MIAMI UNIVERSITY
The Graduate School
Certificate for Approving the Dissertation
We hereby approve the Dissertation
Of
Paromita Banerjee
Candidate for the Degree:
Doctor of Philosophy
______Director (Dr. Thomas C. Dockendorff)
______Reader (Dr. Joyce J. Fernandes)
______Reader (Dr. David G. Pennock)
______Reader (Dr. Kathleen A. Killian)
______Graduate School Representative (Dr. Christopher A. Makaroff)
ABSTRACT
MODELING THE EFFECTS OF FMR1 ALLELES ON BEHAVIORAL AND SYNAPTIC PLASTICITY
Paromita Banerjee
Fragile X syndrome is a common (1 in 6000 births) form of inherited mental retardation that arises from mutation of the FMR1 gene. The fragile X protein (FMRP) is an RNA binding protein that plays a major role in behavioral and synaptic plasticity by regulating local protein synthesis in response to neurotransmitter stimulation. The majority of FMRP analyses come from in vitro biochemical assays and cell culture models and, as a result, little is known about the importance of the individual domains in an intact animal model. The development of the Drosophila model for fragile X syndrome with phenotypes similar to those observed in mice and humans lacking FMR1, underscores the relevance of this model for FMRP function and provides a means to uncover novel functions of FMRP.
A molecular genetics approach was utilized to analyze functions of individual domains within dFMR1. These studies provide evidence that multiple domains present in FMRP are critical for its function, perhaps working both in concert and through separate molecular pathways. The C- terminal region of Drosophila fragile X protein has a domain enriched in glutamine/asparagine (Q/N) residues. Q/N domains are known modules for protein-protein interaction. The Q/N domain deletion alleles of dfmr1 showed that it is essential for regulating behavior and memory phenotypes, presumably through mediating dFMR1 assembly into ribonucleoprotein (RNP) complexes that regulate translation. These alleles will be vital for identification of RNP complexes that contain the dFMR1 Q/N(+) protein. Such RNPs may harbor proteins and RNAs that are critical components for establishment and consolidation of behavioral memory. Analysis of flies expressing the dFMR1 ΔQ/N protein indicates that dFMR1 has a role in regulation of alternative splicing of its own and other target mRNAs, perhaps by helping to confer distinct RNA and/or protein binding profiles to dFMR1. Identification of novel functions for FMRP may uncover new avenues for therapeutic interventions.
MODELING THE EFFECTS OF FMR1 ALLELES ON BEHAVIORAL AND SYNAPTIC PLASTICITY
A DISSERTATION
Submitted to the
Faculty of Miami University
in partial fulfillment of
the requirements for the degree of
Doctor of Philosophy
Department of Zoology
by
Paromita Banerjee
Miami University, Oxford, Ohio
2008
Dissertation Advisor/Mentor:
Dr. Thomas C. Dockendorff
Table of Contents
Chapter I: Introduction 1 Fragile X Syndrome 1 Identification of the fragile X gene and the basis of its mutability 2 FMRP is an RNA binding protein 3 RNA substrates for FMRP 3 FMRP-interacting proteins 5 Mechanisms of FMRP function 5 Synaptic activation of FMRP: the mGluR theory of fragile X syndrome 8 A Drosophila model for study of FMRP function 9 Phenotypes of dfmr1 mutants 10 Behavior patterns of dfmr1 mutants 11 Merits of the Drosophila fragile X model 12 Rationale for these studies 14 References 15 Figures 22
Chapter II: Substitution of Critical Isoleucines in the KH Domains of Drosophila Fragile X Protein Result in Partial Loss of Function Phenotypes. 24 Abstract 24 Introduction 24 Materials and Methods 27 Results 29 Discussion 33 References 38 Figures 43
Chapter III: A Glutamine/Asparagine-rich Domain in Drosophila Fragile X Protein is Essential for Regulating Behavioral Plasticity. 51 Summary 51 Introduction 51 Results 53 Discussion 60 Experimental Procedures 64 References 69 Figures 74
Chapter IV: A Genetic Dissection of the dFMR1 C-terminal Domain Uncovers Novel Functions for Fragile X Protein 84 Chapter 4A: Generation and analysis of fly stocks that produce either the Q/N(-) or Q/N(+) isoform as the sole source of dFMR1 protein 84 Methods 85 Results 87 Discussion 88
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Chapter 4B: Does the dFMR1 Q/N domain exert its effects through interactions with other proteins? 89 Rationale 89 Methods 89 Results and Conclusions 90 Chapter 4C: Are the effects of the dFMR1 Q/N domain deletion the result of interfering with the function of the highly conserved C-terminal peptide of dFMR1? 90 Rationale 90 Methods 91 Results 91 Discussion 92 Chapter 4D: The dFMR1 Q/N domain may be necessary for efficient alternative splicing of its pre-mRNA transcript 92 Rationale 92 Results and Discussion 93 References 94 Figures 96
Chapter V: Summary 105 There may be many mechanisms by which FMRP regulates neuronal function 105 Testing the role of a Q/N rich domain 107 Future prospects for molecular genetic analyses of mental retardation 108 References 109
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List of Tables
Chapter I 1 Table 1 23
List of Figures
Chapter I: Introduction 1 Figure 1 22
Chapter II: Substitution of Critical Isoleucines in the KH Domains of Drosophila Fragile X Protein Result in Partial Loss of Function Phenotypes. 24 Figure 1 43 Figure 2 44 Figure 3 46 Figure 4 48 Figure 5 49
Chapter III: A Glutamine/Asparagine-rich Domain in Drosophila Fragile X Protein is Essential for Regulating Behavioral Plasticity. 50 Figure 1 74 Figure 2 75 Figure 3 77 Figure 4 78 Figure 5 79 Figure 6 81 Figure 7 82
Chapter IV: A Genetic Dissection of the dFMR1 C-terminal Domain Uncovers Novel Functions of Fragile X Protein 84 Figure 1 96 Figure 2 98 Figure 3 100 Figure 4 101 Figure 5 102 Figure 6 103 Figure 7 104
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CHAPTER I
INTRODUCTION
Mental retardation is clinically diagnosed in 1-3% of the population from industrialized countries (reviewed by Inlow and Restifo, 2004). In addition to the humanitarian concerns for affected individuals, mental retardation poses challenges for both the families of those affected and society, since mentally retarded individuals are in need of care and attention throughout their lives. Thus, understanding the molecular and cellular bases of mental retardation as a possible means of therapy or cure is of considerable importance to affected families, clinicians and scientists. Although it is difficult to accurately measure, 25-50% of mental retardation cases are thought to have a genetic basis (McLaren and Bryson, 1987). This underscores the importance of identifying and characterizing genes that mutate to a mental retardation phenotype. While some of these cases arise from multigenic deletions or duplications, many single gene mutations leading to mental retardation have been identified (reviewed by Inlow and Restifo, 2004), and such genes will be of primary interest for achieving the above goals.
Fragile X Syndrome Fragile X Syndrome is the most common (1 in 6000 births) form of inherited mental retardation where the effects result from loss of function of a single gene. While a founder effect is associated with most other inborn errors of metabolism, fragile X syndrome is present in all human populations. Thus, the frequency and global distribution of fragile X syndrome makes it one of the most common genetic disorders in humans. Prominent phenotypes associated with fragile X syndrome are cognitive and behavioral defects characterized by mental retardation, autistic behavior, hyperactivity, attention deficit disorder, and sleep disorders. Other clinical phenotypes include macroorchidism in male patients and mild facial dysmorphology. The penetrance and expressivity of fragile X phenotypes varies greatly between individuals, indicating that the genetic background of the patient has a role in manifestation of the phenotypes. The pleiotropic phenotypes of fragile X syndrome indicate that the fragile X protein regulates a variety of biochemical and physiological functions. Although no abnormalities of gross brain morphology are present in fragile X patients, alterations in dendritic spine
1 morphology are observed (reviewed by de Vries et al., 1998; Irwin et al, 2000). The dendritic spine phenotype is of interest since dendritic spine dysmorphology is associated with other mental retardations of uncharacterized etiology (Purpura, 1974) and suggest that the fragile X protein modulates synaptic maturation and/or maintenance.
Given the cognitive impairment associated with mental retardation, many genes that mutate to such a phenotype may very well play a central role in cellular and molecular processes that contribute to synaptic function, and thus control cognition, learning, and memory. As will be illustrated in the following sections, studies of the fragile X gene are providing insights into mechanisms of local protein synthesis necessary for synaptic plasticity and the identification of genes whose products are needed to modulate synaptic morphology and function.
Identification of the fragile X gene and the basis of its mutability A locus tightly linked to both a fragile site and polymorphisms associated with fragile X chromosomes encoded a single open reading frame that was suggested as a candidate for the fragile X mental retardation gene (Verkerk et al, 1991). The absence of mRNA transcripts from this gene in fragile X patients, coupled with subsequent findings of deletions encompassing this locus in patients diagnosed with fragile X syndrome, provided proof that this gene is associated with fragile X syndrome (reviewed by O’Donnell and Warren, 2002). Limitations to DNA and protein sequence databases at the time of FMR1 cloning made it difficult to predict the function of the gene product. Nonetheless, the cloning of FMR1 helped to provide insights about the primary mechanism of FMR1 mutation and helped in development of tools for expression and functional analysis of the protein product. A detailed examination of FMRP expression revealed that it is present in many developing tissues, more prominently in the nervous system and testis (Hinds et al., 1993; Bakker et al., 2000).
An unusual feature of the FMR1 cDNA was the presence of a repetitive sequence at the 5´ end of the gene that had previously been identified by both hybrid breakpoints and mapping of unstable DNA fragments. A vast majority of individuals (90-95%) suffering from fragile X syndrome have an expansion of CGG repeats in the 5´-untranslated region of the FMR1 gene (reviewed by O’Donnell and Warren, 2002). The FMR1 gene in the general population has in the range of 6-
2 60 CGG repeats. In premutation carriers (nonpenetrant), the CGG triplet increases to 60-200 copies, while in completely penetrant full mutants, the repeat number increases to >200, and may number in the thousands. The premutation alleles are often unstable and have a tendency to expand to the full mutation (perhaps due to replication slippage) during generation of gametes, a process occurring most often in female carriers (reviewed by O’Donnell and Warren, 2002). The
(CGG)n expansion increases the number of CpG islands that are substrates for cytosine methylation, and the subsequent high level of this modification induces transcriptional silencing of the FMR1 gene. The inadequate amount of fragile X protein product results in phenotypes associated with fragile X syndrome.
FMRP is an RNA binding protein The aforementioned limitations to gene and protein sequence databases at the time of FMR1 cloning, coupled with the fact that it was not cloned through function-based means, made initial identification of a biochemical function for the fragile X protein (FMRP) difficult. A breakthrough came from the discovery by Siomi et al (1993) that FMRP has domains that are common to RNA-binding proteins. Two KH domains (hnRNP-K homology) and an arginine and glycine-rich motif (RGG box) are present in FMRP (Figure 1). Biochemical tests showed that in vitro translated FMRP binds RNA homopolymers, with the KH domains and RGG box being necessary for binding, depending upon reaction conditions (Siomi et al., 1993, 1994). Further characterization of FMRP RNA-binding properties showed that FMRP has specificity for RNA substrates, as it associates with only a small fraction of human fetal brain messages (Ashley et al., 1993).
RNA substrates for FMRP Substitution of a highly conserved isoleucine residue with asparagine in the second KH domain of FMRP is the only change in the open reading frame of FMR1 from a patient with unusually severe fragile X phenotypes (De Boulle et al., 1993), and this mutation is associated with a selective loss of RNA binding capacity (Darnell et al., 2005a). Thus to understand FMRP function, it is important to identify its in vivo RNA targets. However, identification of bona fide substrates for RNA binding proteins can be extremely challenging. In vitro experimental conditions (e.g. salt and detergent concentrations) used for protein purification via co-
3 immunoprecipitation can affect the binding of FMRP to its target ligands. Another difficulty faced in such experiments is that FMRP can be part of multiprotein complexes that contain other RNA binding proteins, and thus it would be nearly impossible to discern between RNAs bound to FMRP or to the other RNA binding proteins in the complex (discussed in Darnell et al., 2005b). Moreover, ribonucleoproteins might reassociate with non-specific RNAs after cell lysis, confounding subsequent analyses (Mili and Steitz, 2004). Nonetheless, efforts to identify FMRP RNA substrates have been undertaken. One strategy used is immunoprecipitation of FMRP from mouse brains or select domains of cultured neurons, followed by the extraction of the co- precipitated RNAs and their identification through microarray analysis (Brown et al., 2001; Miyashiro et al., 2003). A second method utilizes in vitro RNA selection, where a library of random RNA substrates is allowed to bind with the RNA-binding protein of interest. Elution of bound RNAs at different stringencies is performed, and tightly bound RNAs are then subject to amplification and subsequent analysis. From such a study done with FMRP, a predicted G- quartet structure was present in the most tightly bound RNA ligands (Darnell et al., 2001). Importantly, many of the RNAs identified through the immunoprecipitation screen also had G- quartet structure, and it is now recognized that this structure interacts with the RGG box (Brown et al., 2001; Darnell et al., 2001; Schaeffer et al., 2001). That two independent screens selected structurally similar RNAs with a specific affinity for the FMRP RGG box provides validation for the G-quartet-RGG box interaction. However, it also illustrates the pitfalls and potential for bias when screening for ligands of RNA binding proteins, as the above interaction was the only one selected and identified, despite the presence of other known RNA binding domains in FMRP. A subsequent RNA selection screen using the second KH domain of FMRP revealed that it recognizes a complex loop-loop pseudoknot RNA structure referred to as a kissing complex (Darnell et al., 2005a). The N-terminal 134 amino acids of FMRP bind RNA homopolymers (Adinolfi et al., 2003), and a stem-loop structure within the non-coding RNA BC1 interacts specifically with the N-terminal 217 amino acids of FMRP (Zalfa et al., 2005). From this data, it can be concluded that the different RNA binding domains in FMRP interact with exclusive sets of target RNAs.
Refinements of the above strategies have been devised to address some of the challenges associated with identifying RNA-protein interactions. To circumvent the problem of non-
4 specific RNAs binding after cell lysis, cross-linking immunoprecipitation (CLIP) has been developed, where the tissue is subjected to UV-B irradiation for in situ covalent cross-linking of proteins to bound RNAs (Ule et al., 2003). The cross-linked protein-RNA complex can then be immunoprecipitated to identify the substrate RNAs. Although the use of a deletion null allele control can help subtract some non-specific background from immunoprecipitations, such alleles will of course not allow for precipitation of the target protein and other possible RNA binding proteins associated with it, and thus not avoid the problems raised when multiple RNA binding proteins are present in a precipitating complex. Generation of fly stocks with FMR1 alleles where individual RNA binding domains are disrupted will be instrumental in refining the list of potential FMRP substrates and in identifying substrates binding to these particular domains. In this approach, tissues expressing either wild type or domain-specific mutant forms of FMRP are put through the CLIP protocol, using antibodies that precipitate both proteins. The RNA-binding profile produced from the mutant FMRP is then subtracted from the wild type profile to produce a list of substrates that are specific for a particular FMRP RNA binding domain.
FMRP-interacting proteins As an RNA binding protein, FMRP exerts at least some of its effects through assembly into messenger ribonuclear protein (mRNP) complexes. Thus, to characterize mechanisms and pathways by which FMRP regulates metabolism and function of its mRNA substrates, biochemical fractionation, yeast two-hybrid screening, and proteomics approaches have been employed to identify FMRP-interacting proteins. A list of FMRP-interacting proteins is presented in Table 1, and based on the biochemical functions of these proteins, it can be inferred that FMRP has the potential to play a broad role in the metabolism and function of its RNA substrates. The following paragraphs provide descriptions of the best-characterized and/or most salient roles of FMRP in regulating synaptic function, along with a novel means by which FMRP may regulate gene expression.
Mechanisms of FMRP function Regulation of translation: The co-purification and/or interaction of FMRP with ribosome proteins, ribosome subunits, and polyribosomes indicate a function in regulating translation (Khandjian et al., 1996; Siomi et al., 1996; Tamanini et al., 1996). More recently, identification
5 of potential FMRP RNA substrates has allowed for direct testing of protein expression levels from candidate transcripts, and these studies support such a role for FMRP (Zalfa et al., 2003; Todd et al., 2003; Lu et al., 2004). Both up- and down-regulation of protein expression are seen in FMR1 mutant tissues; however, the mechanism(s) by which FMRP regulates translation are still rather vague. FMRP from both flies and mammals interact with Argonaute proteins, which are components of RISC complex (Caudy et al., 2002; Ishizuka et al., 2002; Jin et al., 2004a). This suggests that FMRP regulates translation of at least some of its target mRNAs through micro-RNA (miRNA) based mechanisms, presumably by delivering the substrate mRNAs to miRNA machinery for translational silencing (reviewed by Jin et al., 2004b). The role of FMRP in regulating translation can be indirect, as a recent finding indicates that FMRP enhances the stability of PSD-95 mRNA at synapses (Zalfa et al., 2007). Destabilization of mRNA arising from FMR1 mutation and the resulting decrease in protein product from such transcripts gives the appearance of FMRP serving as a positive regulator of translation. FMRP may shift between having activation or repressive effects during development and/or in different parts of the brain via changes in its phosphorylation status (reviewed by Bagni and Greenough, 2005). The phosphorylation state of FMRP is dynamic, with metabotropic glutamate receptor (mGluR) signaling stimulating both stimulation and inhibition of phosphatase activity on FMRP (Narayanan et al., 2007). Phospho-FMRP can fractionate with stalled ribosomes, a state indicative of translation inhibition (Ceman et al., 2003), while dephosphorylated FMRP results in stimulation of activity-dependent protein synthesis that arises through mGluR signaling (Narayanan et al., 2007).
RNA transport: Localized synthesis of proteins in response to neurotransmitter stimulation is necessary for synaptic plasticity (reviewed by Sutton and Schuman, 2006). Since a neuron may have dozens of dendrites projecting from a common soma, this mechanism of regulation allows for a synapse-specific response to stimulation. The polarized nature of neurons means that a transport mechanism is needed to deliver RNAs from the soma to a post-synaptic site. Several lines of evidence implicate FMRP as a component of an RNA transport complex. FMRP and its mRNA co-localize as granules and are found in dendritic processes, including dendritic spines, suggesting a role for FMRP in sub-cellular localization of its bound mRNAs. The movement of FMRP granules into dendrites is enhanced through mGluR signaling (Antar et al., 2004).
6 Whether FMRP is actively engaged with RNA substrates during transport through dendrites has yet to be conclusively determined, but evidence of FMRP acting as a transporter comes from findings that levels of mRNA in dendrites of FMR1 knockout mice are less in comparison to wild type mice (Miyashiro et al., 2003). Proteomic analysis of a kinesin-containing RNA- transporting complex purified from mouse brain reveals the presence of Staufen, a proven RNA transporter, and FMRP (Kanai et al., 2004). Interactions between FMRP and ZBP1/insulin-like growth factor II mRNA-binding protein (IMP1), a protein with a known role in RNA transport in fibroblast and neurons, have been uncovered, suggesting that these two proteins might recruit each other into RNP granules in vivo (Rackham and Brown, 2004). In cultured Drosophila cells, bidirectional transport of GFP-tagged FMRP granules on microtubules is observed (Ling et al., 2004). Whether retrograde transport of FMRP is for recycling purposes or part of a signaling response to the soma is unknown. It is interesting to note that patients have been identified with classic symptoms of fragile X syndrome, yet have no trinucleotide expansion in the FMR1 5´ UTR or mutations in the coding region. Such individuals may suffer from mutations that disrupt the FMRP localization process (reviewed by Bagni and Greenough, 2005).
Nuclear FMRP: FMRP is present in the nucleus, although nuclear-localized FMRP is difficult to detect. A functional nuclear localization signal (NLS) and a nuclear export signal (NES) are present in FMRP as judged by mutagenesis studies, and therefore FMRP may shuttle between nucleus and cytoplasm (reviewed by Bagni and Greenough, 2005). Nuclear functions of FMRP are beginning to be defined, with evidence that it interacts with nucleocytoplasmic transport factors (Zhang et al., 2007). Processing of select RNAs is also a plausible function for nuclear FMRP. A particularly interesting insight towards nuclear FMRP function comes from identification of Agenet domains that are well conserved between all FMRP paralogues and orthologues (Maurer-Stroh et al., 2003). Agenet domains are related to methyl substrate binding Tudor and chromo domains that are implicated in regulation of chromatin structure (Maurer- Stroh et al., 2003). The RNAi pathway also functions in the nucleus to regulate chromatin structure via the activity of Argonaute proteins and small RNAs (reviewed by Grewal and Elgin, 2007). Since FMRP interacts with RNAs as well as Argonaute proteins, another possible role of FMRP in nucleus could be regulation of chromatin structure (discussed by Bagni and Greenough, 2005). The observation that null mutations in the Drosophila fragile X orthologue
7 dfmr1 suppress a position-effect variegation phenotype makes such a role even more compelling (Deshpande et al., 2006). For FMRP to exert a regulatory role at the level of transcription creates an entirely new and wholly uncharacterized group of genes subject to its effects, and such a possibility warrants immediate further experimental scrutiny.
