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FMRP binds Per1 mRNA and downregulates its protein expression in mice

FMRP binds Per1 mRNA and downregulates its protein expression in mice FMRP, an RNA‑binding protein, has previously shown to be involved in regulation of circadian rhythms in flies and mice. However, the molecular mechanism remains elusive. Here we demonstrate that core circadian component Per1 mRNA was a target of FMRP and the association leads to reduced PER1 expression. In Fmr1 KO mice, the oscillation of PER1 protein expression was significantly affected in a temporal and tissue ‑ dependent pattern when compared to WT mice. Our work thus identified Per1 mRNA as a novel target of FMRP and suggested a potential role of FMRP in regulation of circadian function. Keywords FXS, FMRP, Circadian rhythm, Per1 downstream target mRNAs essential for regulation of Introduction neuronal development and function [4]. Models of FXS Fragile X syndrome (FXS) is the most common form in flies and mice exhibit circadian abnormalities in the of heritable intellectual disability and the best-known behavioral rhythm. Mutant flies with dfmr1 deletion are monogenic cause of autism [1]. It is induced by disrup- arrhythmic with respect to the time of eclosion during tion of the fragile X mental retardation 1 gene (FMR1) on the day [5]. In addition, a lack of dfmr1 in adult flies also the X chromosome and the subsequent absence of FMR causes arrhythmic locomotor activity, and overexpres- protein (FMRP) [2]. Patients with FXS suffer from a range sion of dFmr1 leads to long period of circadian rhythms of cognitive and behavioral deficits included social defi - [6]. Mice lacking FMRP protein display a shorter free- cits, anxiety, stereotypic movements, hyperactivity, sei- running period of locomotor activity in total darkness zures, memory deficits, and sleep dysfunction [3]. [7]. FXR2P share above 60% amino acid identity with FMRP is a multifunctional RNA-binding protein FMRP. Interestingly, mice with the absence of both FMRP that regulates the translation, transport and stability of and FXR2P protein completely abolish the rhythmicity of locomotor activity in a light–dark cycle [7]. Recently, *Correspondence: a study reported that a specific loss of FMRP in CA1 Jia‑Da Li lijiada@sklmg.edu.cn pyramidal neurons of the mouse hippocampus results in Renbin Lu circadian-dependent defects in learning and memory [8]. lurenbin@sklmg.edu.cn These studies strongly suggest that FMRP is necessary to Hunan Key Laboratory of Molecular Precision Medicine, Xiangya Hospital, Central South University, Changsha, China maintain the behavioral circadian rhythms and this role Center for Medical Genetics, School of Life Sciences, Central South may be involved in the behavioral alterations observed in University, Changsha 410078, Hunan, China FXS patients. Center for Reproductive Medicine, Women and Children’s Hospital of Chongqing Medical University, Chongqing 400010, China Circadian rhythm of daily variations in many physio- National Clinical Research Center for Geratric Disorder, Xiangya Hospital, logic and behavioral variables, including alertness, blood Central South University, Changsha 410008, China pressure and sleep–wake are driven by endogenous circa- Department of Basic Medical Sciences, Changsha Medical University, Changsha, China dian clocks [9–13]. The circadian clocks are composed of interconnected transcription-translation-based negative © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Tang et al. Molecular Brain (2023) 16:33 Page 2 of 8 feedback loop. In mammals, the basic components of cir- Fmr1-WT forward primer (F1): 5′-AGT AGT TTG GTT cadian locomotor output cycles kaput (CLOCK) forms ACA GTA GTG AAG G-3′; heterodimers with aryl hydrocarbon receptor nuclear Fmr1—Mutant forward primer (F2): 5′-TCA CCA AGG translocator-like protein 1 (ARNTL; also known as TGT GCT ACC AATGC-3′; BMAL1) to activate transcription of Period (Per1, Per2, Fmr1—reverse primer (R): 5′- CTC TAA AAG GGA Per3) and Cryptochrome (Cry1, Cry2) genes via direct AAG CAT CAG GAG -3′. binding to the E-box elements at their promoter regions. All procedures regarding the care and use of animals PER and CRY proteins heterodimerize and translocate were approved by the ethics committee of Center for into the nucleus to interact with CLOCK and BMAL1, Medical Genetics, School of Life Sciences, Central South thus inhibiting their transcriptional activity [14, 15]. The University of China. All methods were performed in rhythmic activation and repression of E-box-driven tran- accordance with approved guidelines. scription generate the endogenous ~ 24-h oscillation of circadian rhythms in mammals [16, 17]. RNA co‑immunoprecipitation In this study, we sought to understand the molecular U2OS Cells were lysed with lysis buffer (20  mmol/L pathogenesis of defects in circadian rhythm in FXS and Tris–HCl pH 7.4, 150  mmol/L NaCl, 5  mmol/L MgCl2, identify potential component of the circadian pathway 1  mmol/L DTT, 1% Triton X-100) supplemented with affected by FMRP. We confirmed that FMRP could bind RNase inhibitor (Takara, Kusatsu, Japan, #2313A) and Per1 mRNA and suppress its expression. In mice, rhyth- proteinase inhibitor cocktail (Sigma-Aldrich, St. Louis, mic expression of PER1 protein in cortex, hypothalamus USA, #P8340). Cleared lysates with 1  mg total protein and liver of Fmr1 KO mice was significantly affected were incubated with Dynabeads Protein G (Invitrogen, compared to WT mice and which was in a temporal and Paisley, UK, #10003D) coated by either anti-FMRP anti- tissue-dependent pattern. However, Fmr1 KO mice show body (Abcam, Cambridge, UK, #ab259335) for U2OS no distinct phenotypes in circadian rhythm of locomotor cells or normal mouse IgGs ( Sigma-Aldrich, Saint Louis, activity. Our data identify Per1 mRNA as a novel target