Neuroscience Letters RNA-Mediated Pathogenesis in Fragile X

Neuroscience Letters 466 (2009) 103–108

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Neuroscience Letters

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Review RNA-mediated pathogenesis in fragile X-associated disorders

Huiping Tan a,b,HeLib, Peng Jin a,∗ a Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA b Division of Histology and Embryology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China article info abstract

Article history: Noncoding RNAs play important and diverse regulatory roles throughout the genome and make major Received 13 May 2009 contributions to disease pathogenesis. The FMR1 gene is involved in three different syndromes: fragile Received in revised form 16 July 2009 X syndrome (FXS), primary ovarian insufﬁciency (POI), and fragile X-associated tremor/ataxia syndrome Accepted 20 July 2009 (FXTAS) in older patients. Noncoding RNAs have been implicated in the molecular pathogenesis of both FXS and FXTAS. Here we will review our current knowledge on the role(s) of noncoding RNAs in FXS and Keywords: FXTAS, particularly the role of the microRNA pathway in FXS and the role of noncoding riboCGG (rCGG) Noncoding RNAs repeat in FXTAS. FMR1 Fragile X syndrome © 2009 Elsevier Ireland Ltd. All rights reserved. FXTAS

Recent genome-wide interrogations of transcribed RNA have 4000 males and 1 in 8000 females [58]. In addition to cognitive yielded compelling evidence for its pervasive and complex tran- deficits, the phenotype of fragile X syndrome includes mild facial scription throughout most of the human genome. Nevertheless, dysmorphology (prominent jaw, high forehead, and large ears), a significant portion of this transcribed RNA appears to be non- macroorchidism in postpubescent males, and subtle connective protein-coding and is currently uncharacterized. In-depth analysis tissue abnormalities [58]. Many patients also manifest attention- of 1% of the human genome (∼30 Mb) performed by the Encyclo- deficit hyperactivity disorder and autistic-like behavior. As the first pedia of DNA Elements (ENCODE) project revealed that 92.6% of condition found to be caused by trinucleotide repeat expansion, the interrogated bases could be detected as primary transcripts and fragile X syndrome is typically the result of a massive CGG trinu- that, among these, there were many novel non-protein-coding tran- cleotide repeat expansion within the 5 untranslated region (UTR) scripts [7]. Similarly, another study found that the majority (64%) of the fragile X mental retardation 1 gene (FMR1), which results of polyadenylated (poly-A+) transcripts ≥200 nucleotides (nt) in in transcriptional silencing of FMR1 [19,39,47,48,57]. The majority length lay outside annotated protein-coding regions [35]. These of affected individuals have expansions of over 200 CGG repeats, and other genome-wide analyses have led to the identification of referred to as the full mutation. Most people in the general pop- tens of thousands of noncoding RNA transcripts expressed from the ulation carry between 5 and 54 repeats, while those individuals human genome, most of which have yet to be functionally char- who carry CGG repeat expansions between 55 and 200 are referred acterized. Along with the revelation that noncoding RNAs in the to as premutation carriers. Over the last several years, male and human genome are surprisingly abundant, there has been a surge to a lesser extent female premutation carriers are found to be at in molecular and genetic data showing diverse and important reg- increased risk of developing an age-dependent progressive inten- ulatory roles for noncoding RNA. It has been shown that both the tion tremor and ataxia syndrome, which is uncoupled from fragile development and proper function of the nervous system require the X syndrome [24] and known as fragile X-associated tremor/ataxia intricate spatiotemporal expression of a wide repertoire of regula- syndrome (FXTAS) (Fig. 1). Besides a progressive action tremor tory RNAs. Misregulation of these regulatory RNAs could contribute with ataxia, patients with FXTAS also show a progressive cogni- to the abnormalities in brain development that are associated with tive decline that ranges from executive and memory deficits to neurodevelopmental disorders. dementia [24]. In addition, female premutation carriers are also Fragile X syndrome (FXS) is the most common form of inher- found to have significantly higher rates of primary ovarian insuffi- ited mental retardation, with an estimated prevalence of 1 in ciency (POI); however, the exact molecular basis of POI has yet to be determined (Fig. 1).