Synaptic activation of FMRP: the mGluR theory of fragile X syndrome The behavior and cognitive defects associated with fragile X syndrome, along with the anomalies in dendritic spine morphology, indicates that FMRP has a role in regulating spine development and synaptic function. FMRP and its mRNA are present in dendritic spines and synaptoneurosome preparations (Feng et al., 1997; Weiler et al., 1997; Antar et al, 2004), while synthesis of FMRP and localization of FMR1 mRNA result from activation of group 1 metabotropic glutamate receptors (mGluR) in synaptoneurosomes and tissue culture cells respectively (Weiler et al., 1997; Antar et al, 2004). The placement of FMRP activity into a signal transduction pathway associated with synaptic plasticity, coupled with the availability of receptor-specific agonists and antagonists, was quickly realized to have significant implications for a fragile X therapy.
Long-term potentiation (LTP) and long-term depression (LTD) are the strengthening or weakening of excitatory synaptic transmission in response to electrical or chemical stimuli (reviewed by Malenka and Bear, 2004). Although studies of LTP and LTD are often performed ex vivo with brain slices, it is believed that biochemical and morphological changes of synapses arising from these stimulations are a measure of the in vivo events necessary to establish and maintain memory. Hippocampal LTD can be induced through activation of group 1 mGluR receptors and requires rapid synthesis of proteins (within 60 minutes), which facilitates internalization of AMPA receptors (Huber et al., 2000; Snyder et al., 2001). It was initially assumed that the FMR1 knockout mouse would exhibit defective (i.e. lack of) mGluR LTD, but subsequent investigation of the knockout mice revealed that mGluR LTD was significantly enhanced compared to wild type littermates (Huber et al., 2002). These results are consistent with FMRP having a repressive effect on protein synthesis that results from mGluR signaling, and led to the proposal of the mGluR theory of fragile X syndrome. This theory states that FMRP is synthesized along with other proteins following stimulation of mGluRs. FMRP in turn functions to inhibit further translation of mRNAs in synaptic regions, thus keeping LTD in check
8 by modulating AMPA receptor internalization and changes in spine structure that may be a prelude to loss of synaptic connectivity (reviewed by Bear et al., 2004). An application of this theory is that antagonists of group1 mGluRs would down regulate mGluR-mediated protein synthesis, providing a counter to excessive synaptic protein synthesis that may occur in fragile X patients.
Validation of the mGluR hypothesis has now been demonstrated in part with rescue of the audiogenic seizure phenotype in FMR1 knockout mice by administration of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) (Yan et al., 2005). If reduction of mGluR activity is indeed the basis for the rescue, loss of mGluR5 should be able to correct fragile X phenotypes in FMR1 mutant mice. This was recently demonstrated, where a 50% reduction in mGluR5 expression achieved through heterozygosity, rescued fragile X phenotypes in mice (Dölen et al., 2007). These studies thus indicate that modulation of signaling through group1 mGluRs by the use of receptor specific antagonists might ameliorate cognitive and behavioral phenotypes of fragile X syndrome.
A Drosophila model for study of FMRP function Most studies of FMRP function have utilized in vitro biochemical approaches and cell culture systems. Although structure-function studies in cultured cells and the identification of FMRP- interacting proteins and RNAs have provided insight into FMRP function, these approaches by themselves are not a direct demonstration on how FMRP contributes to processes of behavior and cognition. A molecular genetics-based approach in a model organism that could identify gene interactions and generate unique alleles to uncover novel functions of FMRP was lacking. Furthermore, the development of an intact animal model for fragile X could confirm or challenge models developed by in vitro assays.
A mouse knockout of FMR1 is available and the phenotypes seen in the mutant mice have some similarities to those observed in fragile X patients (Dutch-Belgian Fragile X Consortium, 1994), although phenotypes using standard learning and memory paradigms may be weak, dependent upon genetic background, or nonexistent (Fisch et al., 1999; Kooy, 2003). Molecular genetics with vertebrate animals has some limitations. Generation of animals with novel alleles is
9 relatively time-consuming, and the facilities and costs required to conduct large-scale screens for gene interactions are prohibitive for most laboratories. In the case of FMR1, the presence of the paralogous FXR1 and FXR2 genes in vertebrates can complicate phenotypic analyses via functional redundancy. Indeed, subtle differences in overlap of function between FMR/FXR proteins of mice and humans may account for the relatively mild learning and memory phenotypes observed in FMR1 mutant mice (O’Donnell and Warren, 2002).
The above considerations spurred efforts to develop an invertebrate genetic model for studies of FMRP function. As a historical aside, it is interesting to note that upon cloning of the human FMR1 gene, a “zoo blot” was performed to assess the commonality of this gene among diverse eukaryotic species. Although such blots are prone to spurious hybridizations, and negative results are merely uninformative, a simple conclusion from the blot presented by Verkerk et al. (1991) was that FMR1 orthologues may be present in yeast and nematodes, but not in the fruit fly D. melanogaster. Fortunately, such results did not deter L. Wan from cloning a fly orthologue of FMR1 (Wan et al., 2000), and the sequencing of yeast and C. elegans genomes have revealed no genes coding for proteins with an organization similar to that of FMRP.
Completion of the Drosophila melanogaster genome sequence showed that only a single orthologue of the FMRP protein family (dFMR1) is present. Both the primary peptide sequence and organization of dFMR1 functional domains are highly conserved with its mammalian counterpart (Wan et al., 2000). Similar to vertebrate orthologues, dFMR1 contains two KH domains and an RGG box, as well as a high level of conservation in the ribosomal association and oligomerization domains. The biochemical functions of the protein are conserved as well. dFMRP possesses RNA binding activity that is mediated in part by the KH domains (Wan et al., 2000). The expression pattern of dFMRP shows a nearly ubiquitous distribution in tissues, with elevated levels present in the nervous system and muscles, similar to what is observed in mammals (discussed in Wan et al., 2000).
Phenotypes of dfmr1 mutants Loss-of-function deletion alleles of dfmr1 have been generated by imprecise excision of a P element transposon insertion, and the resulting neurodevelopmental and behavioral phenotypes
10 have striking parallels to those seen in patients and the mouse model (reviewed by Gao, 2002; Zarnescu et al., 2005; Zhang and Broadie, 2005). Analysis of adult brain morphology in dfmr1 deletion alleles revealed abnormalities in axon development. Both guidance and branching phenotypes are seen with intermediate levels of penetrance and expressivity, but the nature of the defect can differ between neuronal types. While overextension of axon projections are seen in β- lobe neurons of mushroom bodies and ventral lateral neurons (LNv), the axons of dorsal cluster (DC) neurons fail to extend properly (Dockendorff et al., 2002; Morales et al., 2002; Michel et al., 2004). The basis for the opposing phenotypes is not known with certainty, but differences in dFMR1-RNA interactions between the neuronal types are plausible. Excessive branching is observed in both the LNv and DC neurons (Dockendorff et al., 2002; Morales et al., 2002), and a greater number of dendritic arbors are present in the dendritic arborization (DA) neurons in larvae (Lee et al., 2003). An increase in numbers of axon branches and larval neuromuscular junction synaptic boutons observed in dfmr1 mutants is suggested to result from misexpression of the microtubule-associated protein 1B (MAP1B) orthologue futsch (Zhang et al., 2001). The branching defects of the ventral lateral neurons in dfmr1 mutants are reported to arise from misregulation of actin dynamics (Reeve et al., 2005). Activity-dependent changes in dendritic spine morphology arise from modulation of the cytoskeletal network (reviewed by Tada and Sheng, 2006), and it seems likely that the neuronal development phenotypes observed in dfmr1 mutants are related to the dendritic spine morphology anomalies observed in fragile X patients.
Behavior patterns of dfmr1 mutants In addition to cognition defects that contribute towards mental retardation, fragile X patients may display any number of several distinct behavior anomalies that are associated with fragile X syndrome. Such individuals are prone to autistic behaviors, attention deficit-hyperactivity disorder, abnormal sleep patterns, and perseverative speech. An arrhythmic circadian locomotion pattern is present with a high degree of penetrance in dfmr1 mutants (Dockendorff et al., 2002; Morales et al., 2002; Inoue et al., 2002), a phenotype related to the sleep disorders and abnormal melatonin output observed in fragile X patients (Hagerman, 1996; Gould et al., 2000). In contrast to the steady pattern of locomotion activity displayed by mutants of the core circadian clock (period, timeless), dfmr1 mutants have erratic locomotion activity with periods of relative hyperactivity (Dockendorff et al., 2002). Courtship behavior in dfmr1 mutants is reduced, with
11 mutants initiating courtship but being unable to maintain courtship interest (Dockendorff et al., 2002). However, dfmr1 mutant males are able to copulate, and thus maintain an adequate ability to detect and respond to cues from receptive females.
Conditioned courtship is a paradigm for learning and memory in Drosophila, which takes advantage of the observation that while a naive virgin male will actively court a receptive virgin female, a non-virgin, non-receptive female displays negative cues towards would-be mates. The courtship activity of a male steadily decreases when paired with a non-receptive female, indicating that he is detecting and responding to the adverse courtship cues produced by the female. When such “trained” males are subsequently paired with a receptive female, their courtship activity remains depressed for a period of time that is related to the length of time and number of exposures that the male has with a non-receptive female. This behavior is thought to be a form of associative memory, and has been used to measure processes of immediate recall, short-term, and long-term memory (Siegal and Hall, 1979; reviewed by Greenspan and Ferveur, 2000).
Males mutant for dfmr1 have been examined for their performance in the conditioned courtship paradigm (McBride et al., 2005). As is observed with wild type flies, the degree of courtship activity in dfmr1 flies declines with time when paired with a non-receptive female, indicating that the mutant males are able to detect and respond to the negative courtship cues. Both immediate recall and one-hour short-term memory of conditioned courtship is disrupted in males homozygous for a dfmr1 deletion allele, indicating that dFMR1 protein is needed for development of the neuronal circuitry needed for the establishment of the earliest stages of memory formation.
Merits of the Drosophila fragile X model The traditional advantages of model genetic organisms (rapid reproduction, ability to culture large number of animals) make Drosophila an attractive model for probing gene interactions that further illuminate mechanisms of known FMRP functions and uncover novel ones. With the help of clever genetic tools and experimental assays, Drosophila can be useful to reveal physiologically important partners of FMRP in an in vivo context. By eliciting a dominant rough
12 eye phenotype via dfmr1 overexpression, Zarnescu et al., (2005) were able to identify enhancers and suppressors of the eye phenotype. Such loci may encode proteins that regulate dFMR1 activity. Other forward genetic screens for suppressors and enhancers of dfmr1-based behavior patterns and sterility are a promising means to probe FMRP interactions and define functions (Zhang and Broadie, 2005). A near-century as a genetic model has resulted in a vast collection of Drosophila mutants. The ready availability of those stocks can facilitate in vivo confirmation of FMRP interactions uncovered by biochemical means (e.g. Costa et al., 2005).
Conserved function between mammalian and insect FMRP facilitates comparative analyses that can be used to validate proposed models. For example, biochemical interactions inferred with mammalian FMRP have utilized the ready ability to perform gene interaction studies with Drosophila as a means of further validation for a model (Schenck et al., 2003; Jin et al., 2004). Conversely, genetic interactions uncovered with dfmr1 were demonstrated to have a broad significance for FMRP function through studies using cultured mammalian neurons (Zarnescu et al., 2005). The similarity in behavior phenotypes between Drosophila and vertebrate models of fragile X syndrome raises the possibility that flies could be used to screen for drugs or metabolites that modify these phenotypes. As proof of this possibility, antagonists of mammalian group 1 mGluRs have ameliorating effects on the courtship behavior of dfmr1 mutants, including rescue of short-term memory deficits, indicating that the basic elements of the mGluR signaling pathway are conserved between insects and vertebrates (Mc Bride et al., 2005).
Finally, since the majority of human FMR1 alleles arise from a trinucleotide repeat expansion, relatively little insight into FMRP function has been gained from identification and analyses of missense mutations. The study of alleles is a time-honored means to probe biological function (discussed by Sikorski and Boeke, 1991), and the amenability of Drosophila to transgenics facilitates this approach. A 14 kb clone encompassing the dfmr1 locus rescues all neural development and behavior phenotypes observed in the null mutants (Dockendorff et al., 2002), and the availability of this rescue fragment allows for generation of transgenic flies that have specific mutations introduced in the dfmr1 gene. The benefits of acquiring fly stocks with partial loss-of-function or hypomorphic alleles of dfmr1 are many-fold: First, it is possible that phenotypes that illuminate a particular function are masked in a null allele background and are
13 uncovered only by alleles that selectively disrupt a subset of protein function (e.g. Keleman et al., 2007). Second, such stocks can be used to isolate allele-specific suppressors or enhancers, which often provide novel insights into pathways in which the protein of interest participates (see Fortini and Artavanis-Tsakonas, 1994 as an example). Third, animal stocks harboring novel alleles of a gene of interest are highly useful tools for biochemical studies. For example, having domain-specific alleles of dfmr1 can facilitate identification of FMRP RNA substrates, using subtractive measures described in preceding paragraphs. A molecular genetics-based experimental approach is the most significant contribution that the Drosophila model brings to the study of FMRP function.
Rationale for these studies To fully uncover the processes in which a gene participates and the mechanisms of its function, a combined approach of genetics, biochemistry, and cell biology is required. Many of the best- characterized genes were first identified in organisms amenable to genetic analysis, but the fragile X gene is not among these. As a result, models and suppositions regarding FMRP functions have been put forth that have yet to be tested in an intact animal system. Moreover, FMRP is a complex protein with several RNA binding and protein-protein interaction domains. The contribution of these domains to FMRP function in the context of an animal model has not been examined. The biologically and medically relevant phenotypes associated with the Drosophila fragile X model, coupled with the advantages of Drosophila molecular genetic technologies, justify the fly model as a platform to begin addressing these issues.
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21
Tudor/Agenet related KH domains RGG box Q/N rich
Protein-protein interaction
85% identity/similarity 63% identity/similarity
Figure 1: Schematic representation of the Drosophila melanogaster fragile X mental retardation protein. The N-terminal 220 amino acids, KH domains and the RGG box have RNA binding capacity. Several protein-protein interactions map within FMRPs. There is about 63% and 85% protein sequence identity/similarity in the N-terminal region and the KH domains respectively, between Drosophila and vertebrate FMRPs. The C-terminal region of dFMR1 is enriched in glutamine and asparagine (Q/N) residues.
22 Table 1 FMRP interacting Cellular localization Possible function Isolation method/ protein with FMRP validation FXR1P Nucleus and cytoplasm Translation regulation? Yeast two-hybrid and in vitro binding assays FXR2P Nucleus and cytoplasm Translation regulation? Yeast two-hybrid, in vivo studies and in vitro binding assays NUFIP1 Nucleus Not known Yeast two-hybrid
CYFIP1 Cytoplasm Maintenance of neural Yeast two-hybrid, in vitro structures through binding assays, co-localization interaction with Rac1
CYFIP2 Cytoplasm Not known Sequence homology to CYFIP1, Yeast two-hybrid, in vitro binding assays, co-localization 82-FIP Nucleus and cytoplasm Not known Yeast two-hybrid
Nucleolin Nucleus Not known Co-immunoprecipitation
YB1/p50 Nucleus Not known Co-immunoprecipitation
Staufen Cytoplasm mRNA transport and Co-immunoprecipitation regulation of translation. TAP-technology Present in somatic P Bodies
PURα Cytoplasm mRNA transport and Co-immunoprecipitation regulation of translation PURβ Cytoplasm mRNA transport and Co-immunoprecipitation regulation of translation Myosin VA Cytoplasm Dendritic RNA transport Co-immunoprecipitation
Ran BPM Nucleus and cytoplasm Not known Yeast two-hybrid, in vitro binding assays, co-localization elF2C2 Nucleus and cytoplasm Translation regulation? Co-immunoprecipitation
Dicer Nucleus and cytoplasm microRNA based Co-immunoprecipitation mechanisms for translational regulation
PABP1 Cytoplasm Not known TAP-technology immunoprecipitation Kinesin Cytoplasm Dendritic transport TAP-technology immunoprecipitation Dynein Cytoplasm Dendritic transport TAP-technology
23 CHAPTER II
Substitution of Critical Isoleucines in the KH Domains of Drosophila Fragile X Protein Result in Partial Loss of Function Phenotypes1
ABSTRACT Fragile X mental retardation proteins (FMRP) are RNA binding proteins that interact with a subset of cellular RNAs. Several RNA binding domains have been identified in FMRP, but the contribution of these individual domains to FMRP function in an animal model is not well understood. In this study, we have generated flies with point mutations in the KH domains of the Drosophila melanogaster fragile X gene (dfmr1) in the context of a genomic rescue fragment. The substitutions of conserved isoleucine residues within the KH domains with asparagine are thought to impair binding of RNA substrates, and perhaps the ability of FMRP to assemble into mRNP complexes. The mutants were analyzed for defects in development and behavior that are associated with deletion null alleles of dfmr1. We find that these KH domain mutations result in partial loss of function or no significant loss of function for the phenotypes assayed. The phenotypes resulting from these KH domain mutants imply that the capacities of the mutant proteins to bind RNA and form functional mRNP complexes are not wholly disrupted, and are consistent with biochemical models suggesting that RNA binding domains of FMRP can function independently.
INTRODUCTION The fragile X mental retardation protein (FMRP) is an RNA-binding protein necessary for normal neuronal development and behavior in all species where its function has been examined. A general model for FMRP function is that it regulates nucleocytoplasmic transport, subcellular localization, and translation of select RNA transcripts (reviewed by Bardoni and Mandel 2002; Jin and Warren 2003; Jin et al. 2004a; Bagni and Greenough 2005). Biochemical analyses have uncovered several RNA binding motifs associated with FMRP function, including two KH domains (hnRNP-K homology), and an arginine and glycine-rich motif (RGG box) that are
1 This chapter is adapted from Banerjee et al., Genetics 175: 1241-1250 (2007). The Genetics Society of America relinquishes copyright permission for thesis and dissertation composition and publishing. 24 common to RNA binding proteins (Ashley et al. 1993; Siomi et al. 1993). The highly conserved N-termini of FMRPs have RNA binding capacity as well (Adinolfi et al. 1999; 2003). The N- terminal 110 amino acids of FMRPs are similar to Tudor/Agenet domains and are members of an extended family that is referred to as the Tudor domain “Royal Family” (Maurer-Stroh et al. 2003). This domain family is related to methyl-substrate binding proteins that are implicated in regulation of chromatin structure, and includes the chromodomain.
RNA substrates for FMRP have conserved elements in primary sequence and/or higher order structures that interact with the aforementioned RNA binding domains. A G-quartet structure within RNA interacts with the RGG box (Darnell et al. 2001; Schaeffer et al. 2001), and the second KH domain recognizes a loop-loop pseudoknot RNA structure referred to as a kissing complex (Darnell et al. 2005). A stem-loop structure within BC1 RNA is reported to interact specifically with the N-terminal 217 amino acids of FMRP (Zalfa et al. 2005; but see Wang et al. 2005 for a contrary view). These studies demonstrate that individual RNA-binding domains of FMRP have distinct substrates with which they interact and that the ability of these domains to bind substrates is not dependent upon other FMRP RNA binding domains. Loss of function of any of these domains might then result in only a subset of RNA substrates losing the ability to bind FMRP.
Expansion of a CGG trinucleotide repeat in the 5′ UTR of the FMR1 gene, followed by methylation and transcriptional silencing is the basis for the vast majority of fragile X cases in humans (see O’Donnell and Warren, 2002 for a review of human fragile X inheritance patterns). Other alleles result from deletions or nonsense codons, and thus little structure-function information has been obtained from analysis of human FMR1 alleles. One significant exception is the substitution of a highly conserved isoleucine residue in KH domains to asparagine (I304N) within the second KH domain of human FMRP that is associated with unusually severe fragile X phenotypes (De Boulle et al. 1993). Until recently, models to explain the effects of the I304N substitution have been enigmatic. Defects in RNA binding have been proposed, based upon finding that the I304N protein is impaired in binding RNA homopolymers under high salt concentrations (Siomi et al. 1994), and the analysis of a co-crystal structure of a KH domain and RNA substrate (Lewis et al. 2000). Although the I304N protein can bind bulk poly(A) RNA
25 (Feng et al. 1997), in contrast to wild type FMRP, it does not associate with polyribosomes (Feng et al. 1997) or with itself (Laggerbauer et al. 2001). These results suggest that the inability to form proper mRNP complexes is a significant factor contributing to the I304N phenotypes, and have prompted suggestions that the severity of phenotypes associated with the mutation arise from dominant negative or antimorphic effects (Feng et al. 1997). The above biochemical studies have been reconciled by the finding that the second KH domain of FMRP binds kissing complex RNAs, the I304N substitution abolishes this association, and that kissing complex RNAs can compete FMRP off polyribosomes (Darnell et al. 2005). These results imply that FMRP association with polyribosomes is dependent upon an interaction of the second KH domain with RNAs containing a kissing complex structure.