MO, USA, #I5381) overnight at 4  °C, and 10% of the of FMRP and indicate a potential role of FMRP in regula- lysates were saved as input. About 30% of the beads were tion of circadian function. used for Western blot analysis and the rest for mRNA enrichment analysis. RNA was extracted by Trizol (Inv- itrogen, CA, USA, #15596-026) and reverse-transcribed Materials and methods using the Revert Aid First Strand cDNA Synthesis Kit. Cell culture and transfection Quantitative real-time PCR (qRT-PCR) was performed U2OS were cultured in Dulbecco’s modified Eagle’s and the mRNA enrichment was calculated with 18S medium (DMEM) (Sigma-Aldrich, St. Louis, USA, rRNA as an external control and input for normalization. #D5546) supplemented with 10% fetal bovine serum The primers used were as follows:human-Per1-F: 5′-TGA (FBS) (ThermoFisher Scientific, Massachusetts, USA, AGC AAG ACC GGG AGA G-3′;human-Per1-R: 5′-CAC #10099), 100 units/ml penicillin, and 100  μg/ml strepto- ACA CGC CGT CAC ATC A-3′; mycin at 37 °C in 5% C O incubators. Plasmid and siRNA transfections were performed with Lipofectamine 2000 Circadian behavior analysis (Invitrogen) reagents according to the manufacturer’s Mice aged 4–6  months were individually housed within protocol. cages equipped with running wheels and were allowed free access to food and water. Their locomotor activi - Animals ties were recorded as revolutions per 5-min interval. FXS mice were generated by using CRISPR-Cas9 tech- Mice were entrained to an initial LD cycle (light intensity nology. Cas9 mRNA and two guide RNAs (gRNA) target- ∼ 150 lx, lights on at 7:00 AM and lights off at 7:00 PM). ing the upstream and downstream regions of the mouse After 2–3  weeks of activity recording in 12:12-h light– Fmr1 gene were injected into C57BL/6 mouse oocytes, dark conditions, the mice were placed in constant dark- and a mouse with deletion of the 2–5 exon was used as ness (DD) for ∼ 3 weeks. These mice were then subjected a founder. Before behavioral tests, mice of the same sex to a light-induced phase shift at day ~ 20 of DD. Animals were group-housed with 3–5 animals per cage under in their home cages were moved to another room and controlled conditions [temperature, 20 ± 2  °C; relative exposed to a 15-min pulse of white light (∼  150  lx) at humidity, 50–60%; 12:12-h light–dark (LD) cycle, lights circadian time (CT) 16, at which CT12 was designated on at 7:00 AM and lights off at 7:00 PM] and had free as activity onset. The light induced phase-shift ampli - access to food and water. The genotype was confirmed by tude was derived from regression lines drawn through PCR. The primers for genotyping were as following: the activity onset at least 7  days immediately before the T ang et al. Molecular Brain (2023) 16:33 Page 3 of 8 day of stimulation and 7  days after reestablishment of a post hoc Bonferroni multiple comparisons test for three steady-state circadian period after stimulation. The free- or more conditions. P < 0.05 was considered significant. run period was calculated using ClockLab software (Acti- All data are presented as the mean ± SEM; *P < 0.05, metrics, Evanston, IL, USA) in the Matlab environment. **P < 0.01, ***P < 0.001, ****P < 0.0001. The free-run period was measured by a 2 periodogram from days 10 through 25 under DD. Results Western blotting Identification of FMRP targets Mice at the age of 4–8  weeks without any behavioral Given the critical role of FMRP in brain function, numer- test were sacrificed by cervical dislocation. The cortex, ous studies have focused on the transcripts it binds and hypothalamus and liver tissues were dissected at 7am regulates [18, 19]. Cross-linking immunoprecipitation and 7 pm. Cells or tissue samples were lysed in SDS lysis (CLIP) is a highly efficient, commonly used method to buffer (2% SDS, 63  mM Tris–HCl, and 10% glycerol) analyze protein interactions with RNA [20]. In present and the protein concentration was determined using the study, we reanalyzed the data from two previous stud- PierceTM BCA protein Assay kit (Termo Fisher, Waltham ies on identification of potential FMRP target mRNA mass, USA). Proteins in lysates were separated by SDS- in mouse brain by using CLIP technology [8, 21]. As PAGE, transferred to nitrocellulose membranes (PVDF), a result, we identified totally 665 transcripts appeared and immunoblotted with the corresponding antibodies in both studies (Fig.  1A). GO analysis shows that these overnight at 4  °C after blocked in 5% skim milk/Tris- transcripts are mainly enriched in terms of synapse, buffered saline that contained 0.1% Tween 20 at room brain development, social behavior and nervous sys- temperature for 1  h. Membranes were then washed and tem development which consistent with the recognized incubated with horseradish peroxidase conjugate sec- FMRP function (Fig. 1B). Interestingly, 20 transcripts are ondary antibodies. The proteins were visualized using enriched in 3 terms associated with circadian rhythm the Pierce ECL Western Blotting Substrate kit (Thermo including regulation of circadian rhythm, circadian regu- Scientific; 32106). Band intensities were quantified by lation of gene expression, and circadian rhythm (Table 1). ImageJ. The antibodies were listed as following: anti-Per1 When further analyzed the function of these 20 potential antibody-N-terminal (1:500, Abcam, Cambridge, UK, target transcripts and excluded the reported FMRP target #ab136451); anti-FMRP-antibody (1:500, Abcam, Cam- transcripts, we finally selected Per1 as a candidate FMRP bridge, UK, #ab259335); anti-β-actin antibody (1:1000, target transcript. Sigma, USA, A2228). Statistical analysis FMRP interacts with core circadian transcript Per1 mRNA Statistical analyses were performed using GraphPad Per1 is a core component in the mammalian circa- Prism 7 (RRID: RDG_1346427 GraphPad Software, lnc., dian clockwork