∗ Although both FXS and FXTAS involve CGG repeat expansions Corresponding author at: Department of Human Genetics, Emory University in the FMR1 gene, the clinical presentations and molecular mecha- School of Medicine, 615 Michael Street, Suite 301, Atlanta, GA 30322, USA. Tel.: +1 404 727 3729; fax: +1 404 727 5408. nisms underlying each syndrome are distinct. Interestingly, recent E-mail address: [email protected] (P. Jin). studies have implicated noncoding RNAs in the pathogenesis of

nous double-strand transcripts [5]. Typically, miRNAs bind to the 3 UTR of target mRNA, leading to translational repression or mRNA degradation. The roles miRNAs play in diverse biological path- ways have been reviewed extensively. Endogenous miRNAs were found associated with FMRP, and FMRP interacts biochemically and genetically with known components of the miRNA pathway [8,28,61]. Experiments in Drosophila revealed specific biochemical interactions between dFmrp and functional RNA-induced silencing complex (RISC) proteins, including dAGO1, dAGO2, and Dicer [8,28,61]. dFmr1 displays strong genetic interaction with dAGO1, and dAGO1 dominantly interacts with dFmr1 in both dFmr1 overexpression and loss-of-function models [33]. Furthermore, dFmr1 also interacts genetically with AGO2, as exemplified by their ability to coregulate ppk1 mRNA levels [61]. Additional studies provide further evidence for the involvement of FMRP in miRNA-containing RISC and P body-like granules in Drosophila neurons [4]. Recombi- Fig. 1. Mutations in the FMR1 gene can lead to three distinct disorders. The nor- nant human FMRP can also act as an acceptor for Dicer-derived mal human FMR1 gene possesses a CGG trinucleotide repeat size of between 5 miRNAs, and importantly, endogenous miRNAs themselves are and 54 in the 5 UTR. A large expansion of over 200 CGG repeats triggers the tran- associated with FMRP in both flies and mammals [8,28,33].Inthe scriptional silencing of FMR1, which results in fragile X syndrome (FXS). For those adult mouse brain, Dicer and eIF2c2 (the mouse homolog of AGO1) individuals with CGG repeat expansions between 55 and 200, an age-dependent progressive neurodegenerative disorder, called fragile X-associated tremor/ataxia interact with FMRP at postsynaptic densities [45]. Recently, the syndrome (FXTAS), has been recognized among many male carriers in or beyond phosphorylation of FMRP was also shown to increase its association their fifth decade of life. Female carriers, on the other hand, may suffer from primary with miRNA precursors [9,10]. Presumably, this interaction works ovarian insufficiency (POI). to regulate translation of target mRNAs in an activity-dependent manner. Based on these findings, it has been proposed that the RISC both disorders. Here we will review our current knowledge about proteins, including Argonaute (Ago) and Dicer, could interact with the involvement of noncoding RNAs in FXS and FXTAS, particularly FMRP and use the loaded guide miRNA(s) to interact with target the role of the microRNA (miRNA) pathway in FXS and the role of sequences within the 3 UTR of RNA bound to FMRP and suppress noncoding riboCGG (rCGG) repeat in FXTAS. its translation [31,33]. In this model, FMRP facilitates the interaction Identification of other mutations (e.g., deletions in patients with between miRNAs and their target mRNA sequence, ensuring proper the typical phenotype) has confirmed that FMR1 is the only gene targeting of guide miRNA-RISC within the 3 UTRs and proper trans- involved in the pathogenesis of fragile X syndrome and that the loss lational suppression. of the FMR1 product, fragile X mental retardation protein (FMRP), The fact that FMRP is associated with Dicer, miRNAs, and specific causes fragile X syndrome [14,44,60]. FMRP along with its auto- mRNA targets raises the question of whether FMRP is associated somal paralogs, the fragile X-related proteins FXR1P and FXR2P, with specific miRNAs and modulates their processing. To address constitutes a