Many studies demonstrate that the Drosophila fragile X protein shares biochemical functions with its vertebrate counterparts and regulates similar neural and behavioral functions (reviewed by Gao 2002; Jin and Warren 2003; Dölen and Bear 2005; Zhang and Broadie 2005). Previously existing alleles of dfmr1 are strong or null alleles resulting from imprecise P element excisions, or nonsense codons (Zhang et al. 2001; Dockendorff et al. 2002; Lee et al. 2003). Although KH domain mutations exist for dfmr1 as cDNA constructs, the misexpression or overexpression of these alleles in neural and muscle tissues via the GAL4-UAS system results in physiologic abnormalities or cell death (Wan et al. 2000; Zhang et al. 2001). Starting with a genomic rescue fragment encompassing dfmr1, we have created derivatives of this rescue fragment where conserved isoleucine residues in the KH domains have been mutated as a means to assess the importance of these domains in an animal model. These P element-borne transgenes have been recombined onto a chromosome deleted for dfmr1 to produce animals that express only mutant forms of dFMR1 protein. Our analyses of flies with the KH domain mutations show that they result in either partial or no loss of function on the behavior and developmental phenotypes that were examined. These findings show that the mutant proteins retain a significant degree of function in vivo and are consistent with biochemical models that predict FMRP RNA binding domains having some independent functions. These alleles of dfmr1 will be useful tools for both genetic and biochemical screens to identify RNAs and proteins that interact with fragile X protein.
26 MATERIALS AND METHODS Generation of KH domain mutants A subclone of a 14 kb BamH1-Stu1 genomic fragment spanning the dfmr1 locus was subjected to site-directed mutagenesis via the mega-primer technique, using a proof-reading polymerase (methods compiled in Sambrook and Russell 2001). All PCR-amplified fragments were sequenced to confirm the presence of the desired mutation and the absence of secondary mutations resulting from base misincorporations during amplification. Mutant DNA was then substituted for the corresponding wild-type fragment and the resulting mutant rescue fragments were cloned into pCaSpeR-4 (Pirrotta 1988) for subsequent transformation. Transformations were done using conditions described in Spradling and Rubin (1982). We found the
transformation efficiency to be very low, with one transformed fly appearing in about 300 G0 crosses for each of the mutant transgenes. Both mutant transgenes mapped to the third chromosome, and thus the transgene insertions were recombined onto a chromosome harboring the dfmr13 allele, which removes the dfmr1 open reading frame (Dockendorff et al. 2002; Pan et al. 2004). These transgenes were judged to map within two recombination units of the endogenous dfmr1 locus based upon the frequency with which recombination of the two loci occurred. The resulting stocks are of the following genotypes: P[dfmr1I244N]w+ dfmr13/TM6C Tb Sb and P[dfmr1I307N]w+ dfmr13/TM6C Tb Sb. These recombinant stocks were then crossed to flies with the dfmr13 allele to produce animals heterozygous for the transgene insertion and homozygous for the dfmr13 allele. Thus, the only dFMR1 protein produced in such animals is from the mutant allele. For clarity, these stocks will be referred to simply by the nature of the KH domain substitution throughout the text and figures. To test for effects of an increased dosage, we crossed the stocks with the transgenes recombined onto the dfmr13 null chromosome to a stock expressing the Δ2-3 transposase. Flies were selected that had enhanced expression of the mini-white marker and of mutant dFMR1 protein, indicative of a replicative transposition event. For both the transgenes, the second copy of the insertion mapped to the third chromosome, and these stocks were balanced using TM6C Tb Sb. To differentiate these stocks from those with the single copy of the transgene, we refer to them in figures and text with the suffix “2X”.
27 Fly stocks, genetics, and culture All dfmr1 mutant stocks in this study were derived from a w1118 background and maintained on a yeast-cornmeal-molasses medium at 25°C. The third chromosome balancer TM6C Tb Sb was used to maintain dfmr1 alleles.
Courtship and circadian behavior analyses For courtship behavior testing, males of the appropriate genotypes were collected within two hours of eclosion and kept in isolation prior to testing. Female targets were of the genotype XX, y, f (attached X) and collected as virgins for courtship testing. All flies were kept in 12:12 light/dark cycles at 25° and 70-75% relative humidity, and were aged four days prior to analysis. For the naïve courtship analysis, the four-day old male and female were transferred via aspiration to a mating chamber 20 mm in diameter and 5 mm deep. These chambers were kept in humidified conditions throughout the assay. Transferred males were given a five-minute recovery period prior to addition of the female target. All assays were performed within 30 minutes of the change in light cycle. Males were monitored for courtship activity that included following of the female, wing extension and vibration, tapping of female with his foreleg and attempted copulation for a period of 10 minutes, or until copulation occurred. The percentage of time the male spent in active pursuit of the female was recorded as the courtship index. A minimum of 25 animals was tested for each genotype.
Circadian behavior was tested as described in (Dockendorff et al. 2002). Flies were entrained to a 12:12 light/dark cycle, placed into activity monitors (Trikinetics, Waltham, MA) maintained in light/dark cycles, and then placed under constant darkness. Locomotion activity was collected in 30-minute bins. The percentage of flies judged to be rhythmic was assessed by Clocklab software (Actimetrics, Evanston, IL) as follows: Using a confidence level of 0.025, batch analyses were performed for the genotypes tested, monitoring the locomotion activity in constant darkness over seven days. The difference between the power(1) and significance(1) values was calculated for each fly, and a value of less than 10 was the basis for judging an arrhythmic phenotype. Visual analyses of periodograms and actograms were also conducted to confirm the results.
28 Antibodies and immunocytochemistry Larval neuromuscular junction (NMJ) type I boutons were detected by staining third-instar larval fillets with anti-horseradish peroxidase (Cappel, Aurora, OH) at a dilution of 1:200. Mushroom bodies were visualized by staining whole-mounts of brains with anti-FasII at a 1:10 dilution (mAb 1D4 obtained from University of Iowa Developmental Studies Hybridoma Bank). Secondary antibodies conjugated to either HRP or fluorochrome were obtained from Jackson ImmunoResearch (West Grove, PA) and used at a 1:200 dilution. Confocal images were collected on an Olympus FV500 microscope. Western blots were performed as described in Wan et al. (2000) using anti-dFMR1 antibody 5A11 at a 1:1000 dilution and anti-β-tubulin mAb E7 (both from the University of Iowa Developmental Studies Hybridoma Bank) at a 1:400 dilution.
Statistical analyses Courtship indices were arcsin transformed, then analyzed by one-way ANOVA, followed by a Tukey-Kramer post-test. NMJ bouton counts were analyzed by one-way ANOVA with a Tukey- Kramer post-test, or by a Kruskal-Wallis test, followed by a Dunn post-test. The analyses of courtship indices and NMJ bouton counts were conducted using InStat software from GraphPad (San Diego, CA). Comparisons of mushroom body axon midline crossings and circadian rhythmicity were made by a chi-square test for homogeneity.
RESULTS Generation of point mutations in KH domains of dfmr1 A 14 kb genomic rescue fragment has previously been shown to rescue all known behavioral and developmental phenotypes associated with dfmr1 loss-of-function (Dockendorff et al. 2002; Lee et al. 2003; Michel et al. 2004; Costa et al. 2005). Subclones of this fragment were subjected to site-directed mutagenesis to convert highly conserved isoleucine residues in the two KH domains to asparagines (I244N, I307N for the Drosophila protein; Figure 1A). These substitutions are predicted to strongly inhibit binding of cognate RNA substrates (Lewis et al. 2000; Darnell et al. 2005), and may interfere with folding of the KH domain (Musco et al. 1996, 1997). Upon reconstruction of mutated sequences to the rescue fragment, the mutant rescue fragments were introduced to flies via P element transformation and then recombined to chromosomes harboring
29 the dfmr13 allele, a deletion null allele where the entire open reading frame of dfmr1 is removed (Dockendorff et al. 2002; Pan et al. 2004). Crossing such chromosomes to the dfmr13 chromosome results in flies heterozygous for the P element transgenes, and that express only the mutant alleles under control of the endogenous dfmr1 promoter. Since it might be expected that an I244N I307N double mutant could have an additive effect on any phenotypes observed with the single KH domain mutants, we attempted to obtain stocks with such an allele. Despite extensive efforts, we failed to obtain transgenic animals that harbored an I244N I307N double mutation of dfmr1 that was expressed via its endogenous promoter even in an otherwise wild type background. Although UAS-GAL4 overexpression of a dfmr1 cDNA with the I244N I307N double mutation in the developing eye fails to induce a rough eye phenotype (Wan et al. 2000), we have observed that overexpression of the same transgene by myosin heavy chain-GAL4 can be lethal to pupae, indicating that such an allele can have dominant deleterious effects in specific tissues (TCD unpublished observations).
To discern the level of dFMR1 protein expression from the transgenes, Western blotting was performed on male flies harboring the mutant KH domain transgenes as the sole allele of dfmr1. These studies show that flies with a single copy of the transgene with the I244N allele express the mutant protein at a level very similar to that seen with a dfmr1 heterozygote. The single copy of the I307N transgene had about 25% the expression of dFMR1 protein present in a control w1118 background (Figure 1B). To create stocks with increased doses of each transgene, the P elements were mobilized and stocks that had undergone replicative transposition resulting in elevated levels of the mutant proteins were selected. These stocks were judged to express mutant proteins at 97% and 62% of the level seen with w1118 for the I244N and I307N substitutions respectively (Figure 1B). To control for dosage effects, dfmr1 heterozygotes are included in all following analyses. Since the human I304N substitution has been hypothesized to exert dominant effects, we examined our KH domain allele stocks for dominant phenotypes through analysis of flies that expressed both a mutant and wild type allele of dfmr1. These stocks are noted in Figures with both the KH domain allele designation and a “+” for the wild type allele.
30 The NMJ bouton overgrowth phenotype associated with null alleles of dfmr1 is not observed with the I244N or I307N KH domain substitutions Strong or null alleles of dfmr1 result in an overgrowth of larval neuromuscular junction boutons (Zhang et al. 2001; Jin et al. 2004b). To assess the impact of the KH domain mutations on this phenotype, we analyzed the numbers of type I boutons at larval NMJs using flies with wild type and null alleles of dfmr1 as controls. We examined muscles 4, 6/7 and 12 from segment A3 for the analysis of larval NMJs. Results in Figure 2 show that in all muscles examined, there is no significant increase in bouton number over wild type or dfmr1 heterozygote controls from larvae with a single copy of a transgene expressing either the I244N or I307N substitutions as the sole source of dFMR1 protein, while there is the expected pronounced overgrowth of boutons from larvae homozygous for the null allele of dfmr1. These results indicate that neither of the Ile→Asn substitutions in the KH domains affects the ability of dFMR1 to regulate larval NMJ bouton numbers, and thus suggests that other domains of FMRP play a more vital role in this process.
Analysis of midline crossing frequency in mushroom body β-lobe neurons Axon development defects have been reported in the central nervous system of flies homozygous for null alleles of dfmr1. The ventral lateral neurons, dorsal cluster neurons, and neurons of the mushroom body all have visible defects in branching, neurite extension, and/or guidance (Dockendorff et al. 2002; Morales et al. 2002; Michel et al. 2004; Pan et al. 2004; Reeve et al. 2005). A significant fraction of flies homozygous for strong or null alleles of dfmr1 have midline crossings of mushroom body (MB) β-lobe neurons (Michel et al. 2004). To monitor the effects of the KH domain alleles on axon development, we used the mushroom body β-lobe phenotype described by Michel et al. (2004), visualizing MBs in whole brain mounts from 2-day old animals via anti-FasII immunostaining. The results in Figure 3 show that when compared to a wild type allele control, flies expressing dFMR1 with the I244N or I307N substitution had a significant increase in the frequency of midline crossings compared to controls where a wild type allele of dfmr1 was present, but this frequency was not as great as that observed in brains from flies homozygous for a null allele of dfmr1 (Figure 3). Thus, the KH domains of dFMR1 play a role in regulating processes that contribute to normal axon development, and the other dFMR1 domains must contribute functions as well. We also examined the midline crossing phenotype in
31 brains from flies where the dosage of either KH domain allele was increased. The frequency of midline crossings did not change significantly from what was observed with the single dose, indicating that the Ile→Asn substitutions have a strong effect on the function of the KH domains, and that other domains of dFMR1 are not able to compensate for the defects.
The KH domain mutations display a partially penetrant circadian phenotype in constant darkness Flies with null alleles of dfmr1 fail to retain circadian rhythmicity when transferred into constant darkness (DD) with about 80% penetrance (Dockendorff et al. 2002; Morales et al. 2002; Inoue et al. 2002). The KH domain mutants had circadian behavior examined by monitoring rest/activity rhythms in both LD and DD. Flies with either mutant allele are capable of responding to light, as judged by their rhythmic locomotion activity (T. Dockendorff, J. Park, unpublished observations). As was seen with the MB β-lobe phenotype, both KH domain substitutions resulted in a statistically significant increase in the percentage of flies that fail to retain rhythmic locomotion activity in constant darkness when compared to flies with a wild type allele of dfmr1 (Figure 4). Likewise, the percentage of KH domain mutants lacking rhythmic activity is not as great as observed for flies homozygous for the dfmr13 null allele, demonstrating that the KH domain alleles result in partial loss of function. For mutant flies determined to have retained rhythmic locomotion activity in constant darkness, the period of such flies did not significantly differ from flies with a wild type dfmr1 allele (not shown). Flies expressing both the mutant transgene allele and a wild type allele of dfmr1 do not differ from wild type controls in the percentage of animals judged to have retained rhythmic locomotion, demonstrating that the insertions and mutant alleles have no dominant effect. An increase in dosage of either KH domain allele had no effect on the percentage of flies judged to be arrhythmic, again demonstrating a strong loss of function effect on the KH domains, and the inability of other dFMR1 domains to provide compensatory function for this phenotype.
Naïve courtship activity is reduced in flies with either KH domain mutation Courtship in Drosophila consists of stereotyped behaviors by males towards receptive females (reviewed by Hall 1994; Greenspan and Ferveur 2000). The courtship process has thus been used as an ethologically relevant measure of Drosophila behavior. Flies with a null allele of
32 dfmr1 have deficits in naïve courtship activity (Dockendorff et al. 2002), which is measured as the percentage of time a male fly spends in courtship activity towards a receptive virgin female during a given period of time, and defined as the courtship index (CI). We analyzed the naïve courtship activity of flies with the KH domain alleles, measuring the time spent by the male in following the female target, wing extension and vibration, tapping with foreleg, and attempted copulation (Figure 5). Flies that harbor both a copy of the transgene bearing either of the KH domain alleles and a wild type allele of dfmr1 do not significantly differ from a wild type control in the amount of time engaged in courtship behavior, showing that these insertions and alleles do not result in any dominant effect that contributes to the phenotype (Figure 5A, B). Our analyses of courtship activity in flies where the KH domain allele is the only source of dFMR1 show that the Ile→Asn substitutions in either KH domain has a significant adverse effect on the courtship index when compared to flies with a wild type allele. The values for the courtship index of both KH domain mutants are also significantly different from those seen with the null allele of dfmr1, indicating that dFMR1 proteins with the KH domain point mutations still have activity and are behaving as partial loss of function alleles. An increase in the dosage of either KH domain allele results only in a small, statistically insignificant increase in the courtship index. This increase could be accounted for by elevated expression of the mini white marker associated with the second copy of the P element vector, since the white gene is known to positively influence courtship activity (reviewed by Hall, 1994). Thus, as was seen with the morphology of mushroom bodies and circadian locomotion, an increased dosage of the mutant KH domain alleles did not provide rescue of this phenotype.
DISCUSSION The vast majority of human fragile X cases arise through the expansion of a trinucleotide repeat, resulting in silencing of the FMR1 gene. Thus, relatively little insight into structure-function relationships have been gained from analysis of human FMR1 alleles. Biochemical and cell culture-based studies of fragile X proteins have uncovered several RNA binding domains that likely contribute to their in vivo function. The amenability of D. melanogaster to transgenics provides an opportunity to conduct structure-function studies of FMRP in the context of an intact animal. To this end, we have generated flies expressing dfmr1 alleles where a codon for a highly
33 conserved isoleucine residue in each of the two KH domains was mutated to code for asparagine. These mutations are predicted to result in a strong loss of affinity for specific RNA ligands as judged from structural (Lewis et al. 2000; Ramos et al. 2003) and biochemical studies (Darnell et al. 2005), and may interfere with the ability of FMRP to interact with other proteins as well (Feng et al. 1997; Laggerbauer et al. 2001; Ramos et al. 2003). We then examined the effects of these mutations on neural development and behavior phenotypes that are associated with null alleles of dfmr1. Several conclusions can be made from these results. Neither of the KH domain alleles produced a phenotype that matched the degree of severity seen with the dfmr1 deletion null allele. For all phenotypes analyzed, the KH domain alleles were recessive to the wild type dfmr1 allele. The failure of increased dosage of the mutant proteins to provide any significant measure of rescue indicates that the Ile→Asn substitutions are strong loss of function mutations in the KH domains, which is consistent with past biochemical and biophysical analyses of KH domains (Lewis et al. 2000; Ramos et al. 2003; Darnell et al. 2005). These results suggest that the ability of the mutant proteins to bind certain RNA species in vivo is lost, and that other RNA binding domains of dFMR1 cannot compensate for the defect. Prior studies have shown that the individual FMRP RNA binding domains can bind RNA as a discreet unit in vitro (Adinolfi et al. 1999; Darnell et al. 2001; Schaeffer et al. 2001; Darnell et al. 2005), and our results are consistent with the observations from these studies. The partial loss of function phenotypes resulting from these KH domain alleles demonstrates that the mutant proteins retain function and must be able to bind RNA and assemble into at least some mRNP complexes in vivo.
KH domain phenotypes and roles for other RNA-binding domains of FMRPs Why the difference between the lack of phenotype seen with the larval NMJ bouton numbers and the partial loss of function observed for other phenotypes analyzed? It is possible that regulation of a different subset of RNAs is involved in larval NMJ bouton development and that these RNAs are not reliant upon interaction with dFMR1 KH domains to conduct their functions and be appropriately regulated. A number of possibilities can explain the partial loss of function phenotypes associated with mushroom body development, courtship behavior, and circadian locomotion activity. It could be that dFMR1 regulates the activity of multiple genes involved in these processes, and that the KH domains are responsible for modulating the activity of a subset of these genes. Multiple RNA binding domains in dFMR1 could also regulate the activity of any
34 one transcript, and loss of KH domain function could lead to a partial degree of misregulation for such transcripts that result in the degree of phenotype observed.
That we did not observe a null phenotype with either of the KH domain alleles makes it rather probable that the other RNA binding domains of FMRP have a significant contribution to its in vivo function. Along with the G-quartet binding RGG box, the N-terminal 217 amino acids of human FMRP also binds RNA (Adinolfi et al. 1999, 2003; Zalfa et al. 2005). The N-terminal amino acids of FMRP are well conserved between mammals and insects (Wan et al. 2000) and are related to methyl-substrate binding domains that are associated with chromatin regulation (Maurer-Stroh et al. 2003). This is of interest because FMRP can be detected in the nucleus and has biochemical and genetic interactions with Argonaute proteins (Verheij et al. 1993; Caudy et al. 2002; Ishizuka et al. 2002; Jin et al. 2004b; Xu et al. 2004). Mutations of RNAi components affect silencing of heterochromatin in D. melanogaster (Pal-Bhadra et al. 2004; see Lippman and Martienssen, 2004; Matzke and Birchler, 2005; Wassenegger, 2005 for reviews on RNAi-based regulation of chromatin). Indeed, it has recently been reported that a null allele of dfmr1affects white gene expression in centromeric heterochromatin (Deshpande et al. 2006). If FMRP is part of a complex that modulates chromatin structure via RNAi-based mechanisms, the loss of such activity via a null mutation could conceivably affect the expression of many genes that might be part of the fragile X pathway, including those that may contribute to the phenotypes examined in this study.