and is important to maintenance of San Diego, CA, USA). All experiments were repeated at circadian rhythms in cells and tissues [22]. To address least three times and the distribution of data points is whether FMRP directly regulates Per1 mRNA, we first presented as mean ± SEM. Student’s t-test for compari- performed RNA immunoprecipitation (RIP) assay with son of two conditions or ANOVAs were utilized with an antibody against Flag or control IgG in U2OS cells Fig. 1 Identification of FMRP targets. A Venn diagram of potential FMRP targets identified in two previous study conducted by Sawicka et al. and Darnell et al. by using CLIP technology. B Gene ontology in 665 transcripts appeared in both studies Tang et al. Molecular Brain (2023) 16:33 Page 4 of 8 Table 1 Potential targets transcripts of FMRP clustered in circadian rhythm GO‑term Gene count Gene Circadian rhythm 11 SETX, PER1, NTRK2,GSK3B, NCOR1, KCNH7, NRIP1, EP300, ADCY1, PPARGC1A, NPAS2 Circadian regulation of gene expression 9 PER1, NCOA2, EGR1, MAGED1, MYCBP2, NRIP1, HUWE1, PPARGC1A, NPAS2 Regulation of circadian rhythm 8 PRKCG, PER1, GSK3B, MAGED1, USP9X, ADXY1, PPARGC1A, MTOR Fig. 2 FMRP interacts with core circadian transcript Per1 mRNA. A U2OS cells expressing Flag‑FMRP were subjected to RIP assay. Immunoblot confirmed the pull‑ downed Flag‑tagged FMRP. B RT–qPCR analysis shows binding of FMRP to Per1 mRNA in U2OS cells. Data presented as means ± SEM (n = 3). ****P < 0.0001; two‑tailed Student’s t ‑test. C Agarose gel electrophoresis of RT ‑PCR reactions from RIP assay overexpressed Flag-tagged FMRP. Figure  2A confirms FMRP regulates PER1 protein expression in a temporal that the anti-Flag antibodies could specifically immu - and tissue‑dependent patterns in mice noprecipitate Flag-tagged FMRP. The quantitative PCR To elucidate the physiological function of FMR1 in vivo, and RT-PCR results revealed a high enrichment of Per1 we generated a mouse strain with a deletion of Fmr1 mRNA in the immunocomplex pulled-down by Flag gene (Additional file  1: Figure S2). In cortex and hypo- antibody, but not by control IgG (Fig. 2B, C). These data thalamus, PER1 protein expression in Fmr1 KO mice indicated that Per1 mRNA is a potential novel target of was significantly reduced compared to WT mice at ZT0 FMRP protein in cells. (Zeitgeber 0) when FMRP protein was at low-expression level, while PER1 protein expression was increased at ZT12 when FMRP protein was at high-expression level FMRP suppress PER1 protein expression in cells (Fig.  4A–F). However, in peripheral liver tissue, PER1 FMRP can bind its target mRNA and generally act as a protein expression was significantly increased compared to WT mice at ZT0 when FMRP protein was at high- translational repressor. Thus far, our experiments indi - expression level in the WT mice, but no significantly cate that FMRP can interact with the Per1 mRNA, but change was observed between WT and Fmr1 KO mice the functional role of this interaction is not clarified. at ZT12 when FMRP protein was at low-expression level To assess the consequence of this interaction on Per1 in the WT mice (Fig.  4G–I). The above findings indi mRNA level, we knockdown FMRP by transient trans- - fection of siRNAs. Silencing Fmr1 gene significantly cate that FMRP deficiency leads to significant changes increased the Per1 mRNA level as analyzed by qPCR in rhythmic expressions of PER1 protein and this conse- (Additional file  1: Figure S1). We also detect the effect quence shows temporal and tissue-specific effects. of FMRP on Per1 protein expression. Consistantly, To address the function of FMRP in circadian regula- silencing Fmr1 gene significantly increased Per1 protein tion, we monitored the wheel-running activity of Fmr1 expression (Fig.  3A, B) and conversely, overexpression KO mice and their WT littermate controls. Both WT and of Fmr1 decreased Per1 protein expression (Fig.  3C, Fmr1 KO mice entrained to LD cycles and showed no sig- D). These results suggest FMRP suppress PER1 protein nificant differences in daily counts or amplitudes of loco - expression in cells. motor rhythmicity (Additional file  1: Figure S3A). Under T ang et al. Molecular Brain (2023) 16:33 Page 5 of 8 Fig. 3 FMRP suppress PER1 protein expression in cells. A, B Representative immunoblots (A) and statistics data of three independent experiments (B) from U2OS cells transfected with control siRNA (NC) or Fmr1 siRNAs. Data are presented as means ± SEM, **P < 0.01, ***P < 0.001 post hoc Dunnett’s t‑test, one ‑ way ANOVA. C, D Representative immunoblots (C) and statistics data of three independent experiments (D) from U2OS cells transfected with different doses of Fmr1 or empty vector (EV ). Data are presented as means ± SEM, **P < 0.01, post hoc Dunnett’s t‑test, one ‑ way ANOVA DD, both WT and Fmr1 KO mice showed similar free- translation. Thus, our results identified Per1 mRNA as a running periods (Additional file  1: Figure S3A-B). We new target for FMRP. also compared the phase shifts generated by exposure to The main function of FMRP is to interact with target a brief light pulse at CT16 (white light, ∼ 150 lx, 10 min) mRNA and commonly act as a repressor of target mRNA under DD conditions. In response to this treatment, we translation [4, 24]. Our data indicated that deletion of did not see any significant difference between WT and Fmr1 expression led to aberrant Per1 protein expression Fmr1 KO mice (Additional file  1: Figures  S3C-D). Our in cells and tissues, suggesting FMRP affected the trans - data thus demonstrate that Fmr1 KO mice have normal lation of Per1 mRNA. In fact, FMRP regulates mRNA phenotypes in circadian rhythm of locomotor activity. translation in various ways. FMRP can reversibly stall ribosomes specifically on its target mRNAs in the process Discussion of translation. In FMRP loss-function mouse model, ribo- FXS as the