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• W1996285721 abstract "•The RNA-binding protein PUMILIO1 regulates levels of ATAXIN1 protein and mRNA•A modest increase in wild-type Ataxin1 levels is enough to cause neurodegeneration•Pumilio1 haploinsufficiency accelerates SCA1 disease progression•Ataxin1 haploinsufficiency rescues Pumilio1+/− phenotypes Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative proteinopathy, in which a mutant protein (in this case, ATAXIN1) accumulates in neurons and exerts toxicity; in SCA1, this process causes progressive deterioration of motor coordination. Seeking to understand how post-translational modification of ATAXIN1 levels influences disease, we discovered that the RNA-binding protein PUMILIO1 (PUM1) not only directly regulates ATAXIN1 but also plays an unexpectedly important role in neuronal function. Loss of Pum1 caused progressive motor dysfunction and SCA1-like neurodegeneration with motor impairment, primarily by increasing Ataxin1 levels. Breeding Pum1+/− mice to SCA1 mice (Atxn1154Q/+) exacerbated disease progression, whereas breeding them to Atxn1+/− mice normalized Ataxin1 levels and largely rescued the Pum1+/− phenotype. Thus, both increased wild-type ATAXIN1 levels and PUM1 haploinsufficiency could contribute to human neurodegeneration. These results demonstrate the importance of studying post-transcriptional regulation of disease-driving proteins to reveal factors underlying neurodegenerative disease. Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative proteinopathy, in which a mutant protein (in this case, ATAXIN1) accumulates in neurons and exerts toxicity; in SCA1, this process causes progressive deterioration of motor coordination. Seeking to understand how post-translational modification of ATAXIN1 levels influences disease, we discovered that the RNA-binding protein PUMILIO1 (PUM1) not only directly regulates ATAXIN1 but also plays an unexpectedly important role in neuronal function. Loss of Pum1 caused progressive motor dysfunction and SCA1-like neurodegeneration with motor impairment, primarily by increasing Ataxin1 levels. Breeding Pum1+/− mice to SCA1 mice (Atxn1154Q/+) exacerbated disease progression, whereas breeding them to Atxn1+/− mice normalized Ataxin1 levels and largely rescued the Pum1+/− phenotype. Thus, both increased wild-type ATAXIN1 levels and PUM1 haploinsufficiency could contribute to human neurodegeneration. These results demonstrate the importance of studying post-transcriptional regulation of disease-driving proteins to reveal factors underlying neurodegenerative disease. Misfolded proteins underlie the pathogenesis of a number of neurodegenerative conditions, collectively known as proteinopathies. Alzheimer disease (AD), Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), and polyglutamine diseases such as Huntington disease all fall into this category (Ross and Poirier, 2004Ross C.A. Poirier M.A. Protein aggregation and neurodegenerative disease.Nat. Med. 2004; 10: S10-S17Crossref PubMed Scopus (2453) Google Scholar, Soto, 2003Soto C. Unfolding the role of protein misfolding in neurodegenerative diseases.Nat. Rev. Neurosci. 2003; 4: 49-60Crossref PubMed Scopus (1101) Google Scholar). Despite the heterogeneity of their pathogenic mechanisms, in each of these diseases, the misfolded protein accumulates in neurons and exerts toxicity. Somewhat surprisingly, the brain can also be sensitive to elevated levels of wild-type (WT) protein: duplication of the amyloid precursor protein (APP) locus causes autosomal dominant early-onset AD (Rovelet-Lecrux et al., 2006Rovelet-Lecrux A. Hannequin D. Raux G. Le Meur N. Laquerrière A. Vital A. Dumanchin C. Feuillette S. Brice A. Vercelletto M. et al.APP locus duplication causes autosomal dominant early-onset Alzheimer disease with cerebral amyloid angiopathy.Nat. Genet. 