Comparing phenotypes of the human I304N and Drosophila I307N substitutions The failure of the I304N protein to bind certain RNAs and its abnormal mRNP fractionation profile have been suggested to be the basis for the unusually severe fragile X phenotypes observed with a patient expressing the I304N substitution (Feng et al. 1997; Darnell et al. 2005), and have been hypothesized to arise from a dominant effect exerted by the I304N protein (Feng et al. 1997). The studies here provide an opportunity to make comparisons between the human I304N and Drosophila I307N phenotypes. Given the severity of fragile X phenotypes associated with the I304N substitution, our findings of relatively modest phenotypes associated with the analogous I307N substitution in the Drosophila model may seem surprising. The failure to observe a phenotype with the I307N protein that matches the strength of a dfmr1 null allele
35 means that the I307N protein must be able to bind RNA and assemble into at least a subset of the mRNP complexes that the wild type protein does. We feel that several hypotheses are plausible to explain the differences observed between the human I304N and Drosophila I307N phenotypes. Subtle differences in substrate binding by these KH domains is one possibility, with the human I304N protein unable to bind certain critical RNA substrates, while the binding of orthologous substrates (if they exist) to the Drosophila I307N protein is impaired to a lesser degree. In vertebrates, FMRP interacts with FXR1 and FXR2 proteins that are not present in D. melanogaster. Since the I304N protein can interact with both FXR1 and FXR2 in vitro (Laggerbauer et al. 2001; Mazroui et al. 2003), it is possible that I304N FMRP could induce a deleterious gain of function effect on either FXR protein that enhances the severity of the phenotype. Alternatively, since not all I304N protein co-fractionates with FXR2 (Feng et al. 1997), the I304N protein may acquire a deleterious function when not in complex with FXR2. Another consideration is the possibility of unusual contributions from the genetic background of the I304N individual that could enhance his fragile X phenotypes. To recapitulate the I304N substitution in the mouse model could be helpful in discerning whether these last two hypotheses have validity. Finally, none of the above explanations are mutually exclusive, and thus any combination of these scenarios might contribute to the severity of the fragile X phenotypes observed with the I304N patient.
The KH domain alleles as probes for FMRP functions What RNAs do fragile X proteins bind to? What is the composition of a fragile X protein- containing mRNP particle? These are questions that still dominate research on FMRP. Biochemical screens utilizing microarrays, along with yeast two-hybrid screens and proteomics approaches, have been undertaken to address these questions (Ceman et al. 1999; Brown et al. 2001; Darnell et al. 2001; Schenck et al. 2001; Ishizuka et al. 2002; Miyashiro et al. 2003; Costa et al. 2005; Zarnescu et al. 2005), and have uncovered several interacting proteins and dozens of RNA species identified as candidate ligands. In vivo genetics-based analyses of these interactions will be necessary for their final validation. Drosophila has proven to be a significant model for study of fragile X protein function owing to the similarities with vertebrate models in biochemical properties and loss-of-function phenotypes (Wan et al. 2000; Zhang et al. 2001; Dockendorff et al. 2002; McBride et al. 2005). Thus, the tools of Drosophila genetics will be
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42
A
1 KH1 KH2 RGG 681
Tudor/Agenet-related I244N KH2
KH1 I307N
B dfmr13 1118 w + I244N I244N 2X I307N I307N 2X
dFMR1
β-tubulin
100 55 55 96 24 62
Figure 1: Schematic of fragile X protein and expression analysis of dfmr1 KH domain alleles. (A) RNA binding domains of FMRP. Two KH domains, an RGG box, and two tandem copies of a Tudor/Agenet-related domain have all been demonstrated to bind RNA. KH domains with conserved isoleucine residues mutated to asparagine produced for this study are depicted below. (B) Western blot of total fly extracts from wild type, dfmr13 heterozygote, and flies expressing one or two (2X) copies of a transgene harboring a dfmr1 genomic rescue fragment coding for either an I244N or I307N substitution in the KH domains. Extracts were prepared from males aged 2-3 days. Signals were scanned and quantified by ImageQuant software (Molecular Dynamics), and average expression levels compared to a w1118 control from six independent blots are given.
43 A Muscle 6/7 60.0 * 50 .0
40.0
30.0
20.0 Numberboutons of 10 . 0
0.0 1118 3 wW 1118dfmr1 d f mr 13 / +/+ Null N ul lI244N I 2 4 4 N I307N I 3 0 7N w1118 dfmr13/+ Null I244N I307N B Muscle 12 50.0 Muscle 12
40.0 **
30.0
20.0
Numberboutons of 10 . 0
0.0 1118 3 wW 1118dfmr1 d f mr 13 / +/+ Null N ul lI244N I 2 4 4 N I307N I 3 0 7N
C Muscle 4 50.0 ** 40.0
30.0
20.0
Numberboutons of 10 . 0
0.0 1118 3 wW1118W 1118dfmr1 ddfmr13/+ f mr 13 / +/+ Null NNull ul lI244N II244N 2 4 4 N I307N II307N 3 0 7N
Figure 2: Numbers of larval neuromuscular junction (NMJ) boutons are not increased in flies expressing KH domain I244N or I307N substitutions as a sole source of dFMR1 protein. Third- instar larvae were dissected and probed with antibodies against horseradish peroxidase to assess numbers of type I NMJ boutons, which are increased in flies homozygous for strong or null alleles of dfmr1 (Zhang et al. 2001; Jin et al. 2004b). Analyses of several muscle types from segment A3 show that type I boutons are significantly increased in all muscle types examined from animals homozygous for a null allele of dfmr1 compared to all wild type and KH domain alleles examined (p < 0.001 for muscle 4, Kruskal-Wallis test and Dunn post-test; p < 0.01 for
44 muscle 6/7, one-way ANOVA, followed by a Tukey-Kramer post-test; p < 0.001 for muscle 12, Kruskal-Wallis test and Dunn post-test). There are no significant changes in type I bouton numbers when wild type controls are compared with either of the two KH domain mutants. The allele designations denote the sole source of dFMR1 protein. Results are from analysis of at least 20 hemisegments for each genotype.
45 A Non crossing over 100 1 Crossing over
90 2
80
70
60
50
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30 3 Frequency of midline crossing phenotype crossing midline of Frequency 20
10
0 w1118 dfmr13 I244N(2X)/+ I307N(2X)/+ Null I244N I244N(2X) I307N I307N(2X) N = 51 N = 27 N = 36 N = 33 N = 53 N = 23 N = 59 N = 35 N = 63 BCNon-crossing mild αα
NC β mild β DE α α
severe β moderate β
Figure 3: Mushroom body (MB) β-lobe phenotypes of flies with dfmr1 KH domain alleles. (A) Graphical representation of the frequency with which a midline crossing of β-lobe neurons was observed. Anti-FasII staining of mushroom bodies (MBs) from 2-day old flies shows that the I244N and I307N substitutions result in a frequency of midline crossing phenotypes intermediate to flies with a wild type allele of dfmr1 and to those homozygous for a dfmr1 null allele. The genotypes listed denote the allele of dfmr1 being expressed, while the presence of a wild type allele to test for dominant effects of the mutant allele is denoted by a “+”. The frequency with which a no crossing phenotype occurred did not change upon an increase in dosage of either mutant KH domain protein. Expression of either mutant KH domain transgene in a background 46 with a wild type copy of dfmr1 present has no effect on midline crossing frequency, indicating that the transgene insertions and mutant proteins do not elicit a detectable dominant effect. Genotypes grouped under a common numerical designation do not differ from each other in percentage of brains observed with a midline crossing of β-lobe axons, while those under different numerical designations differ from each other as judged by chi-square tests of homogeneity. KH domain alleles differ from the null allele in frequency of midline crossing (p < 0.0001) and from flies with a wild type allele of dfmr1 (p = 0.0152). (B-E) Representative examples of MB morphology illustrating the variety of midline crossing phenotypes observed. Alpha and beta lobes are noted, while arrows point to the midline where crossovers of the β-lobe neurons may occur. NC = no crossover.
47 A Arrhythmic 100 Rhythmic
90 C 80 B
70
60
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40 Percent rhythmic 30
20
10
0 1118 W1118w I244N I244N/+/+ I307N I307N/+/+ Null I244NI244N I244N I244N(2X) 2X I307NI307N I307N I307N(2X) 2X N = 44 N = 21 N = 21 N = 39 N = 29 N = 48 N = 58 N = 50
Figure 4: Analysis of circadian locomotion activity of flies expressing dFMR1 with mutant KH domains in constant darkness. An assignment of rhythmic versus arrhythmic activity for individual flies was determined using ClockLab software as described in Materials and Methods. The percentage of flies from each genotype judged to be rhythmic was compared by a chi-square test for homogeneity. Genotypes that are grouped by a common number do not have any significant difference between them in the percentage of animals displaying a rhythmic locomotion phenotype, while separate groups differ to a confidence level of less than 0.0001. Increasing the dose of mutant dFMR1 protein had no significant effect on the percentage of animals judged to have maintained rhythmic locomotion activity, indicating that other RNA binding domains of the mutant proteins are unable to compensate for the defects in the KH domains. The genotypes listed denote the allele of dfmr1 that is the sole source of dFMR1 protein, while the presence of a wild type allele to test for dominant effects of the mutant allele is denoted by a “+”. Flies that express both a mutant and wild type allele of dfmr1 resemble wild type flies in the percentage of animals judged to be rhythmic, demonstrating that the mutant allele and transgene insertion do not have a detectable dominant effect
48 A 1
45 40 35 2 30 25 20 15
Courtship index 3 10 5 0 ww 11181118 I244N/+I244N/+ dfmr1[3]/+dfmr13/+ NULL Null I244N I244N(2X) I244N(2X)
B 1
45 40 35 2 30 25 20 15
Courtship index 3 10 5 0 ww 11181118 I307N/+I307N/+ dfmr1[3]/+dfmr13/+ NULL Null I307NI307N I307N(2X) I307N(2X)
Figure 5: Analysis of naïve courtship activity of flies expressing dFMR1 KH domain mutations. Naïve courtship was analyzed as described in Materials and Methods. At least 25 flies of each genotype were tested. For each mutant KH domain transgene, expression in a background with a wild type allele of dfmr1 does not result in a phenotype differing from wild type, indicating that the transgene insertion and mutant protein do not induce a detectable dominant effect. (A) Flies expressing dFMR1 with the I244N substitution as the sole source of dFMR1 protein have a significant decrease in naïve courtship activity compared to flies with a wild type allele of dfmr1, but the decrease in courtship activity is not as strong as is observed in flies homozygous for a dfmr1 null allele. Increasing the dosage of the I244N allele does not result in a significant increase in courtship activity. Courtship indices were arcsin transformed and the data was analyzed by a one-way ANOVA, followed by a Tukey-Kramer post-test. The p value for the ANOVA is less than 0.0001. Genotypes under the same numerical heading do not vary from each other to a significant extent, while genotypes under different numerical headings are
49 significantly different from each other (p is less than 0.05). (B) The I307N substitution results in a significant decrease in naïve courtship activity compared to flies expressing wild type dFMR1, but not to the degree observed with flies homozygous for a null allele of dfmr1. As was seen with the I244N allele, an increase in dosage of the I307N allele does not produce a significant increase in naïve courtship activity. The date for these genotypes was processed in the same manner as the I244N flies, and the p value for the ANOVA is less than 0.0001. Genotypes under the same numerical grouping do not differ from each other in courtship activity to a significant extent; while genotypes under different numerical groupings have a significant variation in the courtship index (p is less than 0.01).
50 CHAPTER III
A Glutamine/Asparagine-Rich Domain in Drosophila Fragile X Protein is Essential for Regulating Behavioral Plasticity
SUMMARY Biochemical and morphological changes that modulate synaptic connectivity underlies the process of memory. These changes require regulation of de novo protein synthesis that arises in part from local translation at synapses. The Drosophila fragile X protein (dFMR1) has a domain enriched in glutamine/asparagine (Q/N) residues that is related to those associated with fungal prions. Deletion of the dFMR1 Q/N domain shows that while it is not essential for immediate recall, it is needed for one-hour short-term memory of conditioned courtship training. The dFMR1 Q/N domain can aggregate in vivo, suggesting that it may function as a protein interaction module. These results demonstrate a direct role by a Q/N domain in mediating behavioral plasticity, and are consistent with a model where the dFMR1 Q/N domain facilitates interactions with ribonucleoprotein complexes that regulate local translation in response to stimulation.
INTRODUCTION Persistent forms of synaptic plasticity require de novo protein synthesis, with these products contributing towards morphological and biochemical changes of dendritic spines and synapses that modulate their function. Such changes are postulated to be the molecular basis for establishment and consolidation of memory. Since a common soma is shared between multiple dendrites, the specificity of synaptic response to stimulation is achieved by localized translation of mRNAs transported to these compartments (reviewed by Kelleher et al., 2004; Sutton and Schuman, 2006). Studies of human disorders and animal models show that mutations in genes whose products regulate post-synaptic protein synthesis can result in behavioral anomalies and memory deficits.
Much experimental evidence implicates the RNA-binding fragile X mental retardation protein (FMRP) as a regulator of localized protein synthesis. A model for post-synaptic FMRP function
51 is that it regulates translation of localized mRNAs in response to signaling through metabotropic glutamate receptors (mGluRs; reviewed by Bagni and Greenough, 2005). Stimulation of mGluRs induces changes in the phosphorylation state of FMRP that may influence its ability to regulate RNA function (Narayanan et al., 2007). The protein synthesis that arises from mGluR stimulation enhances internalization of AMPA receptors, and results in a depression of the excitatory post-synaptic current (EPSC; Huber et al., 2000; Snyder et al., 2001). Hippocampal slices from FMR1 knockout mice exhibit exaggerated mGluR-induced long-term depression (LTD) that is proposed to upset a balance in synaptic regulation, resulting in behavioral phenotypes of fragile X syndrome (reviewed by Bear et al., 2004). The nature of ribonucleoprotein (RNP) complexes that FMRP associates with to modulate mGluR-LTD and behavioral memory have yet to be established.
A Drosophila model to study FMRP function has been developed, with loss-of-function for dfmr1 leading to neural development and behavior anomalies that have significant parallels with FMR1 mutant phenotypes in fragile X patients and mouse knockouts (reviewed by Gao, 2002; Zhang and Broadie, 2005). The presence of mGluRs in Drosophila, and the rescuing effects of mGluR antagonists on courtship behavior of dfmr1 mutants (McBride et al., 2005) suggest that mGluR stimulation and FMRP activity are coupled in flies as well. In this study, we report on the role of a dFMR1 domain that is enriched in glutamine and asparagine (Q/N) residues. Q/N- rich domains are implicated in facilitating protein-protein interactions, and some are associated with acquisition of prion-like properties (reviewed by Shorter and Lindquist, 2005). The stability of prions has led to a proposal that regulators of long-term memory could be governed by acquisition of a prion state (Si et al., 2003). As has been observed with other similar domains (Patino et al., 1996; Si et al., 2003; Gilks et al., 2004), the Q/N domain of dFMR1 confers protease resistance and facilitates aggregation of proteins to which it is fused. Deletion of the dFMR1 Q/N domain results in defects for a subset of neural development phenotypes associated with a null allele of dfmr1, demonstrating that it is not essential for all dFMR1-regulated processes. While immediate recall and short-term memory of conditioned courtship training are disrupted in dfmr1 null mutants, the Q/N domain mutants are deficient only in short-term memory. This observation indicates that the development of neuronal connections necessary for immediate recall is normal in flies with the dFMR1 Q/N domain deletion. The subsequent defect
52 in short-term memory is consistent with the timing of regulated protein synthesis that is required for mGluR-mediated hippocampal LTD (Huber et al., 2000). The biochemical properties of the dFMR1 Q/N domain indicate that it can facilitate interactions with other proteins. Such interactions are likely necessary for dFMR1 to assemble into ribonucleoprotein (RNP) complexes, and suggests a means by which dFMR1 could serve as part of a synaptic tag.
RESULTS A glutamine/asparagine-rich domain is present in several isoforms of dFMR1 Four isoforms of the Drosophila fragile X protein (dFMR1) are known or predicted based upon genomics analyses (Figure 1A; FlyBase). Two of these isoforms (PA, PC) are closely related, differing by only a few amino acids that appear in the second KH domain of the PA isoform as a result of alternative splice site selection. The PE isoform arises from alternative translation initiation, and excludes a Tudor/Agenet domain that is well conserved between fly and human FMRP (Maurer-Stroh et al., 2003), but is otherwise identical to the PC isoform. The PB isoform of dFMR1 is smaller, and is derived from an alternative splicing event where a 541 nucleotide intron is retained and then serves as a source for canonical hexanucleotide sequences that can recruit the cleavage and polyadenylation machinery (Figure 1B). The PB isoform is 529 amino acids, and a protein corresponding to its predicted mass can be detected on Western blots of tissue extracts from both genders. An examination of C-terminal amino acids of the PA, PC, PD, and PE isoforms that are coded by the terminal 3′ exon of dfmr1 reveals that this 122 amino acid peptide is unusually enriched in glutamine and asparagine residues (Figure 1C). The series of short glutamine repeats in the dFMR1 C-terminal peptide sequence resembles those of the prion domain associated with Sup35 protein from Saccharomyces cerevisiae, and neuronal paralogs of CPEB from Aplysia and Drosophila. Two independently derived algorithms for the identification of proteins enriched in Q/N residues and thus having the potential for prion-like properties both uncover dFMR1 (Supplemental data from Michelitsch and Weissman, 2000; Harrison and Gerstein, 2003).
53 The Q/N domain of dFMR1 confers properties of aggregation and protease resistance Biochemical properties associated with Q/N-rich domains include the conferral of a metastable activity state, resistance to protease, and the ability to facilitate aggregation of proteins to which they have been fused. To test whether the Q/N domain of dFMR1 has such properties, we fused it to reporter proteins that have previously been used to detect and monitor prion-like activity. When fused to a constitutively active form of the glucocorticoid receptor (GR) transcription factor and expressed in yeast, the Sup35 NM and Aplysia CPEB Q/N domains confer a metastable state of activity upon the GR transcription factor (GR526, Schena and Yamamoto, 1988; Li and Lindquist, 2000; Si et al., 2003). In this assay, yeast colonies plated on X-gal media switch between functional (blue colony) and non-functional (white colony) states of the GR526 transcription factor. When the 122 codons that specify the dFMR1 Q/N domain are fused to GR526, we observe a high level of switching between active and inactive states of the GR526 transcription factor, as judged by scoring blue and white colonies that appear on X-gal plates (Figure 2A). This degree of instability contrasts with what is observed for the prion-forming Sup35 NM domain and the Q/N-rich domain of Aplysia CPEB protein (Li and Lindquist, 2000; Si et al., 2003). The number of glutamine repeats in the dFMR1 domain are fewer than what are present in the Sup35 NM and Aplysia Q/N-rich domains, and may result in a lesser degree of stability for any conformation the dFMR1::GR526 fusion protein adopts.
The ability to visualize green fluorescent protein (GFP) in live cells facilitates detection of protein aggregates in vivo. While native GFP has a uniform diffuse distribution throughout the cytoplasm of a yeast cell, fusions of prion or Q/N domains to GFP can induce its aggregation (Patino et al., 1996; Sondheimer and Lindquist, 2000; Si et al., 2003). We thus tested the ability of the dFMR1 Q/N domain to facilitate GFP aggregation in yeast. Examination of live yeast cells expressing a dFMR1 Q/N::GFP fusion protein shows that about 5% of cells had aggregates of GFP fluorescence, a distribution never observed with native GFP (Figure 2B). This rate is comparable to what has been reported with the Q/N domain of Aplysia CPEB (Si et al., 2003), and the frequency at which defined Saccharomyces prion domains induce GFP aggregation within a homologous host cell can be as low as 10% (Osherovich et al., 2004). It is important to note that GFP aggregated via the dFMR1 Q/N domain or by other prion-like domains is still
54 active, and underscores that protein in a potential amyloid or prion conformation can retain function.
We also examined the ability of the dFMR1 Q/N domain to facilitate protein aggregation in Drosophila tissues. Genes encoding GFP or dFMR1 Q/N::GFP were cloned behind a UAS sequence, and their expression was driven by either myosin heavy chain-GAL4 or 201Y-GAL4, a driver that has strong expression in the mushroom body (O’Dell et al., 1995; Yang et al., 1995). Examination of larval muscles expressing either of the GFP isoforms shows that only the dFMR1 Q/N fusion exhibits aggregation of GFP antigen in these tissues (Figure 2C). Adult brains in which the Q/N::GFP fusion was expressed via the 201Y-GAL4 driver showed occasional GFP aggregates in select neurons of the mushroom body γ-lobe (Figure 2C). These results suggest that elevated concentrations of dFMR1 with the Q/N domain, such as those that might arise from local translation, can facilitate protein aggregation.