small, well-conserved family of RNA-binding pro- this question, the expression and processing of miRNAs were exam- teins (fragile X-related gene family) that share over 60% amino ined in Drosophila dFmr1 mutants; in the fly brain, dFmrp was acid identity and contain two types of RNA-binding motifs: two specifically associated with miR-124a, a nervous system-specific ribonucleoprotein K homology domains (KH domains) and a clus- miRNA [62]. dFmrp is required for the proper processing of pre-miR- ter of arginine and glycine residues (RGG box) [51,65]. Both the 124a, whereas the loss of dFmr1 leads to a reduced level of mature KH domains and RGG box of FMRP have been found to medi- miR-124a and an increased level of pre-miR-124a. These results ate FMRP-RNA interactions both in vitro and in vivo [2,16,17,41,43]. suggest a modulatory role for dFmrp to maintain proper levels of Both motifs contribute to the role of FMRP as a suppressor of tar- miRNAs during neuronal development [62]. In our own studies, we get messenger RNA (mRNA) translation via binding of noncoding have shown that dFmr1, the Drosophila ortholog of the FMR1 gene, RNA structures, including G-quartets and “kissing complexes” (also plays a role in the proper maintenance of germline stem cells in known as loop-loop-pseudoknots), within the untranslated regions Drosophila ovary, potentially through the miRNA pathway [63].To of target mRNAs [2,12,13,16,41,43,50,53]. The ability of FMRP to bind test this, we recently used an immunoprecipitation assay to reveal RNA and suppress translation has definite clinical relevance, as evi- that specific microRNAs (miRNAs), particularly the bantam miRNA denced by a severely affected individual with an I304N missense (bantam), are physically associated with dFmrp in ovary [64].We mutation in the KH2 domain of FMRP [17]. As an RNA-binding pro- found that, like dFmr1, bantam is not only required for repressing tein, FMRP is found to form a messenger ribonucleoprotein (mRNP) primordial germ cell differentiation, it also functions as an extrin- complex that associates with translating polyribosomes [16]. Fur- sic factor for germline stem cell maintenance [64]. Furthermore, thermore, FMRP is known to be involved in translational control we showed that bantam genetically interacts with dFmr1 to reg- and could suppress translation both in vitro and in vivo [41,43].At ulate the fate of germline stem cells [64]. Collectively, our results the cellular level, abnormal dendritic spines are found in the brains support the notion that the FMRP-mediated translational pathway of both human patients with fragile X syndrome and Fmr1 knock- functions through specific miRNAs to control stem cell regulation; out (KO) mice, implying that synaptic plasticity is affected in the however, we saw no effect of dFmrp on the biogenesis of the ban- absence of FMRP [11,46]. Based on these observations, it has been tam miRNA. Whether FMRP is associated with specific miRNAs in proposed that FMRP is involved in synaptic plasticity via regulation mammalian cells remains to be determined. of mRNA transport and local protein synthesis of specific mRNAs All the current findings support the idea that FMRP could at synapses. Due to space limitations, we will focus here on the regulate the translation of its mRNA through miRNA interaction. potential role of the miRNA pathway in fragile X syndrome (for However, the exact mechanism of its action together with the an in-depth discussion of the molecular pathogenesis of fragile X RISC and miRNAs is not yet clear. Thus the relevance of these syndrome, please see the recent review by Bassell and Warren [6]). observations to FMRP-mediated translational regulation and the MicroRNAs (miRNAs) are a new class of small noncoding RNAs possible involvement of miRNAs in mental retardation more gen- that are ∼22 nucleotides (nt) in length and generated from endoge- erally remain to be explored. H. Tan et al. / Neuroscience Letters 466 (2009) 103–108 105