2006; 38: 24-26Crossref PubMed Scopus (967) Google Scholar, Rumble et al., 1989Rumble B. Retallack R. Hilbich C. Simms G. Multhaup G. Martins R. Hockey A. Montgomery P. Beyreuther K. Masters C.L. Amyloid A4 protein and its precursor in Down’s syndrome and Alzheimer’s disease.N. Engl. J. Med. 1989; 320: 1446-1452Crossref PubMed Scopus (546) Google Scholar), and duplications or triplications of α-synuclein (SNCA) are associated with familial PD (Chartier-Harlin et al., 2004Chartier-Harlin M.C. Kachergus J. Roumier C. Mouroux V. Douay X. Lincoln S. Levecque C. Larvor L. Andrieux J. Hulihan M. et al.Alpha-synuclein locus duplication as a cause of familial Parkinson’s disease.Lancet. 2004; 364: 1167-1169Abstract Full Text Full Text PDF PubMed Scopus (1573) Google Scholar, Ibáñez et al., 2004Ibáñez P. Bonnet A.M. Débarges B. Lohmann E. Tison F. Pollak P. Agid Y. Dürr A. Brice A. Causal relation between alpha-synuclein gene duplication and familial Parkinson’s disease.Lancet. 2004; 364: 1169-1171Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar, Singleton et al., 2003Singleton A.B. Farrer M. Johnson J. Singleton A. Hague S. Kachergus J. Hulihan M. Peuralinna T. Dutra A. Nussbaum R. et al.alpha-Synuclein locus triplication causes Parkinson’s disease.Science. 2003; 302: 841Crossref PubMed Scopus (3469) Google Scholar). Along similar lines, it has been shown recently that leucine-rich repeat kinase 2 (LRRK2) mutations, the most common cause of inherited PD, increase overall protein synthesis in Drosophila, and that reduction in dLRRK levels is protective (Martin et al., 2014Martin I. Kim J.W. Lee B.D. Kang H.C. Xu J.C. Jia H. Stankowski J. Kim M.S. Zhong J. Kumar M. et al.Ribosomal protein s15 phosphorylation mediates LRRK2 neurodegeneration in Parkinson’s disease.Cell. 2014; 157: 472-485Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Spinocerebellar ataxia type 1 (SCA1) is paradigmatic of the subgroup of polyglutamine (polyQ) proteinopathies caused by expansion of an unstable CAG repeat in the coding region of the relevant disease gene, in this case ATAXIN1 (ATXN1) (Orr et al., 1993Orr H.T. Chung M.Y. Banfi S. Kwiatkowski Jr., T.J. Servadio A. Beaudet A.L. McCall A.E. Duvick L.A. Ranum L.P. Zoghbi H.Y. Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1.Nat. Genet. 1993; 4: 221-226Crossref PubMed Scopus (1477) Google Scholar). The onset of SCA1 is usually in mid-life, when motor coordination begins to deteriorate because of cerebellar degeneration; patients eventually die of bulbar dysfunction that renders them unable to clear their airway (Zoghbi and Orr, 2009Zoghbi H.Y. Orr H.T. Pathogenic mechanisms of a polyglutamine-mediated neurodegenerative disease, spinocerebellar ataxia type 1.J. Biol. Chem. 2009; 284: 7425-7429Crossref PubMed Scopus (169) Google Scholar). There is clear evidence that the expanded polyQ tract stabilizes ATXN1 and causes it to resist being cleared by the ubiquitin-proteasome pathway, in effect increasing its abundance in neurons (Cummings et al., 1999Cummings C.J. Reinstein E. Sun Y. Antalffy B. Jiang Y. Ciechanover A. Orr H.T. Beaudet A.L. Zoghbi H.Y. Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice.Neuron. 