Resistance to protease treatment is associated with prions and amyloids, and thus the dFMR1 Q/N::GFP fusion protein was tested for resistance to protease. As a control, a fusion of GFP and amino acids 107-168 from dFMR1 was tested as well. Extracts of yeast cultures expressing these fusions were exposed to proteinase K for up to 30 minutes. Figure 2D shows the result from a typical Western blot of extracts probed with anti-GFP antibody. While a fraction of the dFMR1 Q/N::GFP fusion protein is stable out to 30 minutes, the control fusion is essentially undetectable after 3 minutes of exposure to proteinase K. Ponceau-S staining shows that equivalent levels of extract were loaded and demonstrates the efficacy of the protease enzyme. A second control fusion where a cytoplasmic domain of rat α2 Na+/K+ ATPase was fused to GFP was also quickly degraded by the protease (unpublished data). Taken together, the ability of the dFMR1 Q/N domain to induce a reversible change in protein activity, confer resistance to protease, and facilitate protein aggregation in both heterologous yeast cells and endogenous Drosophila tissues indicate that it has properties that are similar to those of previously described Q/N-rich domains.
55 The C-terminal peptide of human FMRP shares some biochemical properties with the dFMR1 Q/N domain While there is considerable amino acid identity/similarity between human FMRP, the paralogous FXR1 and FXR2 proteins, and dFMR1 in the N-terminal 400 amino acids, C-terminal residues of these proteins have significantly diverged (Wan et al., 2000). However, the C-terminal six amino acids between all four proteins are conserved, suggesting that the exons encoding these amino acids may have a common ancestor. To test whether the C-terminal amino acids of hFMRP share any properties observed with the dFMR1 Q/N domain, a fusion was made between 61 codons that specify C-terminal amino acids of hFMRP and GFP. This fusion protein was expressed in yeast and examined for in vivo aggregation, but none was reliably observed (unpublished data). The ability of hFMRP C-terminal amino acids to confer resistance to in vitro protease treatment was tested, and found to result in protection of the GFP moiety from proteinase K in a manner similar to that observed with the dFMR1 Q/N domain (Figures 2D, E). Thus, despite the differences in primary sequence between C-terminal peptides of hFMRP and dFMR1, they may share common properties that contribute towards their function.
Deleting the Q/N domain does not affect dFMR1 stability or spatial expression in the central nervous system A 14 kb genomic rescue fragment encompassing the dfmr1 locus rescues phenotypes associated with the dfmr13 deletion null mutation (Dockendorff et al., 2002). We used this rescue fragment to create a 270 bp deletion derivative that removed most of the codons specifying Q or N from the 3′ exon (Figure 3A; see Materials and Methods). The mutant rescue fragment was introduced by P element transformation and three independent transgene insertions were subsequently analyzed. The mutant rescue fragments were crossed into a dfmr13 null background to generate stocks where the mutant transgene is the sole source of dFMR1 protein. The truncated protein is stable in a steady-state as judged by Western blotting, and whole-mount staining of adult brains with anti-dFMR1 antibody shows that mutant animals have a spatial expression pattern of dFMR1 in the central nervous system that has no discernible difference from the pattern observed in brains of wild type animals (Figures 3B, C). Quantification of signals from Western blots shows that a single copy of the transgene results in a level of dFMR1
56 protein that is either equivalent to that observed in a w1118 control animal (ΔQ/N 8), or at about 1.8-fold overexpression (ΔQ/N 1B, 1C; Figure 3D).
The dFMR1 Q/N deletion protein rescues axon guidance and embryonic development phenotypes associated with dfmr1 null alleles Deletion null alleles of dfmr1 elicit pleiotropic phenotypes that include defects in embryogenesis, neuronal development, and behaviors of larvae and adults (Zhang et al., 2001; Dockendorff et al., 2002; Morales et al., 2002; Lee et al., 2003; Michel et al., 2004; Deshpande et al., 2006; Monzo et al., 2006). The development phenotypes of embryos produced by dfmr1 mutant females include defects in nuclear division cycles, cellularization, and cleavage furrow formation. The breakdown of these embryonic development processes results in hatch rates of embryos from dfmr1 mutant females being about 60% that of embryos from wild type mothers (Monzo et al., 2006). To assess the effects of the Q/N deletion on embryonic development, we measured the hatch rate of eggs from wild type females, those homozygous for the dfmr13 null allele, and females expressing the transgene alleles of dfmr1 where the region coding for the Q/N domain is deleted. The results presented in Figure 4A show that all transgenic lines expressing the mutant dFMR1 provide a significant level of rescue of embryonic hatch rates over that observed with embryos from females homozygous for the dfmr13 deletion null allele.
In wild type flies, the β-lobe axons of mushroom bodies infrequently cross the midline of the central brain. However, flies homozygous for null alleles of dfmr1 display a midline-crossing defect in these axons with a high level of penetrance (Michel et al., 2004; Banerjee et al., 2007). We examined the brains of 2-day old adults harboring wild type, null, or Q/N domain deletion alleles of dfmr1 for this phenotype. All three transgenes expressing dFMR1 lacking the Q/N domain provide significant rescue of the phenotype observed with the null allele control (Figure 4B). These results, along with those observed with the embryonic hatch rate, show that functions and properties conferred by the Q/N domain are not essential for the ability of dFMR1 to execute at least a subset of its normal activities, and that the PB isoform of dFMR1 may have select developmental and physiologic processes that it governs.
57 An increase in larval neuromuscular junction boutons results from deletion of the dFMR1 Q/N domain Larvae homozygous for a null allele of dfmr1 have elevated numbers of neuromuscular junction boutons, which are the swellings associated with synapse formation. The increase in boutons is due in part to excessive branching from the axon track innervating the larval muscles (Zhang et al., 2001). Using larvae expressing a wild type rescue fragment in an otherwise dfmr1 null background (WT rescue), larvae homozygous for a null allele of dfmr1, and larvae expressing dFMR1 with a deletion of the Q/N domain, we examined three muscle types from larval segment A4 for numbers of type 1 boutons. The results from these experiments are presented in Figure 5 and show that the loss of the Q/N domain results in a significant increase in bouton numbers when compared to larvae that are expressing a wild type allele of dfmr1. The strength of the mutant phenotype varies with different mutant rescue fragments and muscle types. However, in two muscles examined, the ΔQ/N 8 rescue fragment, which expresses dFMR1 at a level similar to endogenous dFMR1, elicits bouton numbers that resemble those observed with the null allele (Figure 5) indicating that the Q/N domain is a significant factor for dFMR1 function in this process of synaptic development.
Rhythmic locomotion activity is affected by loss of the dFMR1 Q/N domain A null mutation of dfmr1 results in loss of rhythmic locomotion activity that likely occurs via misregulation of one or more clock-controlled output genes (Dockendorff et al., 2002). We thus tested the Q/N deletion transgenic stocks for their ability to maintain rhythmic locomotion activity in constant darkness. The results in Figure 6 show that about 40% of the animals from each of the three ΔQ/N transgenic lines retain rhythmicity in constant darkness, compared to about 20% of null mutants, and 80% of dfmr13 homozygotes that express a wild type allele of dfmr1. The Q/N domain thus contributes to dFMR1 control of outputs from a biological clock.
Social behavior during courtship is impaired by the Q/N deletion Courtship in Drosophila involves a series of stereotyped behaviors between the courting male and the female mate (reviewed by Greenspan and Ferveur, 2000). Although conduct of these behaviors requires the function of several sensory modalities, their ethological relevance has made analysis of Drosophila courtship a common method to study genes that control behaviors
58 and processes of learning and memory (reviewed by Mehren et al., 2004). When placed with a receptive virgin female, naive males will initiate courtship behaviors that include tapping the female with his foreleg, extension and vibration of a wing, licking the female with his proboscis, and attempts at copulation. The percentage of time engaged in these behaviors over a defined period of time is referred to as the courtship index.
Males homozygous for a null mutation of dfmr1 have a reduction in naive courtship activity compared to wild type flies (Dockendorff et al., 2002), indicating that dFMR1 protein is needed for establishment and/or function of neural pathways needed for efficient courtship behavior. However, dfmr1 mutant males can achieve copulation, indicating that their reduced courtship index is not simply a consequence of a total deficit in sensory detection or processing of courtship cues. We tested the dFMR1 ΔQ/N transgenic stocks for naive courtship behavior, and data in Figure 7A show that removal of the Q/N domain results in naive courtship levels that resemble those of null allele controls. The Q/N domain of dFMR1 is thus necessary for establishment and/or function of neural circuitry that contributes towards naive courtship behavior.
Immediate recall is maintained and short-term memory of conditioned courtship training is defective in flies lacking the dFMR1 Q/N domain Synaptic plasticity can be divided into phases that are distinct relative to the need for protein synthesis and its source. The early phase (0-60 minutes post-induction) of long-term potentiation (E-LTP) is not reliant on protein synthesis, while intermediate (1-3 hours) and late phases (> 4 hours) require protein synthesis provided by local translation and transcription respectively (reviewed by Pfeiffer and Huber, 2006). Likewise, behavioral memory in Drosophila can be dissected into phases that have been defined by genetic and pharmacologic criteria (reviewed by Greenspan, 1995), and include immediate recall, short-, and long-term memory of the training protocol.
Conditioned courtship is a paradigm for memory where unreceptive Drosophila females present both visual and pheromone-based cues towards a courting male that results in a reduction of his courtship activity. Moreover, the inhibition of the male’s courtship activity carries over to other
59 courtship targets, even receptive females, and thus represents a form of associative memory. Depending upon the training regimen with a non-receptive female, this memory can be maintained for periods of a few hours (short-term memory) to long-term memory that lasts several days (Siegel and Hall, 1979; McBride et al., 1999). Prior studies have established that like wild type males, the courtship index of dfmr1 null mutants declines during training, demonstrating that the mutant is able to detect and respond to the negative courtship cues displayed by the female (McBride et al., 2005). Flies homozygous for a null allele of dfmr1 are deficient in both immediate recall (0-2 minutes post-training) and one-hour short-term memory of conditioned courtship training (McBride et al., 2005).
The necessity for the dFMR1 Q/N domain on immediate recall and short-term memory was tested with conditioned courtship. Males with wild type, null, or ΔQ/N alleles of dfmr1 were paired with unreceptive females for one hour. Trained males were removed from the courtship chamber and placed with a receptive female (immediate recall), or left in isolation for one hour, and then paired with a receptive female to measure short-term memory. A comparison of the naive courtship index and the courtship index after training shows that flies expressing the ΔQ/N protein have a significant decrease in courtship at the immediate recall stage, indicating that they have retained memory of the training (Figure 7B). In contrast, there is not a significant difference between the naive courtship index of ΔQ/N flies and the courtship index measured one hour after training (Figure 7C). The results from these experiments show that the Q/N domain of dFMR1 is essential for the formation of short-term memory of conditioned courtship training.
DISCUSSION Local translation at synapses, reorganization of cytoskeletal elements, and changes in transcriptional output help modulate the synaptic growth and development necessary for establishment and maintenance of memory (reviewed by Bailey et al., 2004). The presence of FMRP at synapses, its interactions with translation machinery and cytoskeletal regulators, and the phenotypes that arise from its mutation, place FMRP as a key regulator of synaptic function (reviewed by Bagni and Greenough, 2005). The Drosophila fragile X model facilitates a molecular genetics approach to dissect FMRP function. To this end, we have explored the importance of a Q/N-rich domain present in the C-terminal region of dFMR1. Q/N-rich domains
60 are surprisingly common in eukaryotic proteomes and are implicated in protein-protein interactions and in some cases, prion formation (Michelitsch and Weissman, 2000; Harrison and Gerstein, 2003). The properties of Q/N-rich domains are proposed to be vital for synaptic function (Si et al., 2003; Bailey et al., 2004; Shorter and Lindquist, 2005). Our results show that the dFMR1 Q/N domain is necessary for optimal execution of neural development and behavior patterns that are associated with dFMR1 function.
A connection between mGluR-regulated protein synthesis and the necessity for the dFMR1 Q/N domain in short-term memory of conditioned courtship Signaling through mGluRs induces protein synthesis-dependent long-term depression (LTD) in hippocampal slices (Huber et al., 2000). Inhibition of protein synthesis with an m7GpppG cap analogue has no significant effect on excitatory post-synaptic potentials (EPSPs) early in the time course of LTD (less than 30 minutes after induction). In contrast, at 60 minutes post- induction of these slices, the EPSC returns to what is observed in unstimulated controls. Inhibitors of transcription do not affect the EPSC readings within these time frames, demonstrating the need for de novo protein synthesis is at the level of translation (Huber et al., 2000; reviewed by Bear et al., 2004). mGluR activation leads to internalization of ionotropic glutamate receptors, and protein synthesis is needed to maintain this change (Snyder et al., 2001). The loss of FMRP in a mouse model results in an exaggeration of mGluR-stimulated hippocampal LTD, a finding that is consistent with FMRP having a repressive effect on protein synthesis (Huber et al., 2002).
Flies homozygous for a deletion null allele of dfmr1 are deficient at immediate recall (0-2 min), and short-term memory of conditioned courtship (McBride et al., 2005; this study). We find that flies expressing the dFMR1 ΔQ/N protein are able to perform immediate recall of conditioned courtship, suggesting that this mutant protein is sufficient to support the development and function of neural circuitry needed for the earliest phase of memory formation. In contrast, the dFMR1 ΔQ/N flies are defective in one-hour short-term memory. The timing of this memory deficiency is consistent with the requirement for regulated protein synthesis in mGluR- stimulated hippocampal LTD (Huber et al., 2000). The Q/N domain thus facilitates the activity of dFMR1 in mediating later phases of memory formation and/or consolidation.
61 Comparing the courtship phenotypes of orb2 and dfmr1 Q/N domain mutations The Orb/CPEB (cytoplasmic polyadenylation element binding) proteins are another prominent class of synaptic translation regulators (reviewed by Richter, 2007). Phylogenetic analyses show that these proteins fall into two families, CPEB1 and CPEB2. Both invertebrates and vertebrates have a single member of CPEB1, but while flies have a single CPEB2 family member (orb2), vertebrate genomes encode three CPEB2 members (CPEB2-4; Kurihara et al., 2003; Theis et al., 2003). Although the RNA binding domains of the CPEB2 family are structurally related to those of CPEB1, they have a mechanism of function that is distinct from the CPEB1 family (Huang et al., 2006). N-terminal regions that are presumed to have regulatory functions are diverged between the CPEB2 family members. The D. melanogaster Orb2 protein (a CPEB2 member) has a Q-rich domain and a role for this domain in Orb2 function has been recently reported (Keleman et al., 2007). While deletion of orb2 has a lethal phenotype, flies expressing an orb2 allele where the codons spanning the Q-rich region are missing are viable, and no anomalies in mushroom body morphology are observed in these mutants. Studies of conditioned courtship with flies expressing the Orb2 ΔQ protein reveals no change in short-term memory performance (tested 30 minutes after a 90-minute training session) when compared to the control, but long- term memory (tested 24 hours after a 5-hour training session) is strongly affected by the mutation (Keleman et al., 2007). In contrast, we find that deletion of the dFMR1 Q/N domain disrupts short-term memory, and thus it is possible that dFMR1 and Orb2 regulate distinct phases of memory establishment and consolidation. Consistent with this observation, mutation of mouse CPEB1 affects some forms of protein synthesis-dependent LTP in the hippocampus (Alarcón et al., 2004), while the FMR1 mutant mouse has normal NMDA receptor-stimulated hippocampal LTP (Godfraind et al., 1996).
Why do vertebrate FMR/FXR proteins not have a Q/N-rich domain? The fragile X protein family has three members in vertebrates (FMR1, FXR1, FXR2) and a single member in flies (dFMR1). All of these proteins have highly conserved ∼400 amino acid N-terminal regions, and thus these portions of FMR/FXR proteins likely have similar biochemical properties. In contrast, C-terminal amino acids of these proteins have diverged (Wan et al., 2000). Little function has been ascribed to C-terminal regions of FMR/FXR proteins, but the Ran-binding protein in the microtubule-organizing center (RanBPM) interacts
62 with this region of human FMRP as judged by two-hybrid analysis, in vitro binding studies, and in vivo co-localization of the two proteins (Menon et al., 2004). Although we did not observe the C-terminal amino acids of hFMRP to facilitate aggregation of GFP to an extent seen with the dFMR1 Q/N domain, we found that they protected GFP from in vitro protease digestion. The divergence of C-terminal amino acids between dFMR1 and the vertebrate FMR/FXR proteins may be a means of promoting specialized protein-protein interactions, which then provides functions that are unique for each of the FMR/FXR proteins. It is interesting to note that a similar situation may be the case for the aforementioned CPEB2 subfamily, which like FMR/FXR proteins has a single member in D. melanogaster and three in vertebrates. The N- terminal regulatory regions of these proteins have diverged, with the Aplysia and Drosophila proteins having high Q/N content, while the vertebrate orthologs have at most comparatively vestigial levels of Q residues.
How do Q/N domains work to modulate protein function? High levels of Q/N residues promote protein-protein interaction through the hydrogen bonding that can occur via the side chains of these amino acids (Perutz et al., 1994). Q/N-rich domains can mediate both self-interactions and interactions with heterologous proteins (Bailleul et al., 1999; Tang et al., 2000; Decker et al., 2007; Guo et al., 2007; Shewmaker et al., 2007; Kim et al., 2008). In some cases, the self-interactions result in prion formation (reviewed by Shorter and Lindquist, 2005; Wickner et al., 2007). A characteristic of prions is their structural stability, which can be assessed by fusion to a reporter protein, such as the constitutively active glucocorticoid receptor transcription factor GR526 (Schena and Yamamoto, 1988; Li and Lindquist, 2000). Our observations on the rate at which the dFMR1 Q/N::GR526 fusion protein switches between active and inactive states suggests that the dFMR1 Q/N domain may not have prion-forming capacity. It is important to note that only a few of the approximately 100 S. cerevisiae proteins judged to have a Q/N-rich domain are demonstrated to form prions (Michelitsch and Weissman, 2000; Sondheimer and Lindquist, 2000). The RNA binding capacity of FMRP and its association with RNP complexes are means by which it mediates synaptic function and behavior. Based on the long-term memory deficit arising by deletion of a Q-rich region from the D. melanogaster Orb2 protein, a possible role for the Q/N-rich domains in memory establishment is by acting as a component or regulator of a
63 synaptic tag (discussed by Keleman et al., 2007). The concept of a synaptic tag is derived from the observation that a single E-LTP or E-LTD-inducing tetanus at a synapse, followed by a L- LTP or L-LTD-inducing stimulus at a nearby synapse, can facilitate L-LTP or L-LTD at the synapse that had previously been given the E-LTP or E-LTD-inducing tetanus (Frey and Morris, 1997; Sajikumar and Frey, 2004). This phenomenon suggests that L-LTP or L-LTD-inducing factors can be recruited or captured by a synaptic “tag” that is created through induction of E- LTP or E-LTD. Although components of the tag have yet to be conclusively identified, protein synthesis and cytoskeletal regulation machinery are among the candidates (reviewed by Martin and Kosik, 2002). In the above model, the dFMR1 Q/N domain may mediate interactions with RNP complexes that promote proper regulation of protein synthesis occurring in response to stimulation. The protein and RNA components that constitute these RNP complexes are likely central to the establishment and control of memory formation.
EXPERIMENTAL PROCEDURES Fly stocks, transgene construction, and synthesis of transgenic stocks Except where indicated, all fly stocks are derived from a w1118 background. The dfmr13 allele has been previously described (Dockendorff et al., 2002; Banerjee et al., 2007). A genomic rescue fragment spanning the dfmr1 locus is described in Dockendorff et al. (2002). The terminal exon encoding the glutamine/asparagine domain was mutagenized as follows: PCR primers were devised to amplify a 2.0 kb fragment that resulted in a product that had NcoI and BspEI ends; these restriction sites are unique to the genomic rescue fragment. The amplification product was digested with these enzymes and ligated to the backbone of the rescue fragment that had been treated with the same enzymes. The substitution with the PCR product resulted in a construct where 91 codons of dfmr1 encompassing much of the Q/N domain were removed. The PCR amplification product was produced with a proofreading polymerase (Vent polymerase, New England Biolabs) and was sequenced to ensure the absence of secondary mutations arising from base misincorporations. The mutant transgene was introduced to w1118 flies using standard transformation techniques (Spradling and Rubin, 1982). The mutant rescue fragment was then crossed into the dfmr13 background to produce fly stocks where the mutant rescue fragment was the sole source of dfmr1. All dfmr1 stocks were balanced with the TM6C Tb Sb chromosome.
