced by a strong suppression of the BicD overexpression phenotype in Fmr1 null mutants, with neuronal morphology not significantly different to the Fmr1 mutant alone. Because BicD overexpression does not alter FMRP protein levels, increased branching is likely to be influenced by BicD’s ability to control FMRP motility. Live cell imaging revealed that BicD promotes long-distance bidirectional transport of FMRP complexes on microtubules, thereby facilitating the exploration of neuronal processes. Such a mechanism may increase the probability of encounters of these complexes with factors that activate translation of associated mRNAs, which in some contexts could be responsive to local signaling. Nonetheless, the reduction of overall FMRP protein levels is highly likely to contribute to BicD loss-of-function phenotypes in da neurons, as potentially is the altered transport of FMRP-independent cargoes (Bianco, 2010).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.