most common cause of inherited intellec- somal stalling on FMRP target transcripts is relieved and tual disability, results from the loss of the FMRP protein protein expression is significantly increased in the brain expression [2, 23]. Given the critical role of FMRP in neu- [18]. FMRP can regulate the binding of mRNA to ribo- ronal development, its physiological target transcripts some by binding to target mRNA through the G-quartets were extensively studied. Previous studies suggested that that is ubiquitous on mRNA [25]. Cells derived from Per1 mRNA might bind to FMRP using CLIP technology FXS patients display abnormal polyribosome profiles, in mouse brain tissues [8, 21]. In our study, we provided which indicates that the absence of FMRP alters transla- molecular evidence for that FMRP specifically inter - tion [26]. FMRP can also directly bind to ribosomes to acts with Per1 mRNA and disruption of this interaction inhibit mRNA translation. In details, FMRP binds within results in aberrant Per1 mRNA level and Per1 mRNA the intersubunit space of the 80 s ribosome which would Tang et al. Molecular Brain (2023) 16:33 Page 6 of 8 Fig. 4 FMRP regulates PER1 protein expression in a temporal and tissue‑ dependent patterns in mice. A–C Western blot analysis of cortex lysates from WT and Fmr1 KO mice taken at the ZT0 and ZT12 of a day (A) and the quantification of Per1 (B) and FMRP (C) protein levels; Data are presented as means ± SEM; *P < 0.05; **P < 0.01; two‑tailed Student’s t ‑test; n = 4 mice/genotype/time point. D–F Western blot analysis of hypothalamus lysates from WT and Fmr1 KO mice taken at the ZT0 and ZT12 of a day (A) and the quantification of Per1 (B) and FMRP (C) protein levels; Data are presented as means ± SEM; *P < 0.05; **P < 0.01; two‑tailed Student’s t ‑test; n = 4 mice/genotype/time point. G–I Western blot analysis of liver lysates from WT and Fmr1 KO mice taken at the ZT0 and ZT12 of a day (A) and the quantification of Per1 (B) and FMRP (C) protein levels; Data are presented as means ± SEM; ns: P > 0.05; **P < 0.01; two‑tailed Student’s t ‑test; n = 4 mice/genotype/time point results in a blockage of the binding of tRNA and transla- mGluR stimulation in cortex of Fmr1 KO mice, whereas tion elongation factors on the ribosome, thereby reduc- it was illustrated to be normal in hippocampal tissue ing protein translation [27]. Therefore, although our data [29]. mTOR activity was found normal at synapses of indicated that FMRP binds to Per1 mRNA to regulate its the neocortex of Fmr1 KO mice, whereas it is elevated translation, how FMRP functions in this process requires at synapses of the hippocampus. Interestingly, our data further investigation. shows that PER1 protein expression was significantly There is increasing evidence that the absence of reduced at ZT0 in cortex and hypothalamus, while it is FMRP leads to tissue and cell-type specific deficits. significantly increased in peripheral liver tissue of Fmr1 For example, several studies reported that extracel- KO mice. These results suggest that FMRP may regulate lular signal-regulated kinase (ERK) and mechanistic the expression of Per1 in a tissue-dependent patterns in target of rapamycin (mTOR) signaling was disrupted mice. In addition, our data also indicates that though in the FMRP deficient mice while the effect on these PER1 protein expression was significantly increased pathways was different between hippocampus and the compared to WT mice at ZT0, no significantly change cortex [28]. ERK was aberrant deactivated following was observed between WT and Fmr1 KO mice at ZT12 T ang et al. Molecular Brain (2023) 16:33 Page 7 of 8 Funding when FMRP protein was at low-expression level in the This work was funded by China Postdoctoral Science Foundation (No. WT mice. Our data support the speculation that FMRP 2021T140746) and National Natural Science Foundation of China (82101960 to may affect the expression phase of Per1 protein. RBL and 31972913 to JDL). Two studies reported by Dockendorff et  al. and Data availability Morales et  al. demonstrated that although the rhyth- No data was used for the research described in the article. mic mRNA and protein expressions of the core clock genes per and tim was normal in the FXS Drosophila Declarations melanogaster, FMRP deficiency results in behavioral Ethics approval and consent to participate phenotypes of FXS Drosophila melanogaster including All procedures regarding the care and use of animals were approved by the arrhythmic eclosion and locomotor activity [5, 6]. In ethics committee of Center for Medical Genetics, School of Life Sciences, mammals, mice lacking FMRP display a slightly shorter Central South University of China. All methods were performed in accordance with approved guidelines. free-running period of locomotor activity in total dark- ness [7]. Additionally, loss of FMRP in CA1 pyramidal Consent for publication neurons of the mouse hippocampus results in circa- Not applicable. dian-dependent defects in learning and memory [8]. In Competing interests our study, we have carried out behavioral paradigms to The authors have declared that no competing interests exist. detect the circadian rhythm phenotypes of FXS mice under LD or DD condition, however, no obvious abnor- Received: 27 January 2023 Accepted: 24 March 2023 mality has been detected. In our study, we conducted rhythmic behavior testing with 4–6  months old mice. 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Importance of circadian timing for aging and longevity. Nat Commun. 2021;12:2862. rapid publication on acceptance https:// doi. org/ 10. 1038/ s41467‑ 021‑ 22922‑6. support for research data, including large and complex data types 32. Poggiogalle E, Jamshed H, Peterson CM. Circadian regulation of glucose, • gold Open Access which fosters wider collaboration and increased citations lipid, and energy metabolism in humans. Metabolism. 2018;84:11–27. https:// doi. org/ 10. 1016/j. metab ol. 2017. 11. 017. maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ Learn more biomedcentral.com/submissions lished maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Brain Springer Journals