Fragile X-associated tremor/ataxia syndrome (FXTAS) has been show symptoms of FXTAS [15]. These estimates do not consider a recognized in older males of FXS families and is uncoupled from size bias, which takes into account the correlation between the age the neurodevelopmental disorder, FXS [23]. Although both dis- of onset of symptoms and the size of the repeat. Recent studies orders involve repeat expansions in the FMR1 gene, the clinical have correlated the age of onset of clinical FXTAS symptoms with presentation and molecular mechanisms underlying each are com- the length of expanded repeats and shown that larger CGG repeats pletely distinct. The most common clinical feature of FXTAS is a represent an increased risk factor for the development of FXTAS progressive action tremor with ataxia. More advanced or severe [30,42]. Moreover, the degree of brain atrophy and severity of the cases may show a worsening cognitive decline that ranges from tremor and ataxia are associated with the CGG repeat length [42]. executive and memory deﬁcits to dementia [22]. Patients may also Some female carriers also develop clinical features of FXTAS, but at present with common psychiatric symptoms, such as increased a much lower frequency than males, which may be due to partial anxiety, mood liability, and depression [3,27]. Nearly all case studies protection conferred by random X-inactivation of the premutation from autopsies on the brains of symptomatic premutation carri- allele [25,29,66]. ers demonstrate degeneration in the cerebellum, which includes At the molecular level, the premutation is different from either Purkinje neuronal cell loss, Bergman gliosis, spongiosis of the deep the normal or full mutation alleles. Besides the obvious difference cerebellar white matter, and swollen axons [20,21]. The major neu- of the CGG repeat length itself, there are a number of other dis- ropathological hallmark and postmortem criterion for deﬁnitive tinctions. In cells from premutation carriers over a wide range of FXTAS are eosinophilic, ubiquitin-positive intranuclear inclusions repeat lengths, the level of FMR1 mRNA is elevated some two to located in broad distribution throughout the brain in neurons, eight times above normal levels, while the stability of FMR1 mRNA is astrocytes, and in the spinal column [20]. The inclusions are both unchanged (Fig. 2). Indeed, even in the high-end normal range (∼55 tau and ␣-synuclein negative, which indicates that FXTAS is not a repeats), the FMR1 message level was near double that found in nor- tauopathy or synucleinopathy. Notably, the FXTAS inclusions share mal alleles (∼30 repeats) [36,55]. In addition, some hypomethylated the same ubiquitin-positive hallmark as several other inclusion dis- full mutation alleles produce substantial amounts of FMR1 message eases, such as polyglutamine disorders, yet the inclusions do not (∼4.5-fold normal) [55]. Although the precise mechanism for this stain with antibodies that recognize polyglutamine, which suggests overexpression is unknown, it is likely that the increasing length a defect in the proteasomal degradation pathway [21,24,54]. Fur- of the CGG repeat near the FMR1 promoter proportionally opens thermore, unlike the polyglutamine disorders, there is no known the chromatin, allowing more ready access to transcription factors. structurally abnormal protein associated with FXTAS. Despite this elevation in FMR1 levels, the amount of FMR1 protein is One logical explanation for the variability in the FXTAS clini- modestly reduced in the premutation range (∼80% of normal) and cal phenotype might be variability in the size of the premutation may be largely absent in cells expressing FMR1 message with very alleles of individual patients. Interestingly, all these patients are long repeats (>300 repeats) [18,36]. This paradoxical reduction of carriers of a premutation-size CGG repeat (55–200 triplets) in the translation despite elevated mRNA levels can be explained by the 5 UTR of the FMR1 gene. The repeat is expressed in the mature fact that long CGG repeats in the FMR1 mRNA impede 40S ribo- FMR1 mRNA in premutation carriers, as well as in individuals with somal subunit migration from the 5 cap to the initiating codon. normal CGG repeat lengths. A study of families with known FXS It is unlikely that overexpression of FMR1 mRNA is a response to probands into the penetrance of tremor and ataxia among premu- the reduction of FMRP levels, as I304N mutant cells with 30 CGG tation carriers revealed that more than a third of carriers 50 years repeats, which produce nonfunctional protein, do not show an ele- or older show symptoms of FXTAS, and that the penetrance of this vated FMR1 mRNA [16,36]. disorder exceeds 50% for men over 70 years of age [29]. The preva- An RNA gain-of-function mechanism has been suggested for lence of premutation alleles is approximately 1 in 800 for males FXTAS based on the observation of increased levels of CGG- and 1 in 250 for females in the general population; however, it is containing FMR1 mRNA, along with either no detectable change estimated that 1 in 3000 men over 50 in the general population will in FMRP or slightly reduced FMRP levels in premutation carriers