1999; 24: 879-892Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Notably, the severity of neurodegeneration in fly and mouse models of SCA1 correlates directly with levels of mutant ATXN1 protein (Burright et al., 1995Burright E.N. Clark H.B. Servadio A. Matilla T. Feddersen R.M. Yunis W.S. Duvick L.A. Zoghbi H.Y. Orr H.T. SCA1 transgenic mice: a model for neurodegeneration caused by an expanded CAG trinucleotide repeat.Cell. 1995; 82: 937-948Abstract Full Text PDF PubMed Scopus (522) Google Scholar, Fernandez-Funez et al., 2000Fernandez-Funez P. Nino-Rosales M.L. de Gouyon B. She W.C. Luchak J.M. Martinez P. Turiegano E. Benito J. Capovilla M. Skinner P.J. et al.Identification of genes that modify ataxin-1-induced neurodegeneration.Nature. 2000; 408: 101-106Crossref PubMed Scopus (547) Google Scholar), and massive overexpression of even WT ATXN1 under the Purkinje-cell-specific promoter can produce a mild SCA1-like phenotype in mice (Fernandez-Funez et al., 2000Fernandez-Funez P. Nino-Rosales M.L. de Gouyon B. She W.C. Luchak J.M. Martinez P. Turiegano E. Benito J. Capovilla M. Skinner P.J. et al.Identification of genes that modify ataxin-1-induced neurodegeneration.Nature. 2000; 408: 101-106Crossref PubMed Scopus (547) Google Scholar). Although the artificiality of transgenic models limits their relevance to the human disease, these results from SCA1 transgenic mice, along with the evidence from familial AD and PD patients, led us to ask whether there were post-transcriptional modifications that might increase the levels of WT ATXN1 in a more physiologically relevant way and shed further light on the role of protein levels in neurodegeneration. The extraordinarily long 3′ UTR, approximately 7 kb in ATXN1 mRNA, seemed to promise a rich source of key brain-enriched post-transcriptional regulatory elements. To our surprise, we found that ATXN1 is regulated directly by an RNA-binding protein (RBP), Pumilio1, and that a brain-wide increase in WT Atxn1 levels of only ∼50%, caused by Pum1 haploinsufficiency, is sufficient to cause marked neurodegeneration in mice. Two types of molecules are known to modulate protein levels by binding to the corresponding mRNA: RBPs and microRNAs (miRNAs). RBPs bind to specific sequence motifs or secondary structures in mRNAs and regulate multiple steps in RNA metabolism, such as splicing, nucleus-cytoplasm transport, and translation (Lukong et al., 2008Lukong K.E. Chang K.W. Khandjian E.W. Richard S. RNA-binding proteins in human genetic disease.Trends Genet. 2008; 24: 416-425Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). On the other hand, miRNAs are small non-coding RNAs that control various developmental and physiological processes by suppressing the expression of their target genes via binding of a short (6–8 nucleotide) complementary seed region in the 3′ UTRs of mRNAs (Bartel, 2009Bartel D.P. MicroRNAs: target recognition and regulatory functions.Cell. 2009; 136: 215-233Abstract Full Text Full Text PDF PubMed Scopus (15858) Google Scholar). We first scanned the ∼7 kb-long ATXN1 3′ UTR for potential binding sites for miRNAs by using the TargetScan (Friedman et al., 2009Friedman R.C. Farh K.K. Burge C.B. Bartel D.P. Most mammalian mRNAs are conserved targets of microRNAs.Genome Res. 2009; 19: 92-105Crossref PubMed Scopus (6362) Google Scholar), CoMeTa (Gennarino et al., 2012Gennarino V.A. D’Angelo G. Dharmalingam G. Fernandez S. Russolillo G. Sanges R. Mutarelli M. Belcastro V. Ballabio A. Verde P. et al.Identification of microRNA-regulated gene networks by expression analysis of target genes.Genome Res. 2012; 22: 1163-1172Crossref PubMed Scopus (143) Google Scholar), and HOCTARdb (Gennarino et al., 2011Gennarino V.A. Sardiello M. Mutarelli M. Dharmalingam G. Maselli V. Lago G. Banfi S. HOCTAR database: a unique resource for microRNA target prediction.Gene. 