FMRP binds Per1 mRNA and downregulates its protein expression in mice

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Abstract

FMRP, an RNA‑binding protein, has previously shown to be involved in regulation of circadian rhythms in flies and mice. However, the molecular mechanism remains elusive. Here we demonstrate that core circadian component Per1 mRNA was a target of FMRP and the association leads to reduced PER1 expression. In Fmr1 KO mice, the oscillation of PER1 protein expression was significantly affected in a temporal and tissue ‑ dependent pattern when compared to WT mice. Our work thus identified Per1 mRNA as a novel target of FMRP and suggested a potential role of FMRP in regulation of circadian function. Keywords FXS, FMRP, Circadian rhythm, Per1 downstream target mRNAs essential for regulation of Introduction neuronal development and function [4]. Models of FXS Fragile X syndrome (FXS) is the most common form in flies and mice exhibit circadian abnormalities in the of heritable intellectual disability and the best-known behavioral rhythm. Mutant flies with dfmr1 deletion are monogenic cause of autism [1]. It is induced by disrup- arrhythmic with respect to the time of eclosion during tion of the fragile X mental retardation 1 gene (FMR1) on the day [5]. In addition, a lack of dfmr1 in adult flies also the X chromosome and the subsequent absence of FMR causes arrhythmic locomotor activity, and overexpres- protein (FMRP) [2]. Patients with FXS suffer from a range sion of dFmr1 leads to long period of circadian rhythms of cognitive and behavioral deficits included social defi - [6]. Mice lacking FMRP protein display a shorter free- cits, anxiety, stereotypic movements, hyperactivity, sei- running period of locomotor activity in total darkness zures, memory deficits, and sleep dysfunction [3]. [7]. FXR2P share above 60% amino acid identity with FMRP is a multifunctional RNA-binding protein FMRP. Interestingly, mice with the absence of both FMRP that regulates the translation, transport and stability of and FXR2P protein completely abolish the rhythmicity of locomotor activity in a light–dark cycle [7]. Recently, *Correspondence: a study reported that a specific loss of FMRP in CA1 Jia‑Da Li lijiada@sklmg.edu.cn pyramidal neurons of the mouse hippocampus results in Renbin Lu circadian-dependent defects in learning and memory [8]. lurenbin@sklmg.edu.cn These studies strongly suggest that FMRP is necessary to Hunan Key Laboratory of Molecular Precision Medicine, Xiangya Hospital, Central South University, Changsha, China maintain the behavioral circadian rhythms and this role Center for Medical Genetics, School of Life Sciences, Central South may be involved in the behavioral alterations observed in University, Changsha 410078, Hunan, China FXS patients. Center for Reproductive Medicine, Women and Children’s Hospital of Chongqing Medical University, Chongqing 400010, China Circadian rhythm of daily variations in many physio- National Clinical Research Center for Geratric Disorder, Xiangya Hospital, logic and behavioral variables, including alertness, blood Central South University, Changsha 410008, China pressure and sleep–wake are driven by endogenous circa- Department of Basic Medical Sciences, Changsha Medical University, Changsha, China dian clocks [9–13]. The circadian clocks are composed of interconnected transcription-translation-based negative © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Tang et al. Molecular Brain (2023) 16:33 Page 2 of 8 feedback loop. In mammals, the basic components of cir- Fmr1-WT forward primer (F1): 5′-AGT AGT TTG GTT cadian locomotor output cycles kaput (CLOCK) forms ACA GTA GTG AAG G-3′; heterodimers with aryl hydrocarbon receptor nuclear Fmr1—Mutant forward primer (F2): 5′-TCA CCA AGG translocator-like protein 1 (ARNTL; also known as TGT GCT ACC AATGC-3′; BMAL1) to activate transcription of Period (Per1, Per2, Fmr1—reverse primer (R): 5′- CTC TAA AAG GGA Per3) and Cryptochrome (Cry1, Cry2) genes via direct AAG CAT CAG GAG -3′. binding to the E-box elements at their promoter regions. All procedures regarding the care and use of animals PER and CRY proteins heterodimerize and translocate were approved by the ethics committee of Center for into the nucleus to interact with CLOCK and BMAL1, Medical Genetics, School of Life Sciences, Central South thus inhibiting their transcriptional activity [14, 15]. The University of China. All methods were performed in rhythmic activation and repression of E-box-driven tran- accordance with approved guidelines. scription generate the endogenous ~ 24-h oscillation of circadian rhythms in mammals [16, 17]. RNA co‑immunoprecipitation In this study, we sought to understand the molecular U2OS Cells were lysed with lysis buffer (20  mmol/L pathogenesis of defects in circadian rhythm in FXS and Tris–HCl pH 7.4, 150  mmol/L NaCl, 5  mmol/L MgCl2, identify potential component of the circadian pathway 1  mmol/L DTT, 1% Triton X-100) supplemented with affected by FMRP. We confirmed that FMRP could bind RNase inhibitor (Takara, Kusatsu, Japan, #2313A) and Per1 mRNA and suppress its expression. In mice, rhyth- proteinase inhibitor cocktail (Sigma-Aldrich, St. Louis, mic expression of PER1 protein in cortex, hypothalamus USA, #P8340). Cleared lysates with 1  mg total protein and liver of Fmr1 KO mice was significantly affected were incubated with Dynabeads Protein G (Invitrogen, compared to WT mice and which was in a temporal and Paisley, UK, #10003D) coated by either anti-FMRP anti- tissue-dependent pattern. However, Fmr1 KO