9402 • The Journal of Neuroscience, June 24, 2015 • 35(25):9402–9408 Cellular/Molecular FXR2P Exerts a Positive Translational Control and Is Required for the Activity-Dependent Increase of PSD95 Expression Esperanza Fernández,1,2 Ka Wan Li,3 Nicholas Rajan,1,2 Silvia De Rubeis,1,2 XMark Fiers,1,2 August B. Smit,3 Tilmann Achsel,1,2 and XClaudia Bagni1,2,4 1KU Leuven, Center for Human Genetics and Leuven Institute for Neuroscience and Disease, Leuven, Belgium, 2VIB Center for the Biology of Disease, 3000 Leuven, Belgium, 3Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University Amsterdam, 1081 HV Amsterdam, The Netherlands, and 4University of Rome Tor Vergata, Department of Biomedicine and Prevention, 00133 Rome, Italy In brain, specific RNA-binding proteins (RBPs) associate with localized mRNAs and function as regulators of protein synthesis at synapses exerting an indirect control on neuronal activity. Thus, the Fragile X Mental Retardation protein (FMRP) regulates expression of the scaffolding postsynaptic density protein PSD95, but the mode of control appears to be different from other FMRP target mRNAs. Here, we show that the fragile X mental retardation-related protein 2 (FXR2P) cooperates with FMRP in binding to the 3Ј-UTR of mouse PSD95/Dlg4 mRNA. Absence of FXR2P leads to decreased translation of PSD95/Dlg4 mRNA in the hippocampus, implying a role for FXR2P as translation activator. Remarkably, mGluR-dependent increase of PSD95 synthesis is abolished in neurons lacking Fxr2. Together, these findings show a coordinated regulation of PSD95/Dlg4 mRNA by FMRP and FXR2P that ultimately affects its fine-tuning during synaptic activity. Key words: FMRP; FXR2P; mRNA translation; PSD95; RNA binding proteins Introduction morphology; furthermore, PSD95 has been linked with the Wil- Several forms of synaptic plasticity induce mRNA transport and liams and Angelman syndromes (Feyder et al., 2010; Cao et al., protein synthesis at synapses resulting in the remodeling of local 2013). signaling networks (Xing and Bassell, 2013). Disc, Large Homolog Expression of hundreds of mRNAs localized in dendrites and 4 (Drosophila)(DLG4) mRNA encodes the postsynaptic density axons (Cajigas et al., 2012) is specifically controlled by neuronal protein 95 (PSD95), the most abundant scaffold within the post- RNA-binding proteins (RBPs) that commonly interact with spe- synaptic density compartment (PSD) (Cheng et al., 2006). PSD95 cific mRNA sequences frequently present in the 5Ј and 3Ј un- plays a key role in spine formation (Feyder et al., 2010), as well as translated regions (UTRs). The 3Ј-UTR regions modulate the LTP and LTD via NMDA and AMPA receptor signaling (Migaud translation and longevity of mRNAs by interacting with RBPs et al., 1998; Stein et al., 2003; Be´ïque et al., 2006). The absence of (Gebauer et al., 2012). Loss of expression or mutations in certain PSD95 affects mouse behavioral phenotypes and dendritic spine RBPs results in diverse neurological disorders characterized by deficits in synaptic structure and function (Kapeli and Yeo, 2012). Received Nov. 20, 2014; revised May 10, 2015; accepted May 11, 2015. Author contributions: E.F., K.W.L., A.B.S., T.A., and C.B. designed research; E.F., K.W.L., N.R., and S.D.R. per- Fragile X Mental Retardation Protein (FMRP) governs the formed research; E.F., K.W.L., N.R., S.D.R., M.F., A.B.S., T.A., and C.B. analyzed data; E.F., T.A., and C.B. wrote the fate of many mRNAs in brain as well as in non-neuronal cells (for paper. review, see Pasciuto and Bagni, 2014). Absence or mutations in TheworkissupportedbyFoundationLejeune,AssociazioneItalianaSindromeXFragile,FWO-G.0705.11,VIB,EU FMRP cause the Fragile X Syndrome (FXS), the most common FP7SynSys(242167toC.B.,A.B.S.,andK.W.L.),andMarieCurieITNBrainTrain(FP7)VIBtoC.B.E.F.wasrecipientof Intra European career development fellowship Marie Curie FP7. We thank Nathalie Leysen, Jonathan Royaert, and form of inherited intellectual disability and a leading cause of Karin Jonckers for excellent technical assistance; Eef Lemmens for the great administrative support; Rob Willemsen monogenic autism (Bagni et al., 2012; Gross et al., 2012). Among (Erasmus MC, The Netherlands) for providing us the Fxr2 KO mice; and Jennifer Darnell for advice on the HITS-CLIP the putative FMRP targets identified so far, 240 mRNAs encode data. postsynaptic proteins, including the PSD95 mRNA (Mud- The authors declare no competing financial interests. Correspondence should be addressed to Dr. Claudia Bagni, KU Leuven, Center for Human Genetics and Leuven dashetty et al., 2007; Zalfa et al., 2007; Darnell et al., 2011). In Institute for Neurodegenerative Diseases, Leuven, Belgium. E-mail: [email protected]; mice, FMRP controls steady-state and activity-dependent PSD95 [email protected]. levels (Muddashetty et al., 2007; Zalfa et al., 2007). S. De Rubeis’ present address: Seaver Autism Center for