Untranslated Region Variant in FMR1 Eliminates Neuronal Activity

A3′ untranslated region variant in FMR1 eliminates PNAS PLUS neuronal activity-dependent translation of FMRP by disrupting binding of the RNA-binding protein HuR

Joshua A. Suhla, Ravi S. Muddashettyb, Bart R. Andersona,b, Marius F. Ifrimb, Jeannie Visootsaka, Gary J. Bassellb, and Stephen T. Warrena,c,d,1

aDepartment of Human Genetics, Emory University, Atlanta, GA 30322; bDepartment of Cell Biology, Emory University, Atlanta, GA 30322; cDepartment of Biochemistry, Emory University, Atlanta, GA 30322; and dDepartment of Pediatrics, Emory University, Atlanta, GA 30322

Contributed by Stephen T. Warren, October 14, 2015 (sent for review September 29, 2015; reviewed by Claudia Bagni and Kimberly M. Huber) Fragile X syndrome is a common cause of intellectual disability and and is thought to be a principal cause of the cognitive disabilities in autism spectrum disorder. The gene underlying the disorder, fragile FXS patients (12, 18, 19). Whereas the effects of lacking FMRP X mental retardation 1 (FMR1), is silenced in most cases by a CGG- entirely have been extensively investigated, few studies have focused repeat expansion mutation in the 5′ untranslated region (UTR). Re- on the consequences of still-present but dysregulated translation cently, we identified a variant located in the 3′UTR of FMR1 enriched of FMRP. among developmentally delayed males with normal repeat lengths. Despite significant analysis of the FMR1 gene, only a small A patient-derived cell line revealed reduced levels of endogenous number of conventional genetic mutations, such as point muta- fragile X mental retardation protein (FMRP), and a reporter contain- tions and insertions/deletions, have been reported to be associ- ing a patient 3′UTR caused a decrease in expression. A control re- ated with FXS or developmental delay (20–27). To identify porter expressed in cultured mouse cortical neurons showed an causes of developmental delay attributable to FMR1 variants expected increase following synaptic stimulation that was absent other than the repeat expansion, our group sequenced the FMR1 when expressing the patient reporter, suggesting an impaired re- gene in 963 developmentally delayed males, each of whom tested sponse to neuronal activity. Mobility-shift assays using a control negative for the CGG expansion mutation, and discovered a number NEUROSCIENCE – RNA detected an RNA protein interaction that is lost with the pa- of previously unreported variants (28). However, the molecular tient RNA, and HuR was subsequently identified as an associated consequences, if any, of most of these variants remain unknown. protein. Cross-linking immunoprecipitation experiments identified In this study, we describe the functional impact of a variant the locus as an in vivo target of HuR, supporting our in vitro find- in the 3′UTR of FMR1 (c.*746T>C) using genetic, biochemi- ings. These data suggest that the disrupted interaction of HuR im- cal, and cell biological approaches. The variant is associated pairs activity-dependent translation of FMRP, which may hinder with reduced basal FMRP levels and impairs the normal re- synaptic plasticity in a clinically significant fashion. sponse to activity-dependent synaptic translation in cultured fragile X syndrome | FMR1 | FMRP | HuR | autism primary neurons. Our data suggest that the RNA-binding protein HuR binds the locus normally but that this association is lost when the variant is present, leading to destabilized and ragile X syndrome (FXS) is one of the most common forms of rapidly degraded FMR1 transcript. These findings indicate that inherited intellectual disability, and represents a well-known F the c.*746C variant allele, detected at a frequency of 1 in 160 genetic cause of autism spectrum disorder. FXS is a monogenic disorder characterized by the loss or dysfunction of the fragile X mental retardation protein (FMRP), the product of the fragile X Significance mental retardation 1 (FMR1) gene (1). In a vast majority of FXS patients, FMRP expression is absent due to the expansion of an The fragile X mental retardation protein (FMRP) is most highly unstable CGG repeat in the promoter region of the FMR1 gene. expressed in neurons, and is critical for proper synaptic function- This repeat tract is polymorphic in the population, where 5–45 ing. Fragile X syndrome, a common cause of intellectual disability, CGG repeats are typical. FXS patients have in excess of 200 is the result of absent or dysfunctional FMRP, highlighting its repeats, referred to as the full mutation (2), usually inherited via importance to the processes underlying learning and memory. A an unstable maternal premutation allele (55–200 repeats). At the rapid upregulation of FMRP synthesis at the synapse in response full mutation length threshold of ∼200 repeats, an epigenetic to specific neuronal signals is a key step in maintaining a dynamic event manifests that results in hypermethylation of the FMR1 synapse, although the mechanisms governing this up-regulation promoter region and subsequent silencing of the transcript and are not well-understood. We show that a variant in the 3′UTR protein expression (3–5). of fragile X mental retardation 1 (FMR1) causes the loss of this Studies of FMRP function suggest that it is a selective RNA- characteristic increase in synaptic FMRP synthesis, which may binding protein (RBP) that primarily acts as a negative regulator of lead to developmental delay in patients. These data identify translation (6, 7), and is estimated to associate with about 4–5% of several mechanisms and molecules modulating activity-de- mRNA messages expressed in the brain, including its own transcript pendent translation of FMRP. (8–10). FMRP is also a key regulator of translation downstream of glutamate receptor-mediated signaling in neurons, where it is rap- Author contributions: J.A.S., B.R.A., M.F.I., G.J.B., and S.T.W. designed research; J.A.S., R.S.M., B.R.A., and M.F.I. performed research; R.S.M. and J.V. contributed new reagents/analytic idly inactivated by dephosphorylation upon receptor activation, tools; J.V. provided clinical support; J.A.S., B.R.A., G.J.B., and S.T.W. analyzed data; and thereby allowing protein synthesis of its targets to occur in response J.A.S., G.J.B., and S.T.W. wrote the paper. to the stimulus (11–14). The absence of FMRP uncouples gluta- Reviewers: C.B., Catholic University of Leuven Medical School; and K.M.H., University of mate receptor stimulation from the protein synthesis typically re- Texas Southwestern Medical Center. quired for proper