mice show body (Abcam, Cambridge, UK, #ab259335) for U2OS no distinct phenotypes in circadian rhythm of locomotor cells or normal mouse IgGs ( Sigma-Aldrich, Saint Louis, activity. Our data identify Per1 mRNA as a novel target MO, USA, #I5381) overnight at 4  °C, and 10% of the of FMRP and indicate a potential role of FMRP in regula- lysates were saved as input. About 30% of the beads were tion of circadian function. used for Western blot analysis and the rest for mRNA enrichment analysis. RNA was extracted by Trizol (Inv- itrogen, CA, USA, #15596-026) and reverse-transcribed Materials and methods using the Revert Aid First Strand cDNA Synthesis Kit. Cell culture and transfection Quantitative real-time PCR (qRT-PCR) was performed U2OS were cultured in Dulbecco’s modified Eagle’s and the mRNA enrichment was calculated with 18S medium (DMEM) (Sigma-Aldrich, St. Louis, USA, rRNA as an external control and input for normalization. #D5546) supplemented with 10% fetal bovine serum The primers used were as follows:human-Per1-F: 5′-TGA (FBS) (ThermoFisher Scientific, Massachusetts, USA, AGC AAG ACC GGG AGA G-3′;human-Per1-R: 5′-CAC #10099), 100 units/ml penicillin, and 100  μg/ml strepto- ACA CGC CGT CAC ATC A-3′; mycin at 37 °C in 5% C O incubators. Plasmid and siRNA transfections were performed with Lipofectamine 2000 Circadian behavior analysis (Invitrogen) reagents according to the manufacturer’s Mice aged 4–6  months were individually housed within protocol. cages equipped with running wheels and were allowed free access to food and water. Their locomotor activi - Animals ties were recorded as revolutions per 5-min interval. FXS mice were generated by using CRISPR-Cas9 tech- Mice were entrained to an initial LD cycle (light intensity nology. Cas9 mRNA and two guide RNAs (gRNA) target- ∼ 150 lx, lights on at 7:00 AM and lights off at 7:00 PM). ing the upstream and downstream regions of the mouse After 2–3  weeks of activity recording in 12:12-h light– Fmr1 gene were injected into C57BL/6 mouse oocytes, dark conditions, the mice were placed in constant dark- and a mouse with deletion of the 2–5 exon was used as ness (DD) for ∼ 3 weeks. These mice were then subjected a founder. Before behavioral tests, mice of the same sex to a light-induced phase shift at day ~ 20 of DD. Animals were group-housed with 3–5 animals per cage under in their home cages were moved to another room and controlled conditions [temperature, 20 ± 2  °C; relative exposed to a 15-min pulse of white light (∼  150  lx) at humidity, 50–60%; 12:12-h light–dark (LD) cycle, lights circadian time (CT) 16, at which CT12 was designated on at 7:00 AM and lights off at 7:00 PM] and had free as activity onset. The light induced phase-shift ampli - access to food and water. The genotype was confirmed by tude was derived from regression lines drawn through PCR. The primers for genotyping were as following: the activity onset at least 7  days immediately before the T ang et al. Molecular Brain (2023) 16:33 Page 3 of 8 day of stimulation and 7  days after reestablishment of a post hoc Bonferroni multiple comparisons test for three steady-state circadian period after stimulation. The free- or more conditions. P < 0.05 was considered significant. run period was calculated using ClockLab software (Acti- All data are presented as the mean ± SEM; *P < 0.05, metrics, Evanston, IL, USA) in the Matlab environment. **P < 0.01, ***P < 0.001, ****P < 0.0001. The free-run period was measured by a 2 periodogram from days 10 through 25 under DD. Results Western blotting Identification of FMRP targets Mice at the age of 4–8  weeks without any behavioral Given the critical role of FMRP in brain function, numer- test were sacrificed by cervical dislocation. The cortex, ous studies have focused on the transcripts it binds and hypothalamus and liver tissues were dissected at 7am regulates [18, 19]. Cross-linking immunoprecipitation and 7 pm. Cells or tissue samples were lysed in SDS lysis (CLIP) is a highly efficient, commonly used method to buffer (2% SDS, 63  mM Tris–HCl, and 10% glycerol) analyze protein interactions with RNA [20]. In present and the protein concentration was determined using the study, we reanalyzed the data from two previous stud- PierceTM BCA protein Assay kit (Termo Fisher, Waltham ies on identification of potential FMRP target mRNA mass, USA). Proteins in lysates were separated by SDS- in mouse brain by using CLIP technology [8, 21]. As PAGE, transferred to nitrocellulose membranes (PVDF), a result, we identified totally 665 transcripts appeared and immunoblotted with the corresponding antibodies in both studies (Fig.  1A). GO analysis shows that these overnight at 4  °C after blocked in 5% skim milk/Tris- transcripts are mainly enriched in terms of synapse, buffered saline that contained 0.1% Tween 20 at room brain development, social behavior and nervous sys- temperature for 1  h. Membranes were then washed and tem development which consistent with the recognized incubated with horseradish peroxidase conjugate sec- FMRP function (Fig. 1B). Interestingly, 20 transcripts are ondary antibodies. The proteins were visualized using enriched in 3 terms associated with circadian rhythm the Pierce ECL Western Blotting Substrate kit (Thermo including regulation of circadian rhythm, circadian regu- Scientific; 32106). Band intensities were quantified by lation of gene expression, and circadian rhythm (Table 1). ImageJ. The antibodies were listed as following: anti-Per1 When further analyzed the function of these 20 potential antibody-N-terminal (1:500, Abcam, Cambridge, UK, target transcripts and excluded the reported FMRP target #ab136451); anti-FMRP-antibody (1:500, Abcam, Cam- transcripts, we finally selected Per1 as a candidate FMRP bridge, UK, #ab259335); anti-β-actin antibody (1:1000, target transcript. Sigma, USA, A2228). Statistical