64 Neuroanatomical analyses Larval neuromuscular junction (NMJ) type I boutons were detected by staining third-instar larval fillets with anti-horseradish peroxidase (Cappel, Aurora, OH) at a dilution of 1:200. Mushroom bodies were visualized by staining whole-mounts of brains with anti-FasII at a 1:10 dilution (mAb 1D4 obtained from University of Iowa Developmental Studies Hybridoma Bank). Rabbit polyclonal antiserum against GFP was obtained from Invitrogen and used at a 1:200 dilution. Secondary antibodies conjugated to either HRP or fluorochrome were obtained from Jackson ImmunoResearch (West Grove, PA) and used at a 1:200 dilution. Confocal images were collected on an Olympus FV500 microscope. Western blots were performed as described in Wan et al. (2000) using anti-dFMR1 antibody 5A11 at a 1:1000 dilution and anti-β-tubulin mAb E7 (both from the University of Iowa Developmental Studies Hybridoma Bank) at a 1:100 dilution. Magnifications: Whole brains 200X; Mushroom bodies 400X; gamma lobes 1200X; Larval muscles 900X.
Yeast culture, strains, and plasmids Strain W303 (matα, leu2-3,112 trp1-1 ura3-1 ade2-1 his3-11,15) was provided by Rodney Rothstein and used for all subsequent yeast assays. Transformations were done using lithium acetate as described by Gietz et al. (1992).
Transcription factor activity The 122 amino acids of the dFMR1 Q/N-rich domain were fused to a constitutively active form of the glucocorticoid receptor transcription factor (GR526; Schena and Yamamoto, 1988) expressed via a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. Fusions of GR526 to yeast prions has been used to monitor prion stability and inheritance (Li and Lindquist, 2000). The dFMR1 Q/N::GR526 plasmid was co-expressed with a plasmid harboring a LacZ gene with GR binding sites upstream. Yeast cells carrying these plasmids were plated to X-gal media, and blue colonies selected. These colonies were grown overnight in selective media, diluted, and plated again to X-gal media to monitor colony color. GR526 by itself was used as a control for maximal expression of LacZ. Plates were incubated for 2 days at 30°C and then photographed.
65 Protease Assay The 122 amino acid Q/N-rich domain of dFMR1 and human FMRP C-terminal 61 amino acids were fused to the N-terminal of GFP in p2HGsGFP (His+) plasmids and was expressed via the GPD promoter in yeast cells. Cultures were grown overnight and protein extracts were made in
lysis buffer (0.2% Tween 20, 10 mM Tris pH 7.5, 10 mM MgCl2). Protein extracts were exposed to proteinase K at 16.7 µg/ml, and samples were removed and quenched in protein- loading buffer at time points indicated in the figures. Western blots were performed and the blot was probed with rabbit anti-GFP primary antibody (1:5000) and HRP conjugated donkey anti- rabbit secondary antibody (1:5000). As a control, a nonspecific domain of dFMR1 fused to GFP (amino acids 107-168) and GFP alone was treated under similar conditions to demonstrate specificity by the Q/N-rich domain of dFMR1 and human C-terminal peptide for protease resistance. Ponceau-S staining was performed to demonstrate loading equivalency and efficacy of the protease treatment.
In vivo aggregation of GFP The C-terminal 122 amino acids containing the Q/N-rich domain of dFMR1 was introduced to the N-terminal region of GFP in p2HGsGFP(His+) plasmid. This plasmid was used to transfer w303α leu2-3,112 trp1-1 ura3-1 ade2-1 his3-11,15 yeast cells. The transformed colonies were picked and grown overnight in his- selective media. The cells were plated on polystyrene treated glass slides to observe GFP aggregation in yeast via fluorescent microscopy. Control experiments were performed with GFP alone being expressed in yeast cells.
GFP aggregation in Drosophila tissues was analyzed by driving expression of a dFMR1 Q/N::GFP fusion in third instar larval muscle tissues with the myosin heavy chain GAL4 driver and in adult mushroom bodies by the 201Y mushroom body GAL4 driver (O’Dell et al., 1995; Yang et al., 1995). The larval fillets and whole mount adult brains were stained with rabbit anti- GFP primary antibody (1:200) and FITC labeled anti-rabbit secondary antibody (1:200) to visualize GFP aggregates by confocal microscopy. Expression of native GFP was used as a control for background aggregation.
66 Fly behavior Courtship and circadian behavior analyses For courtship behavior testing, males of the appropriate genotypes were collected within two hours of eclosion and kept in isolation prior to testing. Female targets were of the genotype XX, y, f (attached X) and collected as virgins for courtship testing. All flies were kept in 12:12 light/dark cycles at 25°C and 70-75% relative humidity, and were aged four days prior to analysis. For the naive courtship analysis, the four-day old male and female were transferred via aspiration to a mating chamber 20 mm in diameter and 5 mm deep. These chambers were kept in humidified conditions throughout the assay. Transferred males were given a five-minute recovery period prior to addition of the female target. Males were monitored for courtship activity that included following of the female, wing extension and vibration, tapping of female with his foreleg and attempted copulation for a period of 10 minutes, or until copulation occurred. The percentage of time the male spent in active pursuit of the female was recorded as the courtship index. Immediate recall was tested by pairing a naïve male with a non-receptive female for one hour and then placing him in a second chamber with a receptive female. Short- term memory was assessed by taking a male that had been trained with a non-receptive female and placing him in isolation for one hour prior to pairing with a receptive female. At least 17 animals were tested for each genotype during analyses of naive courtship, immediate recall, and short-term memory. Courtship observers were blind to the genotypes of the animals.
Circadian behavior was tested as described in (Dockendorff et al. 2002). Flies were entrained to a 12:12 light/dark cycle, placed into activity monitors (Trikinetics, Waltham, MA) maintained in light/dark cycles, and then placed under constant darkness. Locomotion activity was collected in 30-minute bins. The percentage of flies judged to be rhythmic was assessed by Clocklab software (Actimetrics, Evanston, IL) as follows: Using a confidence level of 0.025, batch analyses were performed for the genotypes tested, monitoring the locomotion activity in constant darkness over seven days. The difference between the power(1) and significance(1) values was calculated for each fly, and a value of less than 10 was the basis for judging an arrhythmic phenotype. Visual analyses of periodograms and actograms were also conducted to confirm the results.
67 Statistical analyses: Courtship indices were arcsin transformed, then analyzed by one-way ANOVA, followed by a Tukey-Kramer post-test. NMJ bouton counts were analyzed by one- way ANOVA with a Tukey-Kramer post-test, or by a Kruskal-Wallis test, followed by a Dunn post-test. The analyses of courtship indices and NMJ bouton counts were conducted using InStat software from GraphPad (San Diego, CA). Comparisons of mushroom body axon midline crossings and circadian rhythmicity were made by a chi-square test for homogeneity.
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73 A C IKVSAIA dFMR1 560-681 36% Q/N
684 aa PA NGAVANNNNKPQSAQQPQQQQPPAPGNKAALNAGDASKQNSGN ANAAGGASKPKDASRNGDKQQAGTQQQQPSQVQQQQAAQQQQP 681 aa PC KPRRNKNRSNNHTDQPSGQQQLAENVKKEGLVNGTS 643 aa PE Aplysia CPEB 1-160 48% Q/N 529 aa DTN PB MQAMAVASQSPQTVDQAISVKTDYKDNQQEHIPSNFEIFRRIN ALLDNSLEANNVSCSQSQSQQQQQQTQQQQQQQQQQQQQQHL α-dFMR1 B QQVQQQRLLKQQQQQAQRQQIQQQLLQQQQQKQQLQQQQQQE QLQQQQLQLQQQLQQQLQHIQKEPSSHTYTPGP PA/C Q/N PB S. cerevisiae Sup35 1-123 45% Q/N
MSDSNQGNNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQAQP AGGYYQNYQGYSGYQQGGYQQYNPDAGYQQQYNPQGGYQQY GGTGAGTTCCGCGAATCATGATGCCAATTAACCAATTAAGTTCAATCAAT NPQGGYQQQFNPQGGRGNYKNFNYNNNLQGYQAGFQPQSQG TTATTTCGCGCGTCACACAAGATACAAATTGAATACTCGATCCGATTCCC TCTCCCCTAAATATGGACATCATGTGGGGTCAAGAATACGAAAATTTGTA Drosophila Orb2B 168-221 54% Q/N CAGCAAGTTTATTAGTTATTAAACAGTATAAAGTATTACATTAAGGACTG TTTCATAAAATCCCTATAATTATAGCATTCTGAATAAAATAAATGAAATT NLNLNKPPQLHQQQHQQQHQQHQQHQQQQQLHQHQQQLSPNL ATCTTGGTTTATTTTGTGTAAAGTATGATAATAAACTTTTCAAAAGTAAA SALHHHHQQQQQ ACATTATACATTATTGAATCTTAACTTTCGCTTTCATTGCCAACAATATA ATATATAAAATAGAAACACTGACCTGGGCTTTGTATTTAAATAAATATCA AAATATATATTTTTTCATTTAATTGAAGTGCGCAGGGGCACAGACAATGA ATTTTTGCTGATGAAGTTATTTCCTTTTTATCGTCATCTGTAATCTCTGC TAGTATACTATGATAACTTATCCATTATCCTTTTCTCCACAG
Figure 1. Isoforms of the Drosophila fragile X mental retardation protein (dFMR1) have a glutamine/asparagine-rich domain (A) Schematic of dFMR1 protein isoforms identified or predicted by genomics analyses. Variance between the isoforms results from alternative splicing or alternative translation initiation. dFMR1 shares several domains with mammalian FMRP that bind RNA. A tandem array of KH domains (highlighted in black) and an RGG box (dark blue) are common to all dFMR1 isoforms, and a pair of Tudor/Agenet domains is present in the amino-terminal region of insect and vertebrate FMRPs (light blue). The PA isoform of dFMR1 is 684 amino acids and has three extra amino acids within the second KH domain that are not present in other isoforms. The PE isoform arises from an alternative translation initiation site, and lacks one of the Tudor/Agenet domains, while the PB isoform lacks a glutamine/asparagine (Q/N) domain as a result of alternative splicing. (B) Depiction of the alternative splicing event that produces the PB isoform. A 541-nucleotide intron is alternatively spliced, with the resulting PB isoform having a carboxy-terminal end of DTN that is derived from codons within the retained segment of the intron (bold, italics). The remaining intronic sequence codes for three consensus hexanucleotide sequences present in the pre-mRNA (AAUAAA) that facilitate recruitment of the cleavage and polyadenylation machinery (bold, underlined). (C) Comparison of Q/N domains from dFMR1, a neuronal paralog of CPEB from Aplysia, the prion-forming domain from the S. cerevisiae Sup35 protein, and a Q-rich region of the D. melanogaster Orb2 protein. Q/N residues are highlighted in green.
74 A. C. GPD GR526 GPD Q/N-rich GR526 UAS GFP UAS Q/N-rich GFP Blue colony Blue colony White colony
dFMR1 Q/N::GFP B. GFP
D. Protease exposure time (min) Protease exposure time (min) 0 3 10 0 3 10 0 3 1030 0 3 10 30 kDa kDa 52 52 37 37 28 28 19 19
dFMR1 Q/N::GFP dFMR1 NS::GFP dFMR1 Q/N::GFP dFMR1 NS::GFP E. 0 3 1030 0 3 10 30 0 3 1030 0 3 10 30 kDa kDa
52 52 37 37 28 28 19 19
hFMRP C61::GFP GFP hFMRP C61::GFP GFP
Figure 2. The dFMR1 Q/N domain facilitates aggregation and confers protection against protease treatment The 122 codons encompassing the dfmr1 Q/N domain were fused to reporter proteins to assess whether these amino acids could confer properties of aggregation and protease resistance that are associated with other Q/N-rich domains. (A) Fusion of the dFMR1 Q/N domain to a constitutively active glucocorticoid receptor transcription factor elicits a metastable LacZ+ 75 phenotype. The dFMR1 Q/N domain from Figure 1C was fused to GR526. The fusion plasmid and a lacZ reporter plasmid with GR binding sites were sequentially transformed to yeast. Cultures were grown in selective conditions, and plated to X-gal media. While colonies from a control GR526 protein produced blue colonies, the dFMR1 Q/N::GR526 fusion resulted in colonies that varied considerably in the strength of LacZ activity. The white colony phenotype was unstable, as picking a white colony, culturing in selective medium, followed by culture on X-gal media, resulted in a mix of blue and white colonies appearing on the plates. (B) The dFMR1 Q/N domain can aggregate GFP in yeast cells. A dFMR1 Q/N::GFP fusion was expressed in yeast and GFP fluorescence examined. While native GFP was present in a uniform distribution in the cytosol and never observed to aggregate, about 2-5% of dFMR1 Q/N:: GFP cells were observed to have aggregates of GFP. (C) The dFMR1 Q/N domain can aggregate GFP in Drosophila tissues. dFMR1 Q/N::GFP was expressed via the UAS-GAL4 system, using myosin heavy chain-GAL4 to drive expression in larval muscles or the 201Y-GAL4 driver to express the fusion protein in the mushroom body. Tissues were fixed and stained with anti-GFP antibody. In contrast to the diffuse staining of GFP in the control, aggregates of dFMR1 Q/N::GFP are observed throughout larval muscle tissue. In about 10% of adult brains examined, aggregates of dFMR1 Q/N::GFP can be seen in the gamma lobe of mushroom bodies when the fusion is expressed via the 201Y-GAL4 driver. Scale bars equal 20 µm. (D) The dFMR1 Q/N domain confers resistance to protease. Yeast cultures expressing either dFMR1 Q/N::GFP or a non- specific peptide of dFMR1 fused to GFP (dFMR1 residues 110-173) were lysed, and protein extracts exposed to proteinase K for varying times. Extracts were then processed for Western blotting and detection with an anti-GFP polyclonal antibody. While the non-specific dFMR1::GFP fusion was degraded within minutes by protease, peptides of the dFMR1 Q/N::GFP fusion could still be detected at least 30 minutes after exposure to protease. A Ponceau-stained blot demonstrates efficacy of protease treatment. (E) The C-terminal 61 amino acids of human FMRP confers resistance to protease in a manner similar to that observed with the dFMR1 Q/N domain.
76 A.
B.
C.
Figure 3. Generation and characterization of a dfmr1 Q/N domain deletion mutation (A) Schematic of a dfmr1 Q/N deletion allele. A 14 kb genomic rescue fragment spanning the dfmr1 locus was the source for allele construction, and a derivative where ninety codons encompassing much of the Q/N-rich region were deleted was generated as described in Experimental Procedures. The Q/N domain peptide sequence lost through the deletion is shaded in gray. (B) Western blots from adult head extracts to assess expression levels and stability of the mutant dFMR1 protein from three independent transgene insertion stocks that express the mutant allele as the sole source of dFMR1 protein. Expression levels range from equal to that of wild type to about 1.8-fold over wild type. In all cases the mutant protein is stable in a steady state. (C) The dFMR1 ΔQ/N protein has a spatial expression pattern in the adult central brain similar to that of wild type dFMR1 protein. dFMR1 is present throughout the central brain, with enhanced expression in the antennal lobes, lateral horn, and ventro-lateral protocerebrum. Scale bar equals 100 µm.
77 p<0.0001 p<0.0001 A. B.
100 100 90 90 80 80 70 70 60 60 50 50 40 40
30 30
Percentage hatched Percentage 20 20
10 10 % no crossover phenotype crossover no %
0 0 ww11181118 ΔQ/N 1B 1B ΔQ/N 1C 1C ΔQ/N 8 8 Null Null w1118w1118 WTWT rescue Δ 1BQ/N ΔQ/N 1C ΔQ/N 8 Null Null rescue 1B 1C 8
Figure 4. Embryonic and mushroom body development phenotypes are rescued by the dfmr1 Q/N deletion allele. Lack of dFMR1 protein in embryos impairs hatching, and results from defects in cellularization. Axon guidance defects are present in brains of flies that are null for dfmr1, and both developmental anomalies are associated with misregulation of actin dynamics (Reeve et al., 2005; Deshpande et al., 2006; Monzo et al., 2006). Flies expressing the dFMR1 ΔQ/N protein as the sole source of dFMR1 were tested for rescue of these phenotypes. (A) Hatch rates of embryos with a dfmr1 ΔQ/N allele are significantly higher than embryos with a null allele of dfmr1. Females expressing wild type, null, or ΔQ/N alleles of dfmr1 were mated to wild type males. Eggs were collected and scored for hatching within 36 hrs of deposition. The transgene insertion for stock 1C elicits a reduction in hatch rate, even when a wild type allele of dfmr1 is present (unpublished results). Statistical analysis was performed by chi-square test for homogeneity. The ΔQ/N alleles of dfmr1 differ from the null alleles in percentage of hatched embryos (P < 0.0001). N is ≥ 290 for all tested genotypes. (B) Axon guidance defects in mushroom body β-lobes are rescued by the dFMR1 ΔQ/N protein. Flies expressing wild type, null, or ΔQ/N alleles of dfmr1 had brains dissected and stained to visualize mushroom bodies as described in Materials and Methods. Mushroom bodies were scored for the presence of β-lobe axons that crossed the midline of the CNS. Statistical analysis was performed by chi-square test for homogeneity and the ΔQ/N alleles of dfmr1 differ from the null alleles in frequency of midline crossing (P < 0.0001) and from flies with wild type allele of dfmr1 (P = 0.0874). N is ≥ 30 for all tested genotypes.
78 70.0 P<0.05
60.0
50.0
40.0
30.0 Muscle 6/7 Bouton numbers Bouton
Number of Boutons of Number 20.0
10.0
0.0 w1118w1118 WTWT 8/+8/+ 1B/+ Null Null 8 1B 1C rescue 60.0 P<0.01
50.0
40.0
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Muscle 12 Bouton numbers Bouton 20.0
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0.0 w1118w1118 WTWT 8/+ 1B/+ Null Null 81B 1B 1C rescue 70.0 P<0.05 60.0
50.0
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10.0
0.0 ww11181118 WT 8/+8/+ 1B/+ 1B/+ Null Null 8 81B 1B 1C 1C rescuerescue
Figure 5. The dFMR1 ΔQ/N protein fails to rescue the larval neuromuscular junction bouton overgrowth phenotype associated with null alleles of dfmr1. Third-instar larvae expressing wild type, null, or ΔQ/N alleles of dfmr1 were filleted, stained with anti-horseradish peroxidase antibody (which cross-reacts with NMJ boutons), and examined for the presence of type 1 boutons at three muscle types. Larvae expressing the mutant transgenes in a background of dfmr1 heterozygosity were also examined to test for possible
79 dominant effects of the mutant alleles. ANOVA shows that for all muscle types tested, the ΔQ/N allele results in a statistically significant increase in bouton numbers over larvae that have a wild type allele of dfmr1, muscle 6/7 (P < 0.05), muscle 12 (P < 0.01) and muscle 4 (P <0.05). N is ≥ 30 for all tested genotypes.
80
p<0.0001
100
90
80
70
60
50
40 Percent rhythmic Percent
Percent rhythmic Percent 30
20
10
0 ww11181118 WT WT rescuerescue Null NullΔQ/N 8 8 ΔQ/N 1B 1B ΔQ/N 1C 1C
Figure 6. Rhythmic circadian locomotion activity is only partially rescued by the dFMR1 ΔQ/N protein. Flies with wild type, null, or ΔQ/N alleles of dfmr1 were entrained to a 12:12 light-dark cycle, then placed in constant darkness for seven days. About 80-90% of wild type flies, and about 20% of null mutants retained rhythmic locomotion activity. The ΔQ/N flies have an intermediate phenotype, with about 40% of the flies judged rhythmic, demonstrating the importance of the Q/N domain in regulating an output of the circadian pacemaker. The ΔQ/N alleles of dfmr1 differ from the wild type allele of dfmr1 (P < 0.0001). N is ≥ 30 for all tested genotypes.