signal transduction at the synapse (15). These The authors declare no conflict of interest. molecular defects are associated with impaired synaptic plasticity, 1To whom correspondence should be addressed. Email: [email protected]. widely believed to underlie the processes of learning and memory, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. which requires tightly controlled synaptic protein synthesis (16, 17) 1073/pnas.1514260112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1514260112 PNAS Early Edition | 1of9 Downloaded by guest on September 29, 2021 developmentally delayed male patients tested (28), may repre- conserved evolutionarily, as indicated by PhyloP (31) (2.76), GERP sent an unrecognized genetic basis of developmental delay via (32) (5.52), and PhastCons (33) (1.00) scores. Last, the locus is FMR1 dysregulation. thymine- and uridine-rich at the RNA level, which we hypothesized could serve as a site of interaction for a class of U-rich RBPs Results (Fig. S1). Based on these data, we explored whether the variant Clinical Assessment of a Patient with the c.*746T>C Variant. One of had any effect on FMRP expression in the patient. A lympho- the patients identified by Collins et al. (28) as harboring the blastoid cell line was established from the patient’sbloodand c.*746T>C variant was clinically evaluated by the Emory University Western blot analysis revealed a modest, but significant, reduction Medical Genetics Clinic. The patient (pictured in Fig. 1A) was of the patient’s endogenous FMRP level compared with two ∼10.5 y old at evaluation and was born full-term at 10 lb, 1 oz healthy lymphoblastoid control lines (Fig. 1B). To corroborate without complication. He was reported to have sat independently these results, luciferase reporter vectors were constructed to in- at 17 mo [typically achieved by 9 mo of age (29)] and first walked clude the full-length FMR1 3′UTR of the patient or a healthy at 24 mo [typically achieved by 18 mo of age (29)]. At examination, control downstream of the firefly luciferase gene. We observed a he was nonverbal and exhibited stereotypic behavior consisting of significant decrease in normalized luciferase signal with the patient rocking, spinning, rubbing his fingers, and repetitively touching his reporter compared with the control in two different cell lines (P = shirt collar. The patient had previously been diagnosed with au- 0.007 in HEK293FT; P = 0.004 in Neuro2a; Fig. 1C). Additionally, tism spectrum disorder and attention deficit hyperactivity disorder the 3′UTR from the patient’s affected half-sibling brother, who (ADHD), and he attends special education classes. Cognitive abil- harbored the c.*746C allele as well, showed a similar reduction in ities were assessed using the Stanford–Binet Intelligence Scales luciferase activity compared with the control (P < 0.001; Fig. 1D). (5th Ed) (30), revealing moderate intellectual disability (IQ score Steady-state levels of luciferase transcript were equivalent between 47). In terms of his growth parameters, the patient is within the 50– the patient and control vectors (Fig. S2), suggesting a posttran- 75th percentiles for weight (88 lb), height (60 in), and head cir- scriptional mechanism underlying the reduction in patient reporter cumference (55 cm). A physical examination was performed activity. However, other mechanisms, such as mRNA instability, where bilateral flat feet with inversion were noted but no other may be responsible for the observed decrease in luciferase ac- significant findings. The patient’s half-brother was also evaluated, tivity, which these quantitative (q)RT-PCR assays cannot evalu- and genotyping revealed that he also possesses the c.*746C variant. ate. Together, these data indicate that the patient 3′UTR is He was reported to have sat independently at 17 mo old, taken his associated with a decrease in endogenous FMRP and reporter ex- first steps at 24 mo, and spoken his first word at 5 y of age. Ap- pression, and that the FMR1 gene may be regulated posttranscrip- proximately 17 y old at examination, he speaks in short sentences, tionally via the 3′UTR at the c.*746 locus. attends some regular 10th-grade classes with specialized classes for reading and math, is mildly intellectually disabled (IQ score 67), and The c.*746 Locus Is Important for Translational Regulation. To identify has previously been diagnosed with ADHD. whether the c.*746 locus is the specific site responsible for the observed decrease in reporter activity because the 3′UTR of Patient 3′UTR Reduces Translation of FMRP and a Reporter. In ad- FMR1 exhibits frequent sequence variation, we used site-directed dition to the developmental delay and intellectual disability in the mutagenesis to change the variant 746C nucleotide in the patient patient, several noteworthy lines of evidence led us to investigate reporter to the reference thymine. This single-nucleotide change the molecular impact of the FMR1 c.*746C allele. First, the var- restored luciferase activity to the level of the control. Conversely, iant was significantly enriched in unrelated developmentally delayed mutagenesis of the control vector to the patient allele caused a male patients compared with gender-matched controls [found in 6 reduction in reporter signal, indicating that the variant is specific of 963 patients and 0 of 1,260 controls; P = 0.007 (28)]. Addition- in causing the diminished translation (Fig. 2A). Given that the ally, the c.*746 position and broader genetic element are highly variant does not completely abolish reporter activity, we examined

Fig. 1. Patient 3′UTR is associated with a significant reduction in endogenous FMRP and reporter activity in multiple cell types. (A) Photograph of a patient harboring the FMR1 c.*746C allele. (B)(Top) Representative Western blot of FMRP and eIF4e from two unrelated control male lymphoblastoid cell lines and the patient-derived lymphoblastoid cell line. (Bottom) The band density on the Western blot of three independent protein preparations was digitally quantified by ImageJ software (NIH). Data shown are the mean ± SD. (C) Luciferase assay in HEK293 and Neuro2a cell lines using vectors with a control 3′UTR or the patient 3′UTR. Each of three independent experiments was normalized to cotransfected Renilla luciferase activity; data shown are the mean ± SD. (D) Luciferase assay results of three independent experiments in HEK293 cells using vectors of the control, patient, and the patient’s half-brother. Data shown are the mean ± SD. Unpaired two-tailed t test, *P < 0.05; a.u., arbitrary units; n.s., not significant.