analysis FMRP interacts with core circadian transcript Per1 mRNA Statistical analyses were performed using GraphPad Per1 is a core component in the mammalian circa- Prism 7 (RRID: RDG_1346427 GraphPad Software, lnc., dian clockwork and is important to maintenance of San Diego, CA, USA). All experiments were repeated at circadian rhythms in cells and tissues [22]. To address least three times and the distribution of data points is whether FMRP directly regulates Per1 mRNA, we first presented as mean ± SEM. Student’s t-test for compari- performed RNA immunoprecipitation (RIP) assay with son of two conditions or ANOVAs were utilized with an antibody against Flag or control IgG in U2OS cells Fig. 1 Identification of FMRP targets. A Venn diagram of potential FMRP targets identified in two previous study conducted by Sawicka et al. and Darnell et al. by using CLIP technology. B Gene ontology in 665 transcripts appeared in both studies Tang et al. Molecular Brain (2023) 16:33 Page 4 of 8 Table 1 Potential targets transcripts of FMRP clustered in circadian rhythm GO‑term Gene count Gene Circadian rhythm 11 SETX, PER1, NTRK2,GSK3B, NCOR1, KCNH7, NRIP1, EP300, ADCY1, PPARGC1A, NPAS2 Circadian regulation of gene expression 9 PER1, NCOA2, EGR1, MAGED1, MYCBP2, NRIP1, HUWE1, PPARGC1A, NPAS2 Regulation of circadian rhythm 8 PRKCG, PER1, GSK3B, MAGED1, USP9X, ADXY1, PPARGC1A, MTOR Fig. 2 FMRP interacts with core circadian transcript Per1 mRNA. A U2OS cells expressing Flag‑FMRP were subjected to RIP assay. Immunoblot confirmed the pull‑ downed Flag‑tagged FMRP. B RT–qPCR analysis shows binding of FMRP to Per1 mRNA in U2OS cells. Data presented as means ± SEM (n = 3). ****P < 0.0001; two‑tailed Student’s t ‑test. C Agarose gel electrophoresis of RT ‑PCR reactions from RIP assay overexpressed Flag-tagged FMRP. Figure  2A confirms FMRP regulates PER1 protein expression in a temporal that the anti-Flag antibodies could specifically immu - and tissue‑dependent patterns in mice noprecipitate Flag-tagged FMRP. The quantitative PCR To elucidate the physiological function of FMR1 in vivo, and RT-PCR results revealed a high enrichment of Per1 we generated a mouse strain with a deletion of Fmr1 mRNA in the immunocomplex pulled-down by Flag gene (Additional file  1: Figure S2). In cortex and hypo- antibody, but not by control IgG (Fig. 2B, C). These data thalamus, PER1 protein expression in Fmr1 KO mice indicated that Per1 mRNA is a potential novel target of was significantly reduced compared to WT mice at ZT0 FMRP protein in cells. (Zeitgeber 0) when FMRP protein was at low-expression level, while PER1 protein expression was increased at ZT12 when FMRP protein was at high-expression level FMRP suppress PER1 protein expression in cells (Fig.  4A–F). However, in peripheral liver tissue, PER1 FMRP can bind its target mRNA and generally act as a protein expression was significantly increased compared to WT mice at ZT0 when FMRP protein was at high- translational repressor. Thus far, our experiments indi - expression level in the WT mice, but no significantly cate that FMRP can interact with the Per1 mRNA, but change was observed between WT and Fmr1 KO mice the functional role of this interaction is not clarified. at ZT12 when FMRP protein was at low-expression level To assess the consequence of this interaction on Per1 in the WT mice (Fig.  4G–I). The above findings indi mRNA level, we knockdown FMRP by transient trans- - fection of siRNAs. Silencing Fmr1 gene significantly cate that FMRP deficiency leads to significant changes increased the Per1 mRNA level as analyzed by qPCR in rhythmic expressions of PER1 protein and this conse- (Additional file  1: Figure S1). We also detect the effect quence shows temporal and tissue-specific effects. of FMRP on Per1 protein expression. Consistantly, To address the function of FMRP in circadian regula- silencing Fmr1 gene significantly increased Per1 protein tion, we monitored the wheel-running activity of Fmr1 expression (Fig.  3A, B) and conversely, overexpression KO mice and their WT littermate controls. Both WT and of Fmr1 decreased Per1 protein expression (Fig.  3C, Fmr1 KO mice entrained to LD cycles and showed no sig- D). These results suggest FMRP suppress PER1 protein nificant differences in daily counts or amplitudes of loco - expression in cells. motor rhythmicity (Additional file  1: Figure S3A). Under T ang et al. Molecular Brain (2023) 16:33 Page 5 of 8 Fig. 3 FMRP suppress PER1 protein expression in cells. A, B Representative immunoblots (A) and statistics data of three independent experiments (B) from U2OS cells transfected with control siRNA (NC) or Fmr1 siRNAs. Data are presented as means ± SEM, **P < 0.01, ***P < 0.001 post hoc Dunnett’s t‑test, one ‑ way ANOVA. C, D Representative immunoblots (C) and statistics data of three independent experiments (D) from U2OS cells transfected with different doses of Fmr1 or empty vector (EV ). Data are presented as means ± SEM, **P < 0.01, post hoc Dunnett’s t‑test, one ‑ way ANOVA DD, both WT and Fmr1 KO mice showed similar free- translation. Thus, our results identified Per1 mRNA as a running periods (Additional file  1: Figure S3A-B). We new target for FMRP. also compared the phase shifts generated by exposure to The main function of FMRP is to interact with target a brief light pulse at CT16 (white light, ∼ 150 lx, 10 min) mRNA and commonly act as a repressor of target mRNA under DD conditions. In response to this treatment, we translation [4, 24]. Our data indicated that deletion of did not see any significant difference between WT and Fmr1 expression led to aberrant Per1 protein expression Fmr1 KO mice (Additional file  1: Figures  S3C-D). Our in cells and tissues, suggesting FMRP affected the trans - data thus demonstrate that Fmr1 KO mice have normal lation of Per1 mRNA. In fact, FMRP regulates mRNA phenotypes in