81 WT A. FS B. WT PrionWT del 8 FS WT PrionFS del C Prion del 8 PrionFS del 1B5 20 PrionΔ Q/N del 81B5 20 Δ Q/N 8 Δ Q/N 1C 50 50 50 50 Δ Q/N 1B Δ Q/N 1B5
40 *** 40 40 40 48 48 *** 19 *** 19 30 26 30 30 26 *** 30 *** 17 23 20 20 20 20 *** 20 19 *** COURTSHIP INDEX COURTSHIP INDEX 19 10 10 10 10
0 0 0 0 Naive Naive 0 minutes TIME INTERVAL AFTER TRAINING TIME INTERVALImmediate AFTER recall TRAINING of courtship training
WT C. FSWT Prion del 8 FS 20 Prion del 1B5 50 Δ Q/N 8 50 Δ Q/N 1B
40 40 48 44 30 19 26 20 30
21 20 *** 20 20 COURTSHIP INDEX 10 10
0 0 Naive 60 minutes TIME Short-termINTERVAL AFTER memory TRAINING
Figure 7. dFMR1 ΔQ/N protein facilitates immediate recall, but not one-hour short-term memory of conditioned courtship training. Courtship behavior in flies with wild type, null, or ΔQ/N alleles of dfmr1 were analyzed by observing naive courtship activity, and both immediate recall and one hour short-term memory of conditioned courtship training. (A) Naive courtship is reduced in flies expressing a dfmr1 ΔQ/N allele. Males were captured after eclosion, kept in isolation for four days, and then paired with a virgin female. Courtship activity was monitored as described in Dockendorff et al. (2002). (B) Flies expressing the dFMR1 ΔQ/N protein display immediate recall of conditioned courtship training. Single males were paired with a non-receptive female for one hour, then immediately transferred to a chamber with a receptive female. The courtship index was measured and compared to that of a naive male of the same genotype. The courtship index of trained males that have either wild type or ΔQ/N alleles of dfmr1 is depressed to a level that is significantly different from the naive controls. (C) Flies expressing the dFMR1 ΔQ/N protein 82 are defective in one hour short-term memory. Single males were paired with a non-receptive female for one hour, then transferred to a chamber and kept in isolation for one hour. These flies were subsequently paired with a receptive female and the courtship index measured. While flies with a wild type allele of dfmr1 have a significant suppression of courtship activity towards the receptive female, those with null or ΔQ/N alleles of dfmr1 have courtship indices at one hour post-training that do no differ from naive controls, indicating a short-term memory deficit. *** - p < 0.001. ANOVAs were performed on pair-wise comparisons of arcsin transformed data to obtain critical p values. Data was generously provided by Sean McBride, Albert Einstein College of Medicine.
83 CHAPTER IV
A Genetic Dissection of the dFMR1 C-terminal Domain Uncovers Novel Functions for Fragile X Protein
Results from the previous chapter demonstrate the Q/N domain is vital for the full range of dFMR1 function. As noted, analysis of cDNAs shows that the dfmr1 locus produces multiple Q/N(+) isoforms and a single Q/N(-) [designated as PB by FlyBase] isoform of dFMR1 protein. What then, is the purpose of the Q/N(-) isoform? What happens when the Q/N(-) isoform cannot be produced? What is the significance of the highly conserved C-terminal peptide of FMR/FXR proteins? These questions can be answered only via generation and analysis of alleles that are designed to express a single isoform or mutation of interest.
Chapter 4A: Generation and analysis of fly stocks that produce either the Q/N(-) or Q/N(+) isoform as the sole source of dFMR1 protein The dFMR1 protein exists in several isoforms, some of which arise from alternative splicing events. The PB isoform is a 529 amino acid peptide lacking the Q/N domain present in other dFMR1 isoforms, and is derived from use of an alternative splice site that results in retention of a ∼540 nucleotide intron that has consensus sequences for 3′ end formation (see Figure from Chapter 3). The splicing reaction results in the PB isoform having a C-terminal peptide that is encoded from part of the retained intron, and terminates with the amino acid sequence DTN (asp- thr-asn).
The availability of cDNA and genomic clones of the dfmr1 locus allows for construction of an allele where the Q/N(-) isoform is no longer produced. Substitution of the alternative spliced intron that specifies the Q/N(-) isoform with dfmr1 cDNA results in such an allele (see Figure 1B). Gene engineering strategies described in the Methods section can be used to produce a dfmr1 allele that expresses only the Q/N(-) isoform.
84 Methods: Construction of a dfmr1 allele that expresses only the Q/N(+) isoform; A 14 kb BamHI-StuI fragment encompassing the dfmr1 locus was cloned to pBluescript and digested with NcoI and BspEI. This releases the region where the alternative splicing occurs to produce the Q/N(-) isoform. The vector and flanking dfmr1 sequences were then gel purified and ligated to an NcoI-BspEI fragment of dfmr1 cDNA to produce an allele that produces only the Q/N(+) isoform. This altered dfmr1 rescue fragment was then cloned to the white-marked pCaSpeR-4 transformation vector (Pirotta, 1988), and introduced to flies via P element-mediated germline transformation (Spradling and Rubin, 1982). The transgene insertions were mapped and then introduced into a dfmr1 null background where they served as the sole source of dFMR1 protein.
Construction of a dfmr1 allele that expresses only the Q/N(-) isoform: The 14 kb dfmr1 rescue fragment described above was used as starting material. To begin, a 450 bp segment of dfmr1 was PCR amplified, using a proofreading polymerase (Vent polymerase, New England Biolabs) and the following primer sequences:
Forward: 5′ TCAGGAGAAGATGGAGATTG 3′ Reverse: 5′ GGAATCCGGATCAATTTGTATCTCTCTCCACGCTGCTCATTTCACG 3′
The purpose of this PCR product is to create a terminal exon sequence that incorporates codons specifying the C-terminal peptide of the PB isoform. The reverse primer has codons for the carboxyl-terminal amino acids (DTN), a stop codon, and a BspEI site incorporated into the sequence. The amplified product was digested with NcoI and BspEI, and cloned to dfmr1 genomic DNA as depicted in Figure 1C. The intron specifying 3′ end formation for the PB isoform was then amplified using primers with BspEI or SacI restriction sites present. This 520 base pair sequence was amplified using Vent polymerase using the following primers:
Forward: 5′ GGAATCCGGAATACTCGATCCGATTCCCTC 3′ Reverse: 5′ GGAAGAGCTCGTTATCATAGTATACTAGCAG 3′
85 The resulting amplification product covers most of the intron, except for the splice site junctions. This product was digested with BspEI and SacI, then ligated to the above clone as depicted in Figure 1C. Clones were then sequenced to ensure fidelity of the amplification. These steps create an allele where the Q/N(-) isoform is expressed and utilizes the intron sequence for 3′ end formation. This dfmr1 allele was cloned to the pCaSpeR4 transformation vector and introduced to flies via P element-mediated germline transformation as described previously. The transgene insertions were mapped and then introduced into a dfmr1 null background where they served as the sole source of dFMR1 protein.
Neuroanatomical analyses Larval neuromuscular junction (NMJ) type I boutons were detected by staining third-instar larval fillets with anti-horseradish peroxidase (Cappel, Aurora, OH) at a dilution of 1:200. Mushroom bodies were visualized by staining whole-mounts of brains with anti-FasII at a 1:10 dilution (mAb 1D4 obtained from University of Iowa Developmental Studies Hybridoma Bank). Secondary antibodies conjugated to either HRP or fluorochrome were obtained from Jackson ImmunoResearch (West Grove, PA) and used at a 1:200 dilution. Confocal images were collected on an Olympus FV500 microscope. Western blots were performed as described in Wan et al. (2000) using anti-dFMR1 antibody 5A11 at a 1:1000 dilution and anti-β-tubulin mAb E7 (both from the University of Iowa Developmental Studies Hybridoma Bank) at a 1:100 dilution.
Circadian locomotion activity Circadian behavior was tested as described in (Dockendorff et al. 2002). Flies were entrained to a 12:12 light/dark cycle, placed into activity monitors (Trikinetics, Waltham, MA) maintained in light/dark cycles, and then placed under constant darkness. Locomotion activity was collected in 30-minute bins. The percentage of flies judged to be rhythmic was assessed by Clocklab software (Actimetrics, Evanston, IL) as follows: Using a confidence level of 0.025, batch analyses were performed for the genotypes tested, monitoring the locomotion activity in constant darkness over seven days. The difference between the power(1) and significance(1) values was calculated for each fly, and a value of less than 10 was the basis for judging an arrhythmic
86 phenotype. Visual analyses of periodograms and actograms were also conducted to confirm the results.
Results Numbers of larval neuromuscular junction boutons from Q/N(+) and Q/N(-) mutant stocks are similar to those observed in wild type animals Previous studies have shown that larvae homozygous for a null allele of dfmr1 have an increase in the number of neuromuscular junction (NMJ) boutons (Zhang et al., 2001). To test for NMJ development phenotypes that may result from expression of the Q/N(+) or Q/N(-) isoforms, larvae expressing these transgenes were analyzed for numbers of type 1 boutons from three different muscle types. Results from this experiment are presented in Figure 2, and show that the Q/N(+) and Q/N(-) transgenes expressed in a dfmr1 null background completely rescues the null phenotype, with bouton numbers being not statistically different as observed in wild type animals. This result shows that the Q/N(-) isoform performs all functions necessary for regulation of NMJ development and that a balance between the Q/N(+) and Q/N(-) isoforms is not needed to properly regulate bouton numbers.
The Q/N(+) and Q/N(-) transgenic stocks rescue mushroom body axon guidance phenotypes associated with dfmr1 null alleles Axon guidance defects are associated with null alleles of dfmr1 (Dockendorff et al., 2002; Morales et al., 2002; Michel et al., 2004). In wild type flies, the β lobe axons of mushroom bodies infrequently cross the midline of central brain, while in animals with null alleles of dfmr1, a higher percentage of brains show axon projections overextending in β lobe neurons (Michel et al., 2004). Two day-old adults expressing either wild type dFMR1, the Q/N(+) and Q/N(-) isoforms as sole dFMR1source, or null alleles were analyzed. All transgenic stocks expressing either the Q/N(+) or Q/N(-) isoforms provided rescue of the phenotype observed in the null allele (Figure 3). This shows that the two isoforms are each capable of providing the function needed to rescue this phenotype, and that other domains common to all dFMR1 isoforms are essential for proper development of the mushroom body β lobe.
87 Rhythmic circadian locomotion activity is affected when the Q/N(-) isoform is the sole source of dFMR1 A failure to maintain rhythmic locomotion activity in constant darkness is observed with a high degree of penetrance in flies homozygous for a null allele of dfmr1 (Dockendorff et al., 2002; Morales et al., 2002; Inoue et al., 2002). The transgenic stocks expressing Q/N(+) and Q/N(-) isoforms were tested for their ability to maintain rhythmic locomotion activity in constant darkness along with wild type and null controls. The results showed that the expression of Q/N(+) isoform was able to rescue the circadian phenotype, but fly stocks expressing Q/N(-) isoform as the sole source resulted in a partially penetrant arrhythmic circadian locomotion phenotype that resembles the one observed with the ΔQ/N stocks (Figure 4; see Figure from Chapter 3). The penetrance of this phenotype was not as high as that seen with null alleles of dfmr1. Based upon biochemical properties associated with Q/N domains, these results suggest that the PB isoform lacking the Q/N domain is unable to assemble into protein complexes that are needed for proper circadian locomotion behavior.
Discussion It was expected that the Q/N(-) isoform of dFMR1 would exhibit phenotypes quite similar to those arising form the ΔQ/N deletion. While this held true for the mushroom body axon guidance and rhythmic circadian locomotion phenotypes, it did not hold for the numbers of boutons observed at larval neuromuscular junctions. Why then do ΔQ/N mutants have an abnormal NMJ bouton number phenotype and the Q/N(-) isoform that also lacks a Q/N domain does not?
A number of explanations are possible for the differences in NMJ bouton number phenotypes between the ΔQ/N mutant and the Q/N(-) isoform. One is the matter of protein expression levels. Numbers of NMJ boutons are sensitive to the amount of dFMR1 protein expressed, with higher amounts of dFMR1 correlating with a decrease in boutons, and lesser amounts of dFMR1 associated with an increase in NMJ boutons (Zhang et al., 2001). The endogenous Q/N(-) isoform and the Q/N(-)-expressing transgene synthesized for this study utilize a 3′ UTR that is different from the one associated with other dFMR1 isoforms, and this may be an explanation for the relatively high level of expression from this transgene. An alternative explanation for the
88 difference between the Q/N(-) and ΔQ/N proteins is the evidence (discussed later in Chapter 4D) that expression of the Q/N(-) isoform is suppressed in the ΔQ/N background, and may result from defective regulation of alternative splicing needed to produce the Q/N(-) isoform. If this second scenario is true, then the Q/N(-) isoform, with its unique C-terminal peptide, may have a special role in regulating axon development and NMJ formation.
Chapter 4B: Does the dFMR1 Q/N domain exert its effects through interactions with other proteins? Rationale: Several studies indicate that Q/N domains act as protein-protein interaction platforms, and can facilitate both self-interactions and interactions with heterologous proteins (see Guo et al., 2007; Decker et al., 2007; Shewmaker et al., 2007; Kim et al., 2008, as just a few examples). The dFMR1 PB isoform (Q/N-) provides a partial rescue of rhythmic circadian locomotion activity over the dfmr1 null allele, but does not rescue to the level observed when the Q/N domain is present. If the dFMR1 Q/N domain functions to interact with other proteins, the replacement of one Q/N domain with another might not be expected to provide a complete rescue of phenotypes associated with deletion of the dFMR1 Q/N domain. To test this possibility, a chimeric gene where the codons specifying the dFMR1 Q/N domain are replaced with ones that also code for a Q/N domain from the orb2 gene of D. melanogaster. The Orb2 Q/N domain is essential for long-term memory of conditioned courtship training (Keleman et al., 2007). The peptide sequences of the dFMR1 and Orb2 Q/N domains are presented in Figure 1 D. Transgenic flies expressing this chimeric protein as the sole source of dFMR1 peptide have been analyzed for phenotypes described in the following paragraphs.
Methods: Construction of a dfmr1-orb2 chimeric transgene: The dfmr1 ΔQ/N transgene is left with a single BspEI site from deletion of the codons specifying Q/N residues. This plasmid was cut with BspEI, and then orb2 cDNA (clone LP05645, Berkeley Drosophila Genome Project) was used to amplify a 309 base pair segment spanning the codons that encode the Orb2 Q-rich region. The proofreading Vent polymerase (New England Biolabs) was used for the amplification, along with the following primers:
89 Forward: 5′ GGTTTCCGGAGGTGGCCTGCCGAATCTCAATC 3′ Reverse: 5′ GGTTTCCGGAGGGCGACACGCCCATCTGGGG 3′
The PCR product was cut with BspEI and ligated to BspEI digested dfmr1 ΔQ/N DNA. Plasmids were examined for the presence of the insert, and sequenced to check orientation and fidelity of the amplification. The resulting chimera construct was cloned to the pCaSpeR4 transformation vector and injected to embryos for germline transformation as described by Spradling and Rubin (1982).
Circadian behavior analyses These studies were performed as described in the preceding section
Results and conclusions A transgenic stock expressing the dFMR1-Orb2 chimeric protein was tested for its ability to maintain rhythmic locomotion activity in constant darkness, using flies with dfmr1 null alleles or ones with a wild type allele as comparative controls. The results show the chimeric protein supports a partial rescue of the dfmr1 null allele circadian phenotype (Figure 5), a finding that is not surprising, given the ability of the Q/N(-) isoform of dFMR1 to support rescue over the null allele. However, no significant difference in rescue capacity between the Q/N(-) isoform and the chimeric allele is seen, suggesting that an interaction with a heterologous protein by the dFMR1 Q/N domain is needed for normal circadian rhythmicity.
Chapter 4C: Are the effects of the dFMR1 Q/N domain deletion the result of interfering with the function of the highly conserved C-terminal peptide of dFMR1? Rationale: The majority of carboxyl terminal amino acids between the vertebrate FMR/FXR proteins have diverged, and could be a means by which these proteins exert unique functions. The corresponding portion of the dFMR1 protein has little sequence identity or similarity to FMR/FXR proteins as well. In contrast, the terminal six amino acids from the vertebrate FMR/FXR proteins and dFMR1 are quite highly conserved, suggesting that the exons encoding the C-terminal amino acids shared a common ancestor (Siomi et al., 1995; Zhang et al., 1995; Wan et al., 2000). Whether the terminal six amino acids of the above proteins have a function
90 distinct from the rest of the C-terminal peptide, or if there simply has been no selective pressure for these amino acids to diverge, is not clear. If the effects of the Q/N domain deletion are simply a consequence of interfering with function of the C-terminal peptide, abolishing the function of the C-terminal peptide should elicit the same phenotypes as the dfmr1 ΔQ/N domain allele.
Methods Generation of a dfmr1 allele that fails to express the conserved C-terminal peptide: A segment of genomic DNA encompassing the exons encoding the dFMR1 C-terminal region was amplified by PCR. The reverse primer sequence was designed with a termination codon immediately upstream of the codons for the conserved C-terminal peptide. The primer sequences are:
Forward: 5′ TCAGGAGAAGATGGAGATTG 3′ Reverse: 5′ CTTATCCGGATTAGCCCTCCTTTTTGACATTCTC 3′
The resulting PCR product has NcoI and BspEI sites at the ends that allows for substitution of the wild type NcoI-BspEI fragment with the mutant one (Figure 1E). The PCR product was sequenced to ensure no secondary mutations were introduced, and the mutant rescue fragment was cloned to the pCaSpeR4 vector and transformed to flies as described previously in this Chapter.
Neuroanatomical and circadian locomotion analysis These assays were done as described earlier in this Chapter.
Results The numbers of larval NMJ boutons from the C-terminal deletion mutants do not differ significantly from wild type Third-instar larvae expressing wild type dFMR1, those expressing the dFMR1 C-terminal deleted protein, and larvae homozygous for a null allele of dfmr1 (Figure 6A) were analyzed for neuromuscular junction bouton numbers. Three muscle types were analyzed for type 1 boutons in larval segment A4. The results from this experiment is presented in Figure 6B and show that 91 the C-terminal deletion mutation does not result in an significant increase of bouton numbers relative to wild type control in any muscle type. The results indicate that loss of these conserved C-terminal amino acids do not affect the proper development of axons and neuromuscular junctions, and thus the NMJ development phenotypes observed with the dFMR1 ΔQ/N protein are not simply the result of the C-terminal peptide being unable to perform a critical function.
Normal rhythmic circadian locomotion is observed in the C-terminal deletion mutants A transgenic stock expressing the C-terminal deleted protein was tested for its ability to maintain rhythmic locomotion activity in constant darkness. The results showed that the expression of the mutant protein was able to rescue the circadian phenotype to a level similar to that conferred by the wild type rescue fragment (Figure 6C), further suggesting that if the C-terminal six amino acids has a function, it is separate from that of the Q/N domain.
Discussion The dFMR1 C-terminal mutation does not affect any of the phenotypes that are associated with the Q/N domain deletion. It is possible that the dFMR1 C-terminal peptide has functions in other processes (e.g. courtship behavior) that are not analyzed here, or that if the peptide has a function, it may be distinct from that of the Q/N domain. Another possibility is that the C- terminal six amino acids do not participate in any specific function, and has remained conserved simply because there has been no selective pressure for the codons that specify these amino acids to diverge.
Chapter 4D: The dFMR1 Q/N domain may be necessary for efficient alternative splicing of its pre-mRNA transcript Rationale: An RNA binding protein has the potential to participate in all aspects of biogenesis, function, and turnover of RNA molecules. These steps include RNA processing, nucleocytoplasmic transport, subcellular localization of RNA, regulation of translation, and degradation. Both human and fly FMRP are reported to bind their mRNA transcript (Ashley et al., 1993; Zhang et al., 2001). The dfmr1 transcript may thus serve as a model for how its protein product (dFMR1) can affect RNA metabolism.
92 As discussed in previous sections, the dfmr1 pre-mRNA is subject to alternative splicing, and the Q/N domain may facilitate interactions that allow dFMR1 to assemble into ribonucleoprotein (RNP) complexes. If the dFMR1 Q/N domain is needed for interactions that modulate alternative splicing pathways, it is possible that the dFMR1 ΔQ/N protein is ineffective at promoting the splicing pattern necessary for synthesis of the dFMR1 PB isoform. The dFMR1
ΔQ/N protein has a Mr that is slightly higher than the native PB isoform, and these species can be resolved via SDS-PAGE.
Results and Discussion: A Western blot of whole fly extracts from w1118 (wild type) and a dFMR1 ΔQ/N mutant show that the endogenous PB isoform is poorly expressed in the dFMR1 ΔQ/N mutant (Figure 7). This result indicates that dFMR1 protein participates in regulation of alternative splicing, and that the Q/N domain is needed for the process. Further experimentation (Northern blotting or RT-PCR analyses) is needed to confirm this observation. If confirmed, this finding will demonstrate a novel role for dFMR1, and it is quite probable that such a function will extend to the vertebrate FMR/FXR proteins as well.