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Fig. 2. FMR1 c.*746 site modulates steady-state and activity-dependent reporter activity. (A) Results of three independent luciferase assays using mutagenized vectors in HEK293 cells. Reference allele at c.*746, T; patient allele, C. Data shown are the mean ± SD. (B) A 17-bp deletion in the vectors that deletes the U-rich locus including the 746 site (indicated by a horizontal bracket and red lettering, respectively) was used in luciferase assays. Results are the mean of three independent experiments ± SD. (C) Luciferase assay in WT mouse primary cortical neurons (E17.5) using various vectors with and without DHPG treatment (100 μM, 5 min) ∼24 h posttransfection. Data shown are the mean of four or five independent experiments ± SD. (D) Luciferase assay in Fmr1 KO mouse primary cortical neurons (E17.5) using various vectors with and without DHPG treatment (100 μM, 5 min) ∼24 h posttransfection. Data shown are the mean of four or five independent experiments ± SD. Unpaired two-tailed t test, *P < 0.05. C, control; Del, deletion; P, patient.

whether mutagenesis of the broader U-rich motif would cause a 3,5-dihydroxyphenylglycine (DHPG), a glutamate analog that more pronounced effect to determine the magnitude of trans- stimulates synaptic activity via the group I metabotropic glutamate lational misregulation caused directly by the c.*746C variant. We receptors (mGluRs) (12, 34, 35). The addition of DHPG to cul- deleted 17 bp from the locus, including the variant site, in both the tures expressing the control reporter caused an increase in trans- control and patient vectors (Fig. 2B). When expressing the control lation in WT neurons, but not in Fmr1 KO neurons, as expected vector with the deletion, we observed a significant decrease in re- (Fig. 2 C and D, respectively). However, when expressing the porter activity compared with the control (P = 0.004) but not a patient reporter in WT neurons, there was no increase in trans- more severe reduction than that observed with the patient reporter. lation after DHPG treatment in addition to the steady-state re- The same deletion in the patient vector did not significantly alter duction (Fig. 2C). When the mutagenized control reporter was expression from the unchanged patient reporter (P = 0.27). Because expressed in WT neurons, the normal up-regulation of translation the patient vector with the deletion did not show significantly al- elicited by DHPG treatment was lost (Fig. 2C). These data reveal tered expression compared with the unchanged patient vector, the a loss of activity-dependent translation in neurons in response to c.*746 position itself is likely a critical nucleotide in the motif. Taken glutamate signaling by the patient reporter, and identify the c.*746 together, these data implicate the locus, and the c.*746C variant in locus as associated with this deficit. particular, as causative of the diminished reporter expression. Loss of RNA-Binding Protein Association Caused by the c.*746C Variant. The c.*746C Variant Allele Is Refractory to Glutamate Receptor Signaling Several potential mechanisms underlying these data were consid- in Primary Neurons. Although the patient’s lymphoblastoid cell line ered, including disruption of microRNA or RBP interactions showed reduced levels of FMRP, ∼80% of normal levels were still caused by the variant allele. To determine whether an RBP targets present. We questioned whether this minimal reduction in FMRP the locus, we used electromobility-shift assays (EMSAs) to evaluate could cause the developmental and cognitive disabilities displayed whether a 42-nt biotinylated RNA probe encompassing the c.*746 by the patient, and wondered whether there could be a neuron- site interacts with a protein or protein complex from mouse whole- specific mechanism of dysfunction that may not be apparent in brain lysate. We detected a band shift using the control probe that other tissues. To explore this, we tested whether the variant allele was dosage-sensitive and -specific, because an excess amount of affected activity-dependent protein synthesis in primary neurons. unlabeled competitor probe was able to outcompete the labeled C57BL/6 (WT) and Fmr1 knockout (KO) mouse cortical neurons probe for the interaction (Fig. 3 A and B; see also Fig. S3 and Table were isolated, cultured, and transfected with the described lu- S1). To determine whether the c.*746C variant disrupted this in- ciferase reporters to evaluate translation when treated with (RS)- teraction, we generated a biotinylated patient probe that differed

Suhl et al. PNAS Early Edition | 3of9 Downloaded by guest on September 29, 2021 Fig. 3. RNA EMSAs reveal a specific and dose-dependent interaction between a protein and the c.*746 locus that is absent with the patient sequence. (A)A biotinylated control RNA probe incubated with increasing amounts of mouse whole-brain lysate and resolved by 5% native TBE gel. (B) An unlabeled version of the control RNA probe was added in increasing amounts (up to 40× excess by molar concentration) to the whole-brain lysate binding reaction and resolved on a 5% native TBE gel. (C) A biotinylated patient RNA probe containing the c.*746C nucleotide was incubated with increasing amounts of mouse whole- brain lysate in the same manner as the control probe and resolved on a 5% native TBE gel. (D) An unlabeled version of the patient RNA probe was added in increasing amounts, up to 40× molar excess concentrations, to the binding reactions with the labeled control probe and resolved on a 5% native TBE gel.