circadian rhythm of locomotor activity. translation in various ways. FMRP can reversibly stall ribosomes specifically on its target mRNAs in the process Discussion of translation. In FMRP loss-function mouse model, ribo- FXS as the most common cause of inherited intellec- somal stalling on FMRP target transcripts is relieved and tual disability, results from the loss of the FMRP protein protein expression is significantly increased in the brain expression [2, 23]. Given the critical role of FMRP in neu- [18]. FMRP can regulate the binding of mRNA to ribo- ronal development, its physiological target transcripts some by binding to target mRNA through the G-quartets were extensively studied. Previous studies suggested that that is ubiquitous on mRNA [25]. Cells derived from Per1 mRNA might bind to FMRP using CLIP technology FXS patients display abnormal polyribosome profiles, in mouse brain tissues [8, 21]. In our study, we provided which indicates that the absence of FMRP alters transla- molecular evidence for that FMRP specifically inter - tion [26]. FMRP can also directly bind to ribosomes to acts with Per1 mRNA and disruption of this interaction inhibit mRNA translation. In details, FMRP binds within results in aberrant Per1 mRNA level and Per1 mRNA the intersubunit space of the 80 s ribosome which would Tang et al. Molecular Brain (2023) 16:33 Page 6 of 8 Fig. 4 FMRP regulates PER1 protein expression in a temporal and tissue‑ dependent patterns in mice. A–C Western blot analysis of cortex lysates from WT and Fmr1 KO mice taken at the ZT0 and ZT12 of a day (A) and the quantification of Per1 (B) and FMRP (C) protein levels; Data are presented as means ± SEM; *P < 0.05; **P < 0.01; two‑tailed Student’s t ‑test; n = 4 mice/genotype/time point. D–F Western blot analysis of hypothalamus lysates from WT and Fmr1 KO mice taken at the ZT0 and ZT12 of a day (A) and the quantification of Per1 (B) and FMRP (C) protein levels; Data are presented as means ± SEM; *P < 0.05; **P < 0.01; two‑tailed Student’s t ‑test; n = 4 mice/genotype/time point. G–I Western blot analysis of liver lysates from WT and Fmr1 KO mice taken at the ZT0 and ZT12 of a day (A) and the quantification of Per1 (B) and FMRP (C) protein levels; Data are presented as means ± SEM; ns: P > 0.05; **P < 0.01; two‑tailed Student’s t ‑test; n = 4 mice/genotype/time point results in a blockage of the binding of tRNA and transla- mGluR stimulation in cortex of Fmr1 KO mice, whereas tion elongation factors on the ribosome, thereby reduc- it was illustrated to be normal in hippocampal tissue ing protein translation [27]. Therefore, although our data [29]. mTOR activity was found normal at synapses of indicated that FMRP binds to Per1 mRNA to regulate its the neocortex of Fmr1 KO mice, whereas it is elevated translation, how FMRP functions in this process requires at synapses of the hippocampus. Interestingly, our data further investigation. shows that PER1 protein expression was significantly There is increasing evidence that the absence of reduced at ZT0 in cortex and hypothalamus, while it is FMRP leads to tissue and cell-type specific deficits. significantly increased in peripheral liver tissue of Fmr1 For example, several studies reported that extracel- KO mice. These results suggest that FMRP may regulate lular signal-regulated kinase (ERK) and mechanistic the expression of Per1 in a tissue-dependent patterns in target of rapamycin (mTOR) signaling was disrupted mice. In addition, our data also indicates that though in the FMRP deficient mice while the effect on these PER1 protein expression was significantly increased pathways was different between hippocampus and the compared to WT mice at ZT0, no significantly change cortex [28]. ERK was aberrant deactivated following was observed between WT and Fmr1 KO mice at ZT12 T ang et al. Molecular Brain (2023) 16:33 Page 7 of 8 Funding when FMRP protein was at low-expression level in the This work was funded by China Postdoctoral Science Foundation (No. WT mice. Our data support the speculation that FMRP 2021T140746) and National Natural Science Foundation of China (82101960 to may affect the expression phase of Per1 protein. RBL and 31972913 to JDL). Two studies reported by Dockendorff et  al. and Data availability Morales et  al. demonstrated that although the rhyth- No data was used for the research described in the article. mic mRNA and protein expressions of the core clock genes per and tim was normal in the FXS Drosophila Declarations melanogaster, FMRP deficiency results in behavioral Ethics approval and consent to participate phenotypes of FXS Drosophila melanogaster including All procedures regarding the care and use of animals were approved by the arrhythmic eclosion and locomotor activity [5, 6]. In ethics committee of Center for Medical Genetics, School of Life Sciences, mammals, mice lacking FMRP display a slightly shorter Central South University of China. All methods were performed in accordance with approved guidelines. free-running period of locomotor activity in total dark- ness [7]. Additionally, loss of FMRP in CA1 pyramidal Consent for publication neurons of the mouse hippocampus results in circa- Not applicable. dian-dependent defects in learning and memory [8]. In Competing interests our study, we have carried out behavioral paradigms to The authors have declared that no competing interests exist. detect the circadian rhythm phenotypes of FXS mice under LD or DD condition, however, no obvious abnor- Received: 27 January 2023 Accepted: 24 March 2023 mality has been detected. In our study, we conducted rhythmic behavior testing with 4–6  months old mice. 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Journal

Molecular Brain – Springer Journals

Published: Apr 5, 2023

Keywords: FXS; FMRP; Circadian rhythm; Per1

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