Alternative splicing is believed to be more common in the central nervous system (CNS) relative to other tissues, and the transcripts of ion channels and membrane-bound receptors are just a few of the neuronal mRNAs that are regulated in this manner (Yeo et al., 2004; Lipscombe, 2005; Blencowe, 2006). The diversity of protein isoforms resulting from alternative splicing may contribute towards the fine-tuning of synaptic function that occurs in response to stimulation (reviewed by Licatalosi and Darnell, 2006). Many neurologic diseases, including complex behavioral disorders such as schizophrenia, are traced to defects in splicing and its regulation (Clinton et al., 2003; Licatalosi and Darnell, 2006). As FMRPs likely bind hundreds of RNA species (Brown et al., 2001; Darnell et al., 2001), it is easy to imagine that FMR1 mutation could result in perhaps dozens of these RNA substrates having aberrant splicing patterns that prevent the full repertoire of protein isoforms from being expressed. It seems quite probable that such dysregulation would contribute towards the phenotypes of fragile X syndrome.
93 LITERATURE CITED
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Brown, V., Jin, P., Ceman, S., Darnell, J.C., O’Donnell, W.T., et al. (2001). Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell 107, 477-487.
Clinton, S.M., Haroutunian, V., Davis, K.L., Meador-Woodruff, J.H. (2003). Altered transcript expression of NMDA receptor-associated postsynaptic proteins in the thalamus of subjects with schizophrenia. Am. J. Psychiatry. 160, 1100-1109.
Darnell, J.C., Jensen, K.B., Jin, P., Brown, V., Warren, S.T., Darnell, R.B. (2001). Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function. Cell 107, 489-499.
Decker, C.J., Teixeira, D., Parker, R. (2007). Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae. J. Cell. Biol. 5, 437- 449.
Dockendorff, T.C., Su, H.S., McBride, S.M.J., Yang, Z., Choi, C.H., et al. (2002). Drosophila lacking dfmr1 activity show defects in circadian output and fail to maintain courtship interest. Neuron 34, 973- 984.
Guo, L., Han, A., Bates, D.L., Cao, J., Chen, L. (2007). Crystal structure of a conserved N- terminal domain of histone deacetylase 4 reveals functional insights into glutamine-rich domains. Proc. Natl. Acad. Sci. U S A. 104, 4297-4302.
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Keleman, K., Krüttner, S., Alenius, M., Dickson, B.J. (2007). Function of the Drosophila CPEB protein Orb2 in long-term courtship memory. Nat. Neurosci. 10, 1587-1593.
Kim, D.H., Kim, G.S., Yun, C.H., Lee, Y.C. (2008). Functional conservation of the glutamine- rich domains of yeast Gal11 and human SRC-1 in the transactivation of glucocorticoid receptor Tau 1 in Saccharomyces cerevisiae. Mol. Cell. Biol. 28, 913-925.
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Lipscombe, D. (2005). Neuronal proteins custom designed by alternative splicing. Curr. Opin. Neurobiol. 15, 358-363.
94 Michel, C.I., Kraft, R., Restifo, L.L. (2004). Defective neuronal development in the mushroom bodies of Drosofila Fragile X Mental Retardation 1 mutants. J. Neurosci. 24, 5789-5809.
Morales, J., Hiesinger, P.R., Schroeder, A.J., Kume, K., Verstreken, P. et al. (2002). Drosophila fragile X protein, DFXR, regulates neuronal morphology and function in the brain. Neuron 34, 961-972.
Pirrotta, V. (1988). Vectors for P-mediated transformation in Drosophila. Biotechnology 10, 437-456.
Siomi, M.C., Siomi, H., Sauer, W.H., Srinivasan, S., Nussbaum, R.L., Dreyfuss, G. (1995). FXR1, an autosomal homolog of the fragile X mental retardation gene. EMBO J. 14, 2401-2408.
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Spradling, A. C., and Rubin, G. M. (1982). Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218, 341-347.
Wan, L., Dockendorff, T.C., Jongens, T.A., Dreyfuss, G. (2000). Characterization of dFMR1, a Drosophila melanogaster homolog of the Fragile X mental retardation protein. Mol. Cell. Biol. 20, 8536-8547.
Yeo, G., Holste, D., Kreiman, G., Burge, C.B. (2004). Variation in alternative splicing across human tissues. Genome. Biol. 5, R74.
Zhang, Y., O'Connor, J. P., Siomi, M. C., Srinivasan, S., Dutra, A. et al. (1995). The fragile X mental retardation syndrome protein interacts with novel homologs FXR1 and FXR2. EMBO J. 14, 5358-5366.
Zhang, Y.Q., Bailey, A.M., Matthies, H.J., Renden, R.B., Smith, M.A. et al. (2001). Drosophila fragile X-related gene regulates the MAP1B homolog Futsch to control synaptic structure and function. Cell 107, 591–603.
95 Nco1 BspE1
A. Q/N Tudor/Agenet KH1 KH2 related Nco1 BspE1 dfmr1 cDNA
B. Q/N Tudor/Agenet KH1 KH2 related
C. DTN Tudor/Agenet KH1 KH2 Intron derived 3΄ UTR related
Orb2 Q-rich
BspE1 D. Tudor/Agenet KH1KH2 ΔQ/N related
Drosophila Orb2B dFMR1 GGLPNLNLNKPPQLHQQQHQQ NGAVANNNNKPQSAQQPQQQ QHQQHQQHQQQQQLHQHQQ QPPAPGNKAALNAGDASKQNS QLSPNLSALHHHHQQQQQLRE GNANAAGGASKPKDASRNGDK SGGSHSPSSPGGGGGGSPYN QQAGTQQQQPSQVQQQQAAQ GSQAGCSSGGISPIPPQMGVSP QQQPKPRRNKNRSNNHTDQPS GQQQLAENVKKEGLVNGTS
E.
TAA
Q/N LVNGTS Tudor/Agenet KH1KH2 related
Figure 1: Schematic of dfmr1 alleles affecting the presence of C-terminal peptides. A) The wild type allele of dfmr1. Only select introns are depicted for clarity. B) Removal of a segment of dfmr1 genomic DNA spanning an alternatively spliced intron that specifies production of Q/N(+) or Q/N(-) isoforms of dFMR1. Substitution of this region with dfmr1 cDNA creates an allele where only the Q/N(+) isoform is expressed. C) Generation of a dfmr1 allele that expresses only the Q/N(-) isoform. The terminal exon of the coding sequence is modified so that the tripeptide DTN is the C-terminus. The alternatively spliced intron has been modified to remove splice
96 sites, and inserted downstream to provide sequences for 3′ end formation. D) A chimeric dfmr1::orb2 gene is synthesized by replacing the dfmr1 Q/N coding region with a segment of the D. melanogaster orb2 gene that encodes a Q-rich domain. The sequences of the dFMR1 Q/N domain and Orb2 Q-rich peptide sequence present in the dFMR1::Orb2 chimera are shown. E) C-terminal peptides of dFMR1, hFMR1, FXR1, and FXR2 are highly conserved. A stop codon was introduced upstream of the codons specifying this peptide in the dfmr1 genomic rescue fragment, creating a dfmr1 allele that does not express these conserved amino acids.
97 A. C.
dFMR1 dFMR1
β-tubulin β-tubulin w1118 4-9 3-30 w1118 Q/N(-) Q/N(+)
Muscle 6/7 Muscle 6/7 B. Muscle 6/7 D. Muscle 6/7 60 60 *** *** 50 50
40 40
30 30
20 20 Number of Boutons of Number Number of Boutons of Number
10 10 Number of Boutons of Number Number of Boutons of Number
0 0 w1118 3-30 L 4-9 L null w1118 short null w1118 3-30 4-9 Null w1118 Q/N(-) Null
Muscle 12 Muscle 12
40 Muscle 12 Muscle 12 *** 40 *** 35 35
30 30
25 25
20 20
15 15 Number of Boutons of Number Number of Boutons 10 10
5 5 Number of Boutons of Number Number of Boutons of Number 0 0 w1118 3-30 L 4-9 L null w1118 short null w1118 3-30 4-9 Null w1118 Q/N(-) Null
Muscle 4 Muscle 4
40 Muscle 4 Muscle 4 *** 40 *** 35 35
30 30
25 25
20 20
15 15 Number of Boutons of Number Number of Boutons of Number 10 10
5 5 Number of Boutons of Number Number of Boutons of Number
0 0 w1118 3-30 L 4-9 L null w1118 short null w1118 3-30 4-9 Null 1118 Q/N(-) Null w
Figure 2: Expression of either a Q/N(+) or Q/N(-) isoform of dFMR1 rescues the neuromuscular junction (NMJ) bouton overgrowth phenotype associated with null alleles of dfmr1. A) dFMR1 protein levels from two transgenic stocks that express the dFMR1 Q/N(+) isoform. B) Studies of three muscle types show that larvae expressing a Q/N(+) isoform as the sole source of dFMR1 have NMJ bouton numbers that are statistically indistinguishable from wild type larvae. ANOVA shows that a null allele of dfmr1 results in an increase of bouton numbers that is above both wild type and Q/N(+) alleles. P ≤ 0.01 comparing Q/N(+) to null allele, and P for the ANOVA is < 0.0001. N is ≥ 30 for all tested genotypes. C) Expression of a transgene 98 expressing the Q/N(-) isoform of dFMR1. The enhanced expression of this protein may be the result of the unique 3′ UTR, as multiple transgenic lines gave a similar or higher level of expression. D) Larvae expressing a Q/N(-) isoform of dFMR1 have NMJ bouton numbers that are statistically indistinguishable from wild type in three muscle types. ANOVA shows that a null allele of dfmr1 results in an increase of bouton numbers that is above both wild type and Q/N(-) alleles. P is ≤ 0.01 comparing Q/N(-) to the null allele and P for the ANOVA is < 0.0001. N ≥ 30 for all tested genotypes.
99
100
90
80
70
60 ***
50
40
30
20
10 % no crossover phenotype no crossover % 0 WTwt rescuerescue Q/N(+) 3-30 L Q/N(-) short Null null
Figure 3: Expression of either a Q/N(+) or Q/N(-) isoform of dFMR1 rescues axon guidance defects in mushroom body β-lobes that are observed with dfmr1 null alleles. Brains from two- day old adults were dissected and stained with anti-Fascicilin II antibody to visualize mushroom bodies. β-lobe axons were examined for crossing of the CNS midline. Fisher’s Exact Test shows that both transgenes provide a level of rescue indistinguishable from a wild type background (P > 0.7 for wild type and Q/N(+); P = 0.0007 for wild type and null). N ≥ 30 for all genotypes.
100
A. B. dFMR1 p = 0.047
100
β-tubulin 90 p = 0.035 w1118 7 80 Q/N(+) 70 60
50
40
Percent rhythmic Percent 30
20
10
0 wt rescue L isoform S isoform null WT rescue 7 Q/N(-) Null Q/N(+)
Figure 4: The Q/N(-) isoform of dFMR1 does not provide a wild type level of rescue for rhythmic circadian locomotion activity, while the Q/N(+) isoform does. Flies were entrained to a 12:12 light-dark cycle, then placed in constant darkness for seven days. The percentage of flies that retained rhythmic locomotion activity was assessed and compared via Fisher’s Exact Test (P = 0.047 for wild type and Q/N(-); P = 0.2 for wild type and Q/N(+). N ≥ 18 for all genotypes tested.
101
circadian data A. B. 90
80 dFMR1 70 60
50
40
percent rhythmic 30 β-tubulin 20 Percent rhythmic Percent
10 w1118 Ch 26 Ch 13A 0 WTwt rescue chimera PB(Q/N-) null Ch 13A Q/N(-) Null
Figure 5: A dFMR1::Orb2 chimeric protein fails to rescue defective rhythmic circadian locomotion activity observed with flies expressing the dFMR1 Q/N(-) isoform. A) Expression levels of dFMR1::Orb2 chimeras demonstrate that the protein is stable. B) The percentage of dFMR1::Orb2 animals that are judged to retain rhythmic locomotion activity does not differ compared to animals expressing a dfmr1 Q/N(-) transgene (P = 1.0, Fisher’s Exact Test). N ≥ 24 for the two genotypes compared.
102 A. B. Muscle 6/7 muscle 6/7 60 *** dFMR1 50
40 β-tubulin 30
1118 1118 w ΔC-2 w ΔC-32 20 Number of Boutons of Number
Number of Boutons of Number 10
0 w11181118 del-C null Circadian data w ΔC-32 Null C.
100 Muscle 12 Muscle 12 90 40 ** 80 35 70 30 60 25 50 20 40 15 percent rhythmic 30 Number of Boutons of Number Percent rhythmic Percent 10 20 Number of Boutons of Number 10 5
0 0 WTwt rescuerescue Δ del-cC-2 Null null ww11181118 Δ del-CC-32 Null null
Muscle 4 Muscle 4
40
35
30
25
20
15 Number of Boutons of Number 10 Number of Boutons of Number 5
0 w1118 del-C null w1118 ΔC-32 Null
Figure 6: Loss of the conserved C-terminal peptide of FMRPs does not significantly impair dFMR1 function. A) Expression levels of ΔC dFMR1 protein from two transgenic stocks. B) NMJ bouton numbers from larvae of wild type, dfmr1 ΔC and null genotypes. In muscle 6/7 and 12, the null allele results in significantly higher numbers of boutons than that seen in larvae expressing the dFMR1 ΔC protein (P ≤ 0.01; P for the ANOVA is ≤ 0.0001). For muscle 4, the bouton numbers from the dFMR1 ΔC-expressing larvae are not different from either the wild type or null allele larvae. N ≥ 29 for all genotypes. C) The dFMR1 ΔC protein rescues rhythmic circadian locomotion activity to a level indistinguishable from wild type animals (P = 0.7, Fisher’s Exact Test). N ≥ 23 for the two genotypes.
103
dFMR1 Q/N(+)
Δ Q/N 8 dFMR1 Q/N(-)
β-tubulin
Figure 7: The dFMR1 ΔQ/N protein may result in defective regulation of the alternative splicing pathway needed to produce the Q/N(-) isoform (Flybase isoform PB). Protein extracted from whole adult flies were separated on a 6% gel, blotted, and probed with anti-dFMR1 antibody. β- tubulin was detected as a loading control.
104 CHAPTER V
SUMMARY
The ability to generate novel alleles of genes in Drosophila provides an in vivo approach to analyze gene and protein function. The goal of this thesis was to exploit these properties as a means to dissect the function of fragile X protein. This approach is particularly important for studies of FMRP, since most cases of fragile X syndrome arise from expansion of a CGG trinucleotide repeat followed by cytosine methylation that silences the FMR1 gene. As a result, information on FMRP domain function in the context of an intact organism is scarce. Generating alleles to dissect dfmr1 function may help validate or call into question some of the working hypotheses that guide the fragile X field (and perhaps neuroscience) that were developed primarily through in vitro biochemical and cell culture assays.
There may be many mechanisms by which FMRP regulates neuronal function A new consideration on the importance of KH domains towards FMRP function was uncovered in this study. The severity of phenotypes associated with a fragile X patient with an I304N substitution, and the inability of the mutant protein to interact with polyribosomes in vitro led to a supposition that the RNA binding capacity of the second KH domain is critical for FMRP function. Further examination of these suppositions requires recapitulation of the analogous KH domain substitutions in an intact animal model. Analysis of the KH domain mutants revealed partial or no loss-of-function for neural development and behavior phenotypes when compared to deletion null alleles of dfmr1. This observation is consistent with the idea that individual domains of FMRP can contribute in either an additive or independent manner to FMRP function, and shows that other domains of FMRP contribute towards regulatory processes needed for normal neural development and behavior. An association of FMRP with ribosomes is believed to be important for regulation of protein synthesis at synapses that is required for synaptic plasticity. The I307N mutant allele generated in this study will be helpful for testing whether the ribosome association of FMRP is essential for establishment and/or consolidation of memory in the conditioned courtship paradigm.
105 Although several RNA binding and protein-protein interaction domains are present in FMRP, a region that may have a broad impact on FMRP function are the pair of Agenet domains present in the N-terminus of FMRP. Agenet domains are related to methyl substrate binding Tudor and chromo domains that are implicated in regulation of chromatin structure (Maurer-Stroh et al., 2003). The Agenet domains in FMRP are highly conserved between vertebrates and insects (Wan et al., 2000; Maurer-Stroh et al., 2003), suggestive of an important role in FMRP function. Methylated histones and small RNAs play a major role in regulation of chromatin structure (Grewal and Elgin, 2007), and the ability of the FMRP Agenet domains to bind RNA (Adinolfi et al., 1999; 2003) and perhaps methylated substrates, are consistent with a role in this process. To define such a role for Agenet domains of FMRP would provide an important and novel insight into FMRP function.
Likewise, the possibility that FMRPs can modulate alternative splicing provides yet another means by which they regulate gene expression. Both regulators of chromatin structure and alternative splicing can influence the expression of many genes, and the potential for numerous genes to be dysregulated at these levels of control very likely contributes towards the phenotypes of fragile X syndrome. While the enthusiasm towards testing of mGluR antagonists as a therapy for fragile X syndrome is well justified, it should be noted that mGluR antagonists are not able to rescue the circadian locomotion behavior phenotype associated with dfmr1 null alleles (McBride et al., 2005), indicating that control of this FMRP-dependent behavior is independent of mGluR signaling. If fragile X behavior phenotypes arise from dysregulation of chromatin structure or alternative splicing, it is important to note that there are pharmacologic and molecular means developed to modulate these processes. For example, inhibitors of histone deacetylase activity rescue a late phase of long-term potentiation (L-LTP) and long-term memory of contextual fear conditioning in a mouse model for Rubenstein-Taybi syndrome (RTS; Alarcón et al., 2004). RTS is a rare form of mental retardation that arises from heterozygosity for the gene that encodes CREB binding protein (CBP), a known histone acetyltransferase. In a similar vein, several strategies have been developed to correct aberrant splicing patterns (reviewed in Licatalosi and Darnell, 2006). The discovery of novel functions for FMRP and the means by which it carries them out may uncover connections to pre-existing pharmaceutical and/or molecular therapeutic strategies that can be used to treat fragile X syndrome.
106 Testing the role of a Q/N rich domain Long-term synaptic connectivity requires that regulators of the process maintain a stable and active state long after synaptic transmission has taken place. Recently, a glutamine/asparagine (Q/N) rich domain was proposed to adopt a prion-like state to regulate the activity and stability of Aplysia CPEB (Si et al., 2003). Since Aplysia is not a genetic model, these investigators were unable to assess whether the Q/N domain was necessary for long-term facilitation. During the course of this study, we observed that the C-terminal peptide of Drosophila fragile X protein is enriched in Q/N residues, providing an opportunity to test via genetic means whether such a domain is necessary for the role of dFMRP in neural development and memory of conditioned courtship training. While the results from Chapter 3 clearly show that the dFMR1 Q/N domain is important for some dfmr1 phenotypes, the mechanism by which it functions is not completely clear. Although the dFMR1 Q/N domain confers properties of in vivo protein aggregation and resistance to protease in vitro, the high frequency of switching between active and inactive states of the dFMR1 Q/N::GR526 fusion protein indicates that the dFMR1 Q/N domain might not behave as a prion. However, prion propagation can be highly species-specific, and thus it cannot be conclusively ruled out that the dFMR1 Q/N domain can behave as a prion within the context of its host. It should be noted that of the ~100 Saccharomyces proteins judged to have a Q/N- rich domain, only a very few of them were demonstrated to adopt a prion state (Michelitsch and Weissman, 2000; Sondheimer and Lindquist, 2000).
Even though the N-terminal region is highly conserved, there is little primary sequence conservation between C-terminal peptides of human and fly FMRP. However, the last six amino acids between fly and human FMR/FXR proteins are highly conserved, which suggests that codons specifying the C-terminal peptides between these two species are derived from a common ancestral exon. Preliminary tests with the C-terminal peptides of human FMRP show that it exhibits certain properties similar to that seen with the dFMRP Q/N domain. This perhaps is indicative of a conserved FMRP function between arthropods and mammals, where the C- terminal peptide of human FMRP might have similar protein-protein interaction properties as seen in dFMRP Q/N domains. Whether the C-terminal peptide of both fly and human FMRP undergoes self-interaction or interaction with other proteins will be subject to proteomics-based study in the future. Based upon results from the conditioned courtship assays, it is possible that
107 components of RNP complexes that are needed for memory consolidation will be identified through such studies.
Future prospects for molecular genetic analyses of mental retardation Many single gene mutations leading to mental retardation in humans have obvious orthologues in flies (Inlow and Restifo, 2004), and it is highly plausible that approaches similar to the ones presented in this study will be informative and have a beneficial impact on the treatment of mental retardation. It will be of interest to see if genes resulting in mental retardation can be grouped together according to similar or common molecular pathways. Such findings would raise the possibility that seemingly disparate causes of mental retardation could be treated via the same therapeutic approach. The oft-mentioned amenability of Drosophila melanogaster to molecular genetic analyses will be instrumental in successful pursuit of such an endeavor.
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