only at the 746 position and compared this with the control probe control, similar to the level of β-actin enrichment, whereas GAPDH binding. The patient probe lacked the band shift, suggesting a loss of showed no enrichment (Fig. 4B), indicating that HuR indeed as- association with the protein or protein complex due to the 746C sociates with FMR1. To localize this interaction specifically to the variant (Fig. 3C and Table S1). Additionally, an unlabeled com- 746 locus, we performed gel-shift, competition, and supershift as- petitor patient probe could not compete away the interaction from says with purified HuR protein rather than whole-brain lysate. the control, even at concentrations of 40× molar excess, confirming These assays revealed an association between HuR and the control the inability of the protein to bind the patient sequence (Fig. 3D). probe, and confirmed the lack of interaction between the patient These results demonstrate that the locus is a target of an RBP or probe and HuR (Fig. 4C). Altogether, FMR1 is bound by HuR protein complex and that the variant disrupts this association, at the c.*746 locus, and this interaction is disrupted by the identifying a potential mechanism underlying the observed changes variant allele. in the reporter assays. HuR Localizes to Dendrites and Synapses. HuR is typically most The RNA-Binding Protein HuR Associates with the c.*746 Locus. To abundant in the nucleus of cells, but is known to translocate to identify the protein(s) that interacts with this RNA sequence, we the cytoplasm under certain conditions to facilitate mRNA sta- used two approaches, both of which made use of tandem mass bility and translation (36, 37). If HuR is involved in the activity- FMR1 spectrometry (MS-MS) for protein identification. First, we per- dependent translation of , it must be present at the syn- FMR1 formed two biological replicates of a coimmunoprecipitation (co- apse, because previous work has shown that endogenous IP) assay where streptavidin-coated magnetic beads were fixed mRNA localizes to dendrites (38) and can be locally translated in with the biotinylated control RNA probe and then incubated an activity-dependent manner (39). To determine whether HuR with mouse whole-brain lysate. We eluted bound proteins and is present at the synapse, we used immunocytochemistry to vi- subjected the sample to MS-MS peptide sequencing to identify sualize its location in primary cortical neurons. Although the majority of HuR is located in the nucleus, it is also present in the enriched proteins compared with a bead-only control. Second, dendrites distal to the soma (Fig. 4D), a finding supported by we performed the EMSAs as described and then excised the other very recent data (40). To more specifically determine whether shifted band region from the gel and subjected the eluted protein HuR localizes to the synapse, we performed immunoblot analyses sample to MS-MS analysis to directly identify proteins in the from mouse synaptoneurosome preparations and found the protein band-shift region (Table S2). Three candidates were detected by to be present, suggesting that HuR is at the synapse (Fig. 4E). both screening methods: HuR (also known as ELAVL1), PUR-α, Fmr1 β Previous work has shown that mRNA is present in synaptic and PUR- . Whereas the PUR proteins typically bind purine-rich fractions (18), so we investigated whether Fmr1 mRNA was in a motifs, HuR is known to target U-rich elements in the 3′UTR of FMR1 complex with HuR in this compartment. Co-IP experiments in genes. Given the U-rich nature and location of the c.*746 synaptoneurosomes revealed a significant enrichment of Fmr1 locus, we pursued HuR as the top RBP candidate. mRNA in HuR IP fractions, suggesting that these molecules in- To determine whether the band shift we observed in the EMSAs teract at the synapse (Fig. 4F). was indeed HuR, we first performed a gel-supershift assay using an antibody to HuR. As shown in Fig. 4A,theshiftedbanddisappears Patient FMR1 mRNA Decays Rapidly but Is Trafficked to Dendrites with the addition of HuR antibody to the binding reaction, although Normally. HuR serves many functions, including translation the supershifted band was not visible due to the high background regulation, transcript trafficking, and splicing. One of the best- signal inherent to using a whole-brain lysate. Next, we used a HuR studied functions of HuR is that of mRNA stabilization of antibody to immunopurify HuR-bound mRNAs from HEK293 cell transcripts that it binds via U-rich elements in the 3′UTR (36). lysates and then assayed for FMR1 as well as an established target Because HuR binding to the patient sequence appears to be of HuR (β-actin) and a gene not known to interact with HuR impaired, we assessed the stability of endogenous FMR1 mRNA (GAPDH). FMR1 was enriched by HuR IP ∼400-fold over an IgG in the patient lymphoblastoid cell line. We performed mRNA

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Fig. 4. HuR binds FMR1 at the c.*746 locus and is present at the synapse. (A) Supershift assays were performed using 32P-labeled control probe and HuR antibody. Binding reactions were carried out as described previously with the addition of 0.5–1 μg of antibody per 10 μg of mouse whole-brain lysate in each reaction. (B) Immunoprecipitation of HuR protein from HEK293 cell lysate was performed, and copurified mRNAs were assayed by real-time RT-PCR. The results show the fold enrichment of mRNA levels of β-actin (a known target of HuR), GAPDH (not known to interact with HuR), and FMR1 mRNA normalized to IP with IgG alone. All data are displayed as the ratio of IP mRNA levels to input mRNA levels. Data shows the mean of three independent experiments ± SD. (C) RNA EMSA using purified HuR protein with the control or patient probe, unlabeled competitor control probe, and HuR antibody. (Bottom) Free probe bands from a shorter exposure of the same blot to ensure equal probe loading. (D) Image of immunofluorescence staining for HuR (green), PSD-95 (red), and nuclei (blue) in primary cortical neurons. A merged image of all colors is shown (Top Left). (Scale bar, 10 μm.) The white boxes in the merged image are enlarged in the Bottom two rows, with the Top row representing the Left box and the Bottom row representing the Right box. The merged, HuR (green), and PSD-95 (red) images are shown in both Bottom rows from Left to Right, respectively. (Scale bars, 5 μm.) (E) Western blot showing the presence of HuR in a whole-cortical lysate and synaptoneurosomes. GFAP, a glial marker, was used as an indicator of synaptoneurosome purity. An antibody against eiF4e was used as a loading control. C1 and S1, cortical lysate and synaptoneurosomes, respectively, of preparation 1; C2 and S2, cortical lysate and synaptoneurosomes, respectively, of preparation 2. (F) Synaptoneurosome preparations were subjected to HuR immunoprecipitation, and the level of associated Fmr1 was assayed by qRT-PCR. The levels of GAPDH and β-actin mRNA were also assayed in the immunoprecipitate as a negative and positive control, respectively. Data shown are the mean of three independent synaptoneurosome preparations ± SD. (*P < 0.05 using a two- tailed unpaired t test.) (G) Actinomycin D (5 μg/mL) was added to control and patient lymphoblastoid cells to block transcription, and qRT-PCR was performed for FMR1 (Right) and PSD-95 (Left)after1,2,4,and6hofdrugtreatmentaswellasanuntreated control (0 h). Each point shows the mean percentage of mRNA remaining relative to the 0-h control ± SEM in six independent experiments. Nonlinear regression lines were fit to each set of data points, and the slopes were calculated and compared. For FMR1, the difference in slope was significantly different between the control and patient (P = 0.008); for PSD-95, the difference in slope was not significantly different (P = 0.582).