2011; 480: 51-58Crossref PubMed Scopus (43) Google Scholar) prediction tools. As expected, scanning identified dozens of potential miRNA-binding sites (data not shown). Because RNA folding mediates miRNA-RNA interactions by masking or exposing specific binding-site sequences, we analyzed the secondary structure of the ATXN1-3′ UTR (Wan et al., 2014Wan Y. Qu K. Zhang Q.C. Flynn R.A. Manor O. Ouyang Z. Zhang J. Spitale R.C. Snyder M.P. Segal E. Chang H.Y. Landscape and variation of RNA secondary structure across the human transcriptome.Nature. 2014; 505: 706-709Crossref PubMed Scopus (370) Google Scholar) to prioritize the best candidate ATXN1-modulating miRNAs. This revealed a complicated secondary structure that masks the binding sites for almost all of the putative miRNAs that might target the ATXN1-3′ UTR (Figure S1). For miRNAs to act on ATXN1 mRNA, they would likely require the help of RBPs to unfold such a structure. Scan analysis of the human ATXN1-3′ UTR revealed three putative Pumilio1 (PUM1) binding motifs (Wang et al., 2002Wang X. McLachlan J. Zamore P.D. Hall T.M. Modular recognition of RNA by a human pumilio-homology domain.Cell. 2002; 110: 501-512Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar) at positions 682, 2812, and 5275 from the beginning of the UTR (Figure 1A). The RBP PUM1 regulates its target genes by inducing a conformational switch in the 3′ UTR that unmasks specific miRNA-binding sites (Kedde et al., 2010Kedde M. van Kouwenhove M. Zwart W. Oude Vrielink J.A. Elkon R. Agami R. A Pumilio-induced RNA structure switch in p27-3′ UTR controls miR-221 and miR-222 accessibility.Nat. Cell Biol. 2010; 12: 1014-1020Crossref PubMed Scopus (313) Google Scholar, Miles et al., 2012Miles W.O. Tschöp K. Herr A. Ji J.Y. Dyson N.J. Pumilio facilitates miRNA regulation of the E2F3 oncogene.Genes Dev. 2012; 26: 356-368Crossref PubMed Scopus (116) Google Scholar). Interestingly, the motif in position 5275 (Figure 1A, red box) is highly conserved across several species and represents the canonical PUM1-binding motif (5′-UGUAXAUA-3′) (Galgano et al., 2008Galgano A. Forrer M. Jaskiewicz L. Kanitz A. Zavolan M. Gerber A.P. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system.PLoS ONE. 2008; 3: e3164Crossref PubMed Scopus (205) Google Scholar, Wang et al., 2002Wang X. McLachlan J. Zamore P.D. Hall T.M. Modular recognition of RNA by a human pumilio-homology domain.Cell. 2002; 110: 501-512Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Overexpressing PUM1 in HEK293T cells reduced ATXN1 mRNA levels, whereas decreasing PUM1 by two different RNAi increased ATXN1 mRNA levels (Figure 1B). In vitro overexpression of PUM1 consistently decreased the luciferase activity of a reporter construct expressing the full-length ATXN1-3′ UTR (Figure 1C). Mutation of each PUM1-binding motif within the ATXN1-3′ UTR revealed that only the most conserved site, containing the canonical motif, is functional; when mutated, it abolished the effect of PUM1 overexpression on luciferase activity (Figure 1D). To examine the endogenous expression pattern of Pum1 in mice, we performed in situ hybridization assays (ISH) and western blot on 3-week-old mouse brain sections. Pum1 was expressed in all major brain regions in WT mice, almost completely absent in the brain of null mice, and reduced in heterozygous (Pum1+/−) brains (Figure S2A). We also confirmed that Pum1 protein is widely expressed in the brain at 5 weeks of age (Figure S2B). To determine whether Pum1 binds Atxn1 mRNA in vivo, we performed an RNA cross-linking and immunoprecipitation assay (RNA-Clip) on cerebra and cerebella from 