Fig. 2. Genotype–phenotype correlation at the FMR1 locus. In fragile X syndrome, large expansions of the CGG trinucleotide repeat (>200 repeats) in the 5 UTR of the FMR1 gene cause hypermethylation of the FMR1 promoter, which leads to the transcriptional silencing of FMR1 and the loss of the FMR1 product, FMRP. However, the level of FMR1 mRNA is elevated some two to eight times above normal levels in premutation carriers (55–200 CGG repeats), whereas the amount of FMRP appeared to remain at or slightly below normal levels. The observation of increased FMR1 mRNA levels in premutation carriers supports the idea that FXTAS is an RNA-mediated neurodegenerative disorder. 106 H. Tan et al. / Neuroscience Letters 466 (2009) 103–108

[21,29,34,55,59]. The absence of FXS, which results from the loss- of-function of the FMR1 gene product, in patients with FXTAS, along with the absence of FXTAS symptoms in older individuals with FXS also suggests a role for the expanded rCGG repeat in FXTAS pathology. This type of RNA gain-of-function mechanism may also account for some triplet repeat-related ataxias, such as spinocere- bellar ataxia type 8 (SCA8), SCA10, and SCA12, and in myotonic dystrophy (DM) [49]. The untranslated repeat expansion in DM has in fact given us major insight into the underlying molecular mechanisms of FXTAS. DM1 is caused by a CTG repeat expansion in a region of the 3 UTR of the DMPK gene that is transcribed into RNA but not translated into protein. The mutant transcripts sequester MBNL and other proteins, which form ribonuclear foci or inclusions. Indeed, using in situ hybridization, Tassone et al. demonstrated the pres- ence of expanded FMR1 RNA transcripts in the FXTAS inclusions of a 70-year-old male who died with FXTAS [56]. Beyond the observation of increased levels of CGG-containing FMR1 mRNA in fragile X premutation carriers, several other lines of Fig. 3. RNA-binding proteins (RBPs) are involved in FXTAS pathogenesis. Three RNA- evidence further support an RNA-mediated gain-of-function toxi- binding proteins, Pur ␣, hnRNP A2/B1, and CUGBP1, were found to bind rCGG repeats city model for FXTAS. First, in a “knock-in” mouse model, in which either directly (Pur ␣ and hnRNP A2/B1) or indirectly (CUGBP1, through the inter- the endogenous CGG repeats (five CGG repeats in the wild-type action with hnRNP A2/B1). HnRNP A2/B1 forms a complex with CUGBP1 to bind to ␣ mouse Fmr1 gene) were replaced with a ∼100-CGG repeat frag- rCGG repeats, which is independent of Pur . Overexpression of these proteins can suppress neuronal cell death caused by fragile X premutation rCGG repeats, sup- ment, intranuclear inclusions were found to be present throughout porting the model that sequestration of these proteins by the overproduced rCGG the brain, with the exception of cerebellar Purkinje cells [59]. There repeats in FXTAS prevents them from carrying out their normal functions, leading was an increase in both the number and size of the inclusions over to abnormal RNA metabolism and neurodegeneration. the life course, which correlates with the progressive character of the phenotype observed in humans [20]. Second, neuropathologi- cal studies in humans have revealed a highly significant association regulated by these proteins that are altered by the expression of between length of the CGG tract and frequency of intranuclear rCGG repeats and contribute to the pathogenesis of FXTAS (Fig. 3). inclusions in both neurons and astrocytes, indicating that the CGG Interestingly an antisense transcript, ASFMR1, has recently been repeat length is a powerful predictor of neurological involvement identified to overlap the CGG repeat region of the FMR1 gene clinically (age of death) as well as neuropathologically (number of [38,40]. Similar to FMR1, the ASFMR1 transcript is silenced in inclusions) [20]. Third, intranuclear inclusions can be formed in full mutation individuals and up-regulated in premutation carri- both primary neural progenitor cells and established neural cell ers. However, whether ASFMR1 contributes to the pathogenesis of lines, as was revealed using a reporter construct with an FMR1 5 either FXTAS or FXS remains to be determined. UTR harboring expanded (premutation) CGG repeats [1]. Fourth, It is remarkable that the same type of mutation in the FMR1 gene we described a model of FXTAS using Drosophila expressing the is involved in three distinct disorders that affect patients in an age- FMR1 untranslated CGG repeats 5 to the EGFP coding sequence dependent manner (Fig. 1). Interestingly, two of these syndromes, and demonstrated that premutation-length rCGG repeats are both FXS and FXTAS, are known to be caused, at least in part, by altered toxic and sufficient to cause neurodegeneration [34]. And finally, it noncoding RNA-mediated gene regulation. Understanding the role has been found recently that rCGG expressed in Purkinje neurons of noncoding RNAs in these diseases will not only help unravel the outside the context of Fmr1 mRNA can result in neuronal pathol- molecular pathogenesis of fragile X-related disorders, but will also ogy in a mammalian system [26]. These observations led us and tell us much about the role of noncoding RNAs in human diseases in general. others to propose that transcription of the CGG90 repeats leads to an RNA-mediated neurodegenerative disease. We further posited a mechanism by which rCGG repeat-binding proteins (RBPs) may Acknowledgements become functionally limited by their sequestration to lengthy rCGG repeats, mechanistically similar to the pathophysiology of DM1. We would like to thank C. Strauss for critical reading of the To test this model using both biochemical and genetic manuscript. This work was supported in part by NIH grants. 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