Suhl et al. PNAS Early Edition | 5of9 Downloaded by guest on September 29, 2021 decay assays using actinomycin D to block transcription and then HuR at the c.*746 locus in vivo and is positively regulated by the tracked the longevity of the extant transcript over time. We protein, consistent with our in vitro results. observed rapid decay of FMR1 in the patient cell line, which was significantly faster than a control lymphoblastoid cell line (P = Discussion 0.008). Another gene, PSD-95, showed no difference in the rate The variant c.*746T>C in the FMR1 3′UTR reduces the basal of mRNA degradation after actinomycin D treatment between level of FMRP in patient-derived cell lines and in multiple other the patient and control (P = 0.582; Fig. 4G). These results suggest cell types using reporter constructs and, perhaps more importantly, that the stability of endogenous FMR1 in the patient is diminished, eliminates the normal response to glutamate signaling by reporter potentially because of the inability of HuR to bind at the c.*746 assay in primary cultured neurons. Our data suggest that both locus and stabilize the transcript. This finding may explain the defects are the consequence of a disrupted RNA–protein in- steady-state reduction in reporter assays and endogenous FMRP teraction between HuR and FMR1 caused by the c.*746C variant. in the patient cell line, and potentially has implications for the HuR is known to bind U-rich motifs in 3′UTRs and introns, activity-dependent defect observed in cultured neurons. particularly stretches of uridines with interspersed adenines or Another known function of HuR is trafficking of its mRNA guanines (42, 43, 48). The change from uridine to cytosine at the targets to appropriate cellular compartments (41). Because HuR c.*746 position, which interrupts a multiuridine stretch, presumably targets FMR1, we sought to determine whether the patient tran- hinders the normal interaction of HuR at the locus. In support of script was being properly transported to locations distal to the this hypothesis, mutagenesis of the variant locus modulated the soma, as this may be controlled in part by HuR and may explain steady-state expression of the patient and control vectors as well as the defect in activity-dependent translation of the reporter in the activity-dependent translation. The c.*746C patient allele resulted Fmr1 cultured neurons. Here we used fluorescently tagged FMR1 to in a molecular phenotype similar to that observed in KO track and compare the mutant transcript shuttling and localization neurons after glutamate receptor activation, where glutamate-driven in primary neurons compared with a control. Both versions of protein synthesis was absent. Although these molecular findings are compelling, they do not definitively link the c.*746C variant tagged transcript were observed in dendrites and colocalized with ’ HuR and FMRP, suggesting that the patient message is being to the patient s phenotype. Consequently, it will be important to trafficked to the appropriate neuronal compartments (Fig. S4). evaluate more families and individuals with the c.*746C allele to We also tracked the movement of the FMR1 mRNA message over determine whether the variant is truly pathological and observe time to determine whether the rate of localization of the patient the range of severity and disabilities caused by the variant. The patient’shalf-brotherisalsodevelopmentally delayed/intellectually transcript was impaired relative to the control, and did not observe disabled, and his FMR1 3′UTR showed a reduction in expression by any significant difference (Fig. S5). Thus, the c.*746 locus likely reporter assay, which provides a second case (albeit familial) that does not play a role in FMR1 trafficking. may be attributable to the c.*746C allele. However, he is affected to HuR Targets the c.*746 Locus in Vivo, and Knockdown of HuR Reduces a lesser degree compared with the proband, which may indicate that FMR1 and FMRP Levels. We next wanted to determine whether the other genetic or environmental factors specific to the proband play HuR interaction at the c.*746 locus is recapitulated in vivo and a role in the relative severity of the phenotype. The role of synaptically expressed FMRP is hypothesized to whether disruption of the interaction has an effect on FMR1 and function as a negative feedback loop, where existing FMRP is FMRP expression. To do this, we analyzed HuR binding profiles dephosphorylated and degraded after glutamate receptor stimula- generatedinthreepublisheddatasets:twothatusedphoto- tion and newly synthesized FMRP reins in the burst of translation activatable-ribonucleoside–enhanced cross-linking immunoprecipita- after an appropriate amount of time (14, 34, 49). However, the tion (PAR-CLIP) (42, 43) and one study comparing multiple CLIP importance of locally translated FMRP had not been directly protocols (44). CLIP assays capture in vivo interactions by UV-irra- addressed until recently, when it was discovered that a premutation diating live cells or tissue at a wavelength that cross-links the RBP to FXS mouse model exhibited impaired activity-dependent FMRP the target RNA, thereby identifying specific sites or motifs of contact synthesis likely due to the presence of expanded CGG repeats in (45–47). All three studies identified the 3′UTR of FMR1 as a target ′ FMR1– the 5 UTR (50). Iliff et al. (50) exploited a well-characterized of HuR. Although the sites of HuR interaction differ slightly neuronal phenotype in Fmr1 KO mice, enhanced mGluR-medi- between the datasets, the Kishore et al. study (44) detected HuR ated long-term depression (LTD), to evaluate the impact of dys- association at the c.*746 locus through two different CLIP protocols, functional local FMRP translation on synaptic plasticity in linking the patient variant site to HuR binding in vivo. The other two premutation brain tissue. Their results suggest that the reduced studies found HuR binding sites near the c.*746 locus, although the levels of pre-existing FMRP were not sufficient to suppress the CLIP sequence tags do not directly overlap with the c.*746 site enhanced mGluR-LTD occurring in the premutation neurons; lo- (Table S3). Taken together, there is evidence that the c.*746 site is cally synthesized FMRP was necessary for proper LTD. Whereas targeted by HuR in vivo, although more studies will be necessary to most studies of the premutation allele have focused on late-onset confirm these findings. neurodegenerative phenotypes, accumulating evidence suggests To examine the effect of HuR on its target genes, Lebedeva et al. that the premutation may have a demonstrable effect on neuro- (42) measured new protein synthesis and changes in mRNA levels development as well (51–53). Although the genetic defect in using pulsed stable isotope labeling by amino acids in cell culture and mGluR-mediated translation studied by Iliff et al. (i.e., the pre- RNA-sequencing experiments, respectively, in cells depleted of HuR mutation in the 5′UTR) differs from the 3′UTR defect investigated and calculated the log2 fold change compared with a negative con- here, it suggests that the c.*746C variant could hinder neuro- trol for each target gene. Using these data, we plotted the distribu- development and synaptic plasticity through the impairment of ac- tion of changes in mRNA levels after HuR knockdown and tivity-dependent translation, and is a possible reason for the observed discovered that FMR1 levels were significantly reduced compared developmental delay and cognitive disability in the patient. − A with other genes assayed (log2 score 0.87; Fig. 5 ). Additionally, These data implicate the c.*746 locus in the basal and activity- FMRP was the third–least-abundant protein in the absence of HuR dependent expression of FMRP. However, the exact mechanism(s) from over 2,000 proteins analyzed (log2 score −1.37; Fig. 5B). Even by which this deficit occurs remains to be determined. The trans- when normalizing protein synthesis by concomitant changes in lational insufficiencies could be a direct effect of the inability of mRNA levels, FMRP was still among the least-synthesized proteins HuR to bind FMR1 or an indirect consequence of this perturbed (log2 score −0.51; Fig. 5B), indicating a role for HuR in translation interaction, such as rapidly decaying transcript at the synapse that is regulation of FMR1. These data suggest that FMR1 is targeted by insufficient to support activity-regulated protein synthesis. Another