5-week-old WT animals, using Pum1 knockout mice (Pum1−/−) as negative controls (Figure S2C). We found that Pum1 physically interacts with the conserved binding site of the Atxn1-3′ UTR in WT mice (Figure 2A). Consistent with the finding that Pum1 negatively regulates Atxn1, Pum1 heterozygous (Pum1+/−) mice showed increased levels of both Atxn1 protein (Figure 2B) and mRNA (Figure 2C)—approximately 30% in the cerebrum and 50% in the cerebellum—and Pum1−/− mice showed even more pronounced increases (Figures 2B and 2C). These data demonstrate that Pum1 directly regulates Atxn1 levels in the mouse brain. Several mRNA subsets contain target sites for both RBPs and miRNAs, and cooperation between these two types of post-transcriptional regulators has been described (Bhattacharyya et al., 2006Bhattacharyya S.N. Habermacher R. Martine U. Closs E.I. Filipowicz W. Relief of microRNA-mediated translational repression in human cells subjected to stress.Cell. 2006; 125: 1111-1124Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, Fabian and Sonenberg, 2012Fabian M.R. Sonenberg N. The mechanics of miRNA-mediated gene silencing: a look under the hood of miRISC.Nat. Struct. Mol. Biol. 2012; 19: 586-593Crossref PubMed Scopus (686) Google Scholar, Glorian et al., 2011Glorian V. Maillot G. Polès S. Iacovoni J.S. Favre G. Vagner S. HuR-dependent loading of miRNA RISC to the mRNA encoding the Ras-related small GTPase RhoB controls its translation during UV-induced apoptosis.Cell Death Differ. 2011; 18: 1692-1701Crossref PubMed Scopus (71) Google Scholar, Kim et al., 2009Kim H.H. Kuwano Y. Srikantan S. Lee E.K. Martindale J.L. Gorospe M. HuR recruits let-7/RISC to repress c-Myc expression.Genes Dev. 2009; 23: 1743-1748Crossref PubMed Scopus (439) Google Scholar, Kundu et al., 2012Kundu P. Fabian M.R. Sonenberg N. Bhattacharyya S.N. Filipowicz W. HuR protein attenuates miRNA-mediated repression by promoting miRISC dissociation from the target RNA.Nucleic Acids Res. 2012; 40: 5088-5100Crossref PubMed Scopus (138) Google Scholar). This may be particularly relevant for PUM1, as studies have indicated extensive interaction between PUM1 and the miRNA regulatory system (Kedde et al., 2010Kedde M. van Kouwenhove M. Zwart W. Oude Vrielink J.A. Elkon R. Agami R. A Pumilio-induced RNA structure switch in p27-3′ UTR controls miR-221 and miR-222 accessibility.Nat. Cell Biol. 2010; 12: 1014-1020Crossref PubMed Scopus (313) Google Scholar, Galgano et al., 2008Galgano A. Forrer M. Jaskiewicz L. Kanitz A. Zavolan M. Gerber A.P. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system.PLoS ONE. 2008; 3: e3164Crossref PubMed Scopus (205) Google Scholar). To determine whether PUM1 regulates ATXN1 through miRNA by inducing a conformational switch in its 3′ UTR, we overexpressed PUM1 in HEK293T cells along with miR-101a, a miRNA known to modulate ATXN1 levels (Lee et al., 2008Lee Y. Samaco R.C. Gatchel J.R. Thaller C. Orr H.T. Zoghbi H.Y. miR-19, miR-101 and miR-130 co-regulate ATXN1 levels to potentially modulate SCA1 pathogenesis.Nat. Neurosci. 2008; 11: 1137-1139Crossref PubMed Scopus (183) Google Scholar). These conditions significantly reduced levels of ATXN1 protein (Figures 3A and S3A) and mRNA (Figure S3E), but no more than overexpressing miR-101a or PUM1 separately. In fact, overexpression of miR-101a along with PUM1 knockdown consistently decreased levels of ATXN1 protein (Figures 3B and S3B) and mRNA (Figure S3F) to a degree comparable to that of miR-101a overexpression alone. These results suggest that PUM1 regulates ATXN1 in a miR-101a-independent fashion but do not exclude the possibility that other miRNAs bind