Research and Treatment, Icahn School of Medicine at Here, we have isolated a subset of brain RBPs that bind the Mount Sinai, New York, NY 10029. Ј DOI:10.1523/JNEUROSCI.4800-14.2015 3 -UTR of PSD95 mRNA. Among them, we found FMRP, the Copyright © 2015 the authors 0270-6474/15/359402-07$15.00/0 members of the ELAV-like family, and FXR2P (fragile X mental Fernández et al. • FXR2P and Translational Activation J. Neurosci., June 24, 2015 • 35(25):9402–9408 • 9403 retardation-related protein 2). FXR2P associates with PSD95 NCBI databases. “Unique” peptides matching only one protein cluster mRNA in vitro and in vivo, requiring the presence of FMRP for were considered for protein identification. Only proteins identified with full binding activity. We show that absence of FXR2P impairs at least two peptides with a confidence interval Ն95% (AB Sciex, per- PSD95 mRNA translation indicating a role for FXR2P as transla- centage) were considered. The “unused” value is defined as the sum of tional activator. Our results show that the paralogs FMRP and protein scores from all the nonredundant peptide matched to a single FXR2P control the same mRNA by different mechanisms, shed- protein. Proteins identified with the antisense PSD95 mRNA were con- sidered as background. ding new light on the control of the Fragile X-related protein Immunohistochemistry. Primary cortical neurons were fixed and family members on target mRNAs through a cooperative or in- stained as by Napoli et al. (2008). Coronal sections from fixed brains of dependent interaction. postnatal 30 d WT mice were permeabilized/blocked at room tempera- ture for3hinPBS, 0.3% Triton X-100, 3% BSA, and 5% normal goat or Materials and Methods donkey serum, incubated with the primary and secondary antibodies, Animals. Animal care was conducted according to national and interna- and mounted on glass coverslips using Fluoroshield (Sigma). tional guidelines (Belgian law of August 14th, 1986, and the following Western blotting. Western blotting was performed using standard pro- K.B. of November 14th, 1993 and K.B of September 13th, 2004; Euro- tocols and chemiluminescence (ECL Plus or Advance, GE Healthcare pean Community Council Directive 86/609, OJa L 358, 1, December 12, Pharmacia) with Fujifilm LAS-3000 or with the laser scanning Odyssey 1987; National Institutes of Health Guide for the Care and Use of Labo- Infrared Imaging System (LI-COR Biosciences). Quantification was per- ratory Animals, National Research Council, 1996). Four-week-old Fmr1 formed using Aida software. knock-out (KO) C57BL/6 (Bakker et al., 1994) and Fxr2 KO FVB (Bon- Neuronal cultures. Primary mouse neurons were prepared as previ- tekoe et al., 2002) males and wild-type (WT) littermates were used. ously described (Napoli et al., 2008) and used after 12–14 d in vitro. Constructs. The WT and mutated 3Ј-UTR of PSD95 mRNA (full- Neurons were stimulated with 100 ␮M DHPG (Sigma-Aldrich) or vehicle length and 5 fragments as described by Zalfa et al., 2007) were transcribed for 20 min. using standard protocols in presence of biotin-16-UTP (Roche). Polysome-mRNA ribonucleoparticle distribution. Analyses of hip- PSD95 mRNA alignments. Transcript dataset from FMRP and HuD pocampal extracts from WT and Fxr2 KO have been performed as by HITS-CLIP from P11-P25 mouse brain polysomes (Darnell et al., 2011) Zalfa et al. (2007). was downloaded from NCBI GEO (accession GSE45148). Statistics. Comparisons between two groups were performed using the Antibodies. Antibodies against FMRP (rAMII) were described previ- nonparametric Mann–Whitney test unless indicated differently. Signifi- ously (Napoli et al., 2008); antibodies against FMRP (7G1), FXR2P cance was accepted with p Ͻ 0.05. Error bars indicate SEM. (1G2), and GAPDH were from Developmental Studies Hybridoma Bank; antibodies against FXR2P and Vinculin were from Sigma-Aldrich; antibodies against HuR, HuD, Tristetraprolin (TTP), phospho-ERK1/2, Results and ERK1/2 were from Santa Cruz Biotechnology; Alexa-488 anti-rabbit, FMRP-dependent and FMRP-independent complexes Alexa-488 anti-goat, and Alexa-568 anti-mouse secondary antibodies associate to the PSD95 3؅-UTR were from Invitrogen. PSD95 mRNA is localized and translated at synapses (Mud- Brain extracts. Mouse forebrain and cortex or hippocampi were ho- dashetty et al., 2007; Zalfa et al., 2007). We searched to identify mogenized in 100 mM NaCl, 10 mM MgCl2,10mM Tris-HCl, pH 7.4, 1% Triton X-100 (Sigma), 1 tablet/10 ml Roche protease inhibitor, 40 U/ml the trans-activating factors that regulate PSD95 mRNA metabo- Ј Rnasin, incubated as in Napoli et al. (2008). lism in an unbiased approach. The 3 -UTR of PSD95 contains RNA extraction. Total RNA from cortex and hippocampus was ex- numerous cis-acting elements, including the G-rich element in- tracted using TRIzol (Invitrogen). volved in the direct binding to FMRP (Zalfa et al., 2007; Mud- Immunoprecipitation (IP).