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Fig. 5. HuR depletion down-regulates FMR1 mRNA levels and FMRP synthesis. (A) The distribution of mRNA changes after knockdown (KD) of HuR reveals a

significant decrease in FMR1 levels compared with over 2,000 other mRNAs assayed (log2 value −0.87). The red arrow indicates the bin in which FMR1 falls. (B) The distribution of protein-level changes shows a significant decrease in FMRP synthesis (log2 value −1.37). The red arrow indicates the bin in which FMRP falls. (C) The distribution of protein synthesis changes when accounting for concomitant mRNA changes indicates that the decrease in protein synthesis is not

only the result of a reduction in mRNA levels (i.e., −log2 difference between protein and mRNA levels −0.51). The red arrow indicates the bin in which FMRP falls. All histograms were generated using data from Lebedeva et al. (42).

potential mechanism is based on the observation that HuR com- identified by each dataset were inconsistent, it will be important to petes with other binding proteins, of which there are several that confirm these findings with more targeted studies. Additionally, it recognize a similar U-rich motif (36), to modulate the stability of a would be of interest to determine whether the interactions are given target transcript. For example, the binding factor AUF1 (also recapitulated in different cell types, including neuronal cells or known as hnRNP D), which was also pulled down in our co-IP/MS brain tissue. To determine the functional effect of HuR on FMR1, screen (Table S2), is associated with rapid degradation of bound we analyzed data from experiments that tracked the translation of mRNAs, opposing the function of HuR. Indeed, this type of new proteins after knockdown of HuR, which revealed a drastic competitive binding has recently been postulated to occur in vivo reduction in FMR1 mRNA levels and FMRP synthesis. In fact, NEUROSCIENCE between HuR and another U-rich binding protein that negatively FMRP levels were reduced more than all other proteins assayed regulates transcript stability, ZFP36 (54). This presents the possi- except for two, one of which was HuR itself. Together, these re- bility that each is required as an antagonist to the other, and each sults suggest that the locus is targeted by HuR and that FMRP performs their specific functions when cued by the appropriate expression is significantly down-regulated in the absence of HuR. cellular signals. If HuR binding is impaired at the c.*746 site, If HuR-mediated regulation of FMR1 is via the 746 locus, the lack AUF1 or other similar transcript-destabilizing proteins may have of HuR binding to the patient allele may underlie the reduction in an increased opportunity to degrade FMR1, leading to a reduced expression and transcript stability observed in our data. mRNA half-life, which we observed in the patient cell line. BecauseHuRisamemberofasmallproteinfamily(HuR, A recently recognized function of HuR is that it can act as an HuB, HuC, and HuD) and the other Hu paralogs also bind a “anti-RISC” (RNA-induced silencing complex); that is, HuR similar U-rich sequence motif (59), we were interested in whether binds near microRNA (miRNA) sites, oligomerizes along the these other Hu proteins associated with FMR1 at the c.*746 locus. mRNA, and removes or blocks the miRNA machinery from Our co-IP followed by MS-MS assay did identify HuC as being reaching its target, thereby relieving the down-regulation typically copurified with the biotinylated probe, although it was not de- imposed by miRNAs (43, 55, 56). One study has shown that miR- tected in the gel slice/MS-MS assay and, therefore, was not in- 130b, an miRNA that is expressed in the brain and is highly cluded in our top candidates. However, these data do suggest at conserved, interacts at the 746 variant locus (seed sequence binds least some level of interaction between the neuronal Hu and the FMR1 – at c.*755 761), negatively regulates the expression of c.*746 locus. Additionally, the neuronal Hu proteins were found FMR1 in neural progenitor cells, and affects cell-fate specification to bind the c.*746 locus in vivo in mouse brain on actively trans- (57). These findings present an alternate mode of dysfunction lating polyribosomes (10). Taken together, the locus is a target of whereby the inability of HuR to bind and/or oligomerize adjacent posttranscriptional regulation, and may be targeted by different to the miR-130b site hinders the removal of the miRNA, which Hu proteins under specific temporal, spatial, and environmental then continues to repress FMRP translation unchecked and per- conditions, a possibility that should be examined further. haps alters the response to mGluR stimulation. There is evidence The findings presented here illustrate the impact of a single- of an miRNA-mediated response to glutamate signaling, which nucleotide variant in the regulatory region of a gene, which can requires the removal of miR-125a to allow the normal increase in have significant molecular consequences and may be causative of a PSD-95 expression following glutamate receptor activation (58). clinical phenotype. As whole-genome sequencing becomes more If miR-130b is involved in activity-dependent translation of commonplace, sequence data in the UTRs and other regulatory FMR1 FMRP, failure to block or eliminate its association with may regions will be available for analysis and should be explored for prevent the typical up-regulation of FMRP synthesis following FMR1– functional variants like the one described here when studying the glutamate signaling. Although the disrupted HuR interac- genetic defects underlying certain diseases. Together, these data tion may be the basis of the observed dysfunctions, more research identify the FMR1 c.*746 locus as an important site of post- is required to identify the process(es) that is perturbed. transcriptional regulation and shed new light on the mechanisms Multiple different approaches in this study demonstrate that governing activity-dependent translation of FMRP. HuR binds the FMR1 c.*746 locus in vitro. To investigate in vivo binding of HuR to the locus, we analyzed three published datasets Materials and Methods that identified mRNA targets and binding locations of HuR by Human Subject and Animal Research. All experimental procedures requiring PAR-CLIP assays in human cell lines (HEK293 and HeLa) (42– mouse models was approved by the Emory University Institutional Review 44). All datasets showed that multiple 3′UTR and intronic sites of Board (IRB). Informed consent was obtained from all family members de- FMR1 are targeted by HuR. Because the specific binding locations scribed in the study and was approved by the Emory University IRB.

Suhl et al. PNAS Early Edition | 7of9 Downloaded by guest on September 29, 2021 Luciferase Constructs and Site-Directed Mutagenesis. The Dual-Luciferase Western Blotting. Lysates from lymphoblast cell lines were generated by System from Promega was used for reporter assays. In short, the control and resuspending cells in lysis buffer [50 mM Tris·HCl, 300 mM NaCl, 30 mM EDTA, patient 3′UTRs were amplified with a forward primer that included a 5′ XbaI 0.5% Triton X-100, pH 7.6; cOmplete, Mini, Protease Inhibitor Tablet (Roche)] site and a reverse primer that included a 5′ BamHI site. These amplicons were and incubated on ice for 15 min, followed by pelleting of cell debris at ~23,000 × g cloned using the TOPO TA Cloning System (Invitrogen), excised using BamHI for15minat4°C.SDS/PAGE(4–15% gradient) was performed with 10–20 μg and XbaI restriction enzymes, and ligated into a pGL3-promoter vector total lysate as determined by the Bradford assay. Proteins were trans- downstream of the luciferase gene. All constructs were sequenced to verify ferred to PVDF, blocked with T20 PBS blocking buffer (Thermo Scientific) proper orientation and sequence. The pRL-SV40 vector was used as a trans- for 15–30 min, and incubated with primary antibodies [anti-FMRP (Millipore; fection normalization control. The control and patient 3′UTR vectors were used MAB2160) at 1:2,000 dilution and anti-eIF4e (BD Biosciences; 610270) at as templates for all mutagenesis experiments using the Agilent QuikChange 1:2,000 dilution] overnight at 4 °C with gentle agitation. Next, the membrane Lightning Kit as recommended. was incubated with HRP-conjugated anti-mouse and anti-rabbit secondary antibodies at 1:10,000 dilution in 1% (wt/vol) milk blotto for 1 h at room tem- Cell Culture and Transfection. Neuro2a cells were grown in DMEM supple- perature, washed three times with 1% milk blotto, and incubated with HyGLO mented with 10% (vol/vol) FBS and 1× penicillin/streptomycin antibiotics; ECL solution (Denville Scientific) for 1 min with agitation. HEK293FT cells were grown in DMEM supplemented with 10% FBS, 2 mM RNA Probe Generation by in Vitro Transcription. DNA templates containing the L-glutamine, 1× penicillin/streptomycin, 0.1 mM nonessential amino acids, and T7 promoter and probe template sequence were generated by a modified geneticin-selective antibiotic. Both cell lines were grown at 37 °C with 5% CO2. overlap extension PCR strategy (60). Each oligonucleotide (2 μM) was added Transfections in HEK293FT and Neuro2a cells were performed using Lip- to a PCR mix consisting of 1× Herculase II reaction buffer, 0.25 mM each ofectamine 2000 or Lipofectamine LTX (Invitrogen) in 6- or 12-well plates dNTP, 0.5 μL Herculase II fusion enzyme (Agilent Technologies), 3 μL5M according to the manufacturer’s instructions. Lipofection complexes were betaine solution (Sigma-Aldrich), and MilliQ water up to 50 μL. The reaction left in the medium for 4–6 h and then removed and replaced with fresh was cycled under the following parameters: 95 °C for 2 min, followed by 35 medium. Cells were allowed to express the firefly and Renilla luciferase for cycles of 95 °C for 20 s, 45 °C for 30 s, and 72 °C for 30 s. A final extension ∼24 h before harvesting. cycle of 3 min at 72 °C was added. The resulting double-stranded PCR Primary mouse cortical neuron [embryonic day (E)17.5] cultures were isolated products were run on a 1% (wt/vol) agarose gel, extracted from the gel from C57BL/6 and Fmr1 KO embryos and plated in 12-well plates coated with (Qiagen MinElute Gel Extraction Kit), and resuspended in MilliQ water. DNA – 0.2 mg/mL poly L-lysine. Transfections were carried out at 13 d in vitro using templates were used at a concentration of 2 pM in the in vitro transcription NeuroMag Transfection Reagent (OZ Biosciences) following the recommended reaction for 3–4 h at 37 °C (Ambion T7 MEGAshortscript Kit) in the presence protocol. Neurons were stimulated with 100 μM(RS)-3,5-DHPG (Tocris Biosci- of biotin-17-ATP (Enzo Biosciences) or biotin-14-CTP (Life Technologies). The ences) for 5 min before lysis with passive lysis buffer (Promega). resulting 42-nt RNA transcripts were purified using TRIzol LS (Ambion). The same in vitro transcription procedure was used to generate unlabeled RNA Luciferase Assay. Lysis of the cells was performed using the passive lysis protocol probes for 32P-end labeling and competition gel-shift assays. from the Dual-Luciferase Assay Kit (Promega). To detect luciferase signal, 10–20 μL cell lysate and 50 μL Luciferase Assay Reagent II were mixed thoroughly by