the ATXN1 3′ UTR. To obviate testing the effect of PUM1 on all possible miRNAs regulating ATXN1, we knocked down the catalytic engine of the RNA-induced silencing complex (RISC), Argonaute-2 (AGO2), to globally inhibit miRNA binding and retested PUM1’s ability to regulate ATXN1. We found that PUM1 overexpression in the context of AGO2 knockdown still reduced levels of both ATXN1 protein (Figures 3C and S3C) and mRNA (Figure S3G). Conversely, simultaneous RNAi of PUM1 and AGO2 increased levels of both ATXN1 protein (Figures 3D and S3D) and mRNA (Figure S3H), but no more than silencing PUM1 alone. These data establish that PUM1 modulates ATXN1 levels directly by binding its 3′ UTR, without the assistance of the miRNA machinery.Figure S3PUM1 Modulates the Stability of WT ATXN1 Levels in an miRNA-Independent Manner, Related to Figure 3Show full caption(A–D) Western blot quantification of protein lysate from HEK293T cells upon (A) overexpression of both PUM1 and miR-101a, (B) RNAi PUM1 (siPUM1) followed by overexpression of miR-101a, (C) overexpression of PUM1 followed by RNAi AGO2 (siAGO2), and (D) RNAi of both PUM1 and AGO2. The destination-cloning vector (control), RNAi scramble (siScramble) and cel-miR-67 were used as negative controls. All data were normalized to α-tubulin (TUBA).(E) PUM1 overexpression together with miR-101a decreases ATXN1 levels no more than overexpressing PUM1 alone, as shown by qRT-PCR quantification of ATXN1 levels in transfected HEK293 cells.(F) PUM1 knockdown (siPUM1) followed by miR-101a overexpression decreased ATXN1 levels as much as overexpression of miR-101a alone, whereas downregulation of PUM1 increased ATXN1 levels.(G) PUM1 overexpression together with AGO2 RNAi (siAGO2) did not decrease ATXN1 levels more than PUM1 overexpression alone, as shown by qRT-PCR quantification of ATXN1 levels in transfected HEK293 cells.(H) Silencing both PUM1 and AGO2 did not increase ATXN1 levels more than silencing PUM1 alone, as shown by qRT-PCR quantification of ATXN1 levels in transfected HEK293 cells. GAPDH was used as an internal control for all the qRT-PCR experiments.(I) Western blot quantification of PUM1 protein levels at different time points from zero (DRB treatment) to 8 hr in HEK293T cells after transfection of PUM1 ATXN1-3′ UTR WT and Mut binding sites. Data were normalized to GAPDH.(J and K) Knockdown efficiency of PUM1: mRNA (J) and proteins (K) at different time points in HEK293T cells from zero (DRB treatment) to 8 hr after transfection of RNAi of PUM1 (siPUM1) or scramble (siScramble). Data were normalized to GAPDH levels.All experiments were performed in triplicate (data represent mean ± SEM). p values were calculated by Student’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A–D) Western blot quantification of protein lysate from HEK293T cells upon (A) overexpression of both PUM1 and miR-101a, (B) RNAi PUM1 (siPUM1) followed by overexpression of miR-101a, (C) overexpression of PUM1 followed by RNAi AGO2 (siAGO2), and (D) RNAi of both PUM1 and AGO2. The destination-cloning vector (control), RNAi scramble (siScramble) and cel-miR-67 were used as negative controls. All data were normalized to α-tubulin (TUBA). (E) PUM1 overexpression together with miR-101a decreases ATXN1 levels no more than overexpressing PUM1 alone, as shown by qRT-PCR quantification of ATXN1 levels in transfected HEK293 cells. (F) PUM1 knockdown (siPUM1) followed by miR-101a overexpression decreased ATXN1 levels as much as overexpression of miR-101a alone, whereas downregulation of PUM1 increased ATXN1 levels. (G) PUM1 overexpression together with AGO2 RNAi (siAGO2) did not decrease ATXN1 levels more than PUM1 overexpression alone, as shown by qRT-PCR quantification of ATXN1 levels in transfected HEK293 cells. (H) Silencing both PUM1 and AGO2 did not increase ATXN1 levels more than silencing PUM1 alone, as shown by qRT-PCR quantification of ATXN1 levels in transfected HEK293 cells. GAPDH was used as an internal control for all the qRT-PCR experiments. (I) Western blot quantification of PUM1 protein levels at different time points from zero (DRB treatment) to 8 hr in HEK293T cells after transfection of PUM1 ATXN1-3′ UTR WT and Mut binding sites. Data were normalized to GAPDH. (J and K) Knockdown efficiency of PUM1: mRNA (J) and proteins (K) at different time points in HEK293T cells from zero (DRB treatment) to 8 hr after transfection of RNAi of PUM1 (siPUM1) or scramble (siScramble). Data were normalized to GAPDH levels. All experiments were performed in triplicate (data represent mean ± SEM). p values were calculated by Student’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001. To further explore the mechanism by which PUM1 regulates ATXN1 levels, we tested whether PUM1 influences the stability or the translation of ATXN1 mRNA. We transfected HEK293T with a luciferase reporter encoding an ATXN1-3′ UTR harboring either the conserved WT or mutated (Mut) PUM1-binding site. Later, we used treatment with DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole), a drug that inhibits RNA translation by blocking RNA polymerase II in the early elongation stage, to assess the levels of the reporter transcript. Upon the addition of DRB (time-point zero), the relative expression of reporter transcripts containing the ATXN1-3′ UTR Mut binding site is considerably higher than that of transcripts containing the ATXN1-3′ UTR WT binding site (Figure 3E, top panel). This difference remains stable over time until 8 hr after DRB addition. Remarkably, the ATXN1-3′ UTR with Mut binding site reached its half-life after 19 hr, whereas the ATXN1-3′ UTR with WT binding site decreased linearly over time, reaching its half-life at nearly 8 hr (Figure 3E, top panel). Given that the promoter sequences of the ATXN1-3′ UTR constructs carrying either WT or Mut binding sites are exactly the same and that transfection of neither construct affected PUM1 protein levels, we conclude that PUM1 promotes degradation of ATXN1 by binding its 3′ UTR (Figures 3E, bottom panel and S3I). To investigate physiological changes in ATXN1 mRNA, we decided to knock down PUM1 in HEK293T cells and measure the half-life of endogenous ATXN1 mRNA at different time points after DRB treatment. Knockdown of PUM1 (siPUM1) was associated with a significant increase of ATXN1 mRNA from time zero and remained upregulated up to 8 hr after translation inhibition (Figure 3F, top panel). Our calculation consistently showed that the half-life of ATXN1 mRNA was much longer (nearly 12 hr) after siPUM1 than after siScramble transfection (∼4 hr) (Figure 3F, top panel). We confirmed PUM1 downregulation by quantifying mRNA at time zero (Figure S3J) and protein levels at different time points (Figures 3F, bottom panel and S3K). PUM1 thus increases ATXN1 levels by directly regulating the stability of ATXN1 mRNA. Recent studies have shown that Pum1 is an essential regulator of spermatogenesis in mice and promotes differentiation of embryonic stem cells (Chen et al., 2012Chen D. Zheng W. Lin A. Uyhazi K. Zhao H. Lin H. Pumilio 1 suppresses multiple activators of p53 to safeguard spermatogenesis.Curr. Biol. 2012; 22: 420-425Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, Leeb et al., 2014Leeb M. Dietmann S. Paramor M. Niwa