Electromobility-Shift and -Supershift Assays. Gel-shift assays were carried out pipetting and placed in the luminometer, and firefly luminescence readings using the LightShift Chemiluminescent RNA EMSA Kit (Thermo Scientific) were collected. Immediately after, 50 μL Stop & Glo Reagent (Promega) was according to the manufacturer’s protocol. Briefly, biotin-labeled RNA probes added and mixed thoroughly by pipetting, and the Renilla luminescence signal weremixedwithC57BL/6(WT)orFMR1 KO mouse whole-brain lysates and in- was detected. All values reported are the ratio of firefly:Renilla reporter signal. cubated at room temperature for 20–30 min and then electrophoresed through a 5% nondenaturing Tris-borate-EDTA (TBE) gel. The RNA–protein complexes Quantitative RT-PCR. Reverse-transcription reactions were carried out using were transferred to a positively charged nylon membrane and developed to film iScript One-Step RT-PCR or iTaq Universal SYBR Green Kits (Bio-Rad). All qPCR following the RNA EMSA protocol. Supershift assays were carried out in a similar reactions were performed in duplicate or triplicate as technical replicates. All manner with the following exceptions: addition of ∼1 μg of antibody and in- reactions were cycled for 40 cycles using the Bio-Rad CFX96 real-time system, cubation at room temperature for 20 min before the addition of the radiola- followed by a melt curve cycle, and then analyzed with the Bio-Rad CFX96 beled or biotin-labeled RNA probe in a total of 20 μLreactionvolume. software package. mRNA Decay Assay. Lymphoblastoid cells were plated in six-well plates at a 6 Immunoprecipitation. Dynabeads MyOne Streptavidin T1 (Thermo Fisher density of 5 × 10 cells per well in 2 mL medium consisting of RPMI-1640, 10% × Scientific) was used for biotinylated RNA probe IP following the manufac- FBS, 2 mM L-glutamine, and 1 penicillin/streptomycin antibiotic. Cells were μ turer’s recommended protocol. In each of two independent assays, 50 μgof treated with 5 g/mL actinomycin D (Sigma) for various amounts of time and ’ biotinylated RNA probe was immobilized on 200 μL washed Dynabeads sup- then harvested by TRIzol LS (Ambion) extraction according to the manufacturer s plemented with cOmplete, Mini, EDTA-free (Roche) and SUPERase·In (Life recommended protocol. qRT-PCR was used to assess the amount of FMR1 tran- Technologies) and then incubated with 1 mg whole-brain lysate and a 10× ex- script remaining compared with an untreated control. All qRT-PCRs were per- cess of Torula yeast RNA (Sigma-Aldrich) in binding buffer (1 M NaCl, 1 mM formed in duplicate, and the mean level of FMR1 remaining at each time point β EDTA, 10 mM Tris·HCl, pH 7.5) for 1 h at room temperature with rotation. wasnormalizedtothemeanlevelof -actin remaining at each time point. After three washes, coimmunoprecipitated proteins were eluted in 30 μL binding buffer with 0.1% (wt/vol) SDS at 95 °C for 5 min and subjected to ACKNOWLEDGMENTS. We thank Heather Clark for contacting the patient and recording the family history; Duc Duong, Nicholas Seyfried, and the Emory mass spectrometry. University Proteomics Core Facility for mass spectrometry data and advice; HuR antibody (MBL; RN004P) was immobilized on Dynabeads Protein G Tamika Malone for mouse colony maintenance and cortical tissue dissections; (Immunoprecipitation Kit; Life Technologies) and incubated with HEK293 cell Pankaj Chopra for rank aggregation analysis of mass spectrometry assays; Mika lysates at room temperature for 12 min by rotation, along with a protein Kinoshita, Leila Myrick, and Michael Santoro, among others in the S.T.W. and G-only control. After four washes, the coimmunoprecipitated RNA was eluted G.J.B. laboratories, for helpful insight and discussion. We also thank Robert with 50 μL elution buffer at 95 °C for 5 min and then purified with TRIzol LS Darnell and Jennifer Darnell for helpful discussions and data sharing. This work was supported by NIH Award NS091859 from the National Institute (Life Technologies). qRT-PCR was performed using FMR1, β-actin,and of Neurological Disorders and Stroke; Eunice Kennedy Shriver National GAPDH primers for an input sample as well as the total amount of IP sample, Institute of Child Health and Human Development in support of the Emory and the ratio of calculated starting quantity of IP:input was used to de- National Fragile X Research Center (S.T.W.); NIH Award 1R21NS091038 (to termine the enrichment of each mRNA target. G.J.B.); and a FRAXA Research Foundation fellowship (to J.A.S.).

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