H. Smith A. Genetic exploration of the exit from self-renewal using haploid embryonic stem cells.Cell Stem Cell. 2014; 14: 385-393Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), but its role in the mammalian nervous system has not been investigated. We therefore characterized the brain structure and behavior of Pum1 knockout mice (Chen et al., 2012Chen D. Zheng W. Lin A. Uyhazi K. Zhao H. Lin H. Pumilio 1 suppresses multiple activators of p53 to safeguard spermatogenesis.Curr. Biol. 2012; 22: 420-425Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The Pum1 null allele tends to be transmitted with an altered Mendelian ratio (Figure S4A). Compared to WT and Pum1+/− littermates, Pum1−/− mice were significantly smaller in body length, weight, and brain weight and size (Figures 4A and S4B). Surprisingly, the loss of one copy of Pum1 was sufficient to cause impaired performance on the accelerating rotarod assay in 5-week-old mice (Figure 4B): the motor deficit had progressed in severity by 12 weeks (Figure S4C). This motor incoordination was even more dramatic in Pum1−/− age-matched mice (Figures 4B and S4C), which performed equally poorly in the dowel-walking test (Figures S4D and S4E)—as poorly, in fact, as SCA1 mice at this age in both assays (Watase et al., 2002Watase K. Weeber E.J. Xu B. Antalffy B. Yuva-Paylor L. Hashimoto K. Kano M. Atkinson R. Sun Y. Armstrong D.L. et al.A long CAG repeat in the mouse Sca1 locus replicates SCA1 features and reveals the impact of protein solubility on selective neurodegeneration.Neuron. 2002; 34: 905-919Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar).Figure 4Pum1 Mutant Mice Develop Progressive Motor Deficits and Cerebellar DegenerationShow full caption(A) Representative pictures of 3-week-old mice. Body size, brain weight, and brain size are reduced in Pum1−/− animals.(B) Accelerating rotarod analysis. Mice were trained over 4 days with four trials (t) per day. The null mice were significantly different from WT from day 1; by day 2, the difference between WT and both Pum1 null and heterozygotes was statistically significant, as was the difference between the two mutants.(C) Hind-paw clasping analysis in mice: a higher score indicates a more severe phenotype (see Figure S4F, bottom panel for scoring details). By 6 weeks of age, the null mice were statistically different from WT; by 8 weeks, both mutant lines were statistically significantly different from WT.(D) Open-field test measuring the total distance traveled of the Pum1 null mice relative to WT.(E) Representative images of immunofluorescence (IF) confocal microscopy in 3D depth-coding (see Experimental Procedures). Co-staining with α-IP3R1 and -calbindin antibodies was used to label Purkinje cells and to reveal their arborization.(F) Purkinje cell counts at 3, 4, and 10 weeks old for all examined genotypes.(G) IF for Calbindin and IP3R1 were quantified and averaged in selected rectangular cerebellar subsections.All experiments were performed in WT, Pum1+/−, and Pum1−/− mice. More than 12 mice per genotype were considered in (A)–(D) and 6 per genotype in (E)–(G). Data in (A), (D), and (F) represent mean ± SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001. See also Figure S4.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Representative pictures of 3-week-old mice. Body size, brain weight, and brain size are reduced in Pum1−/− animals. (B) Accelerating rotarod analysis. Mice were trained over 4 days with four trials (t) per day. The null mice were significantly different from WT from day 1; by day 2, the difference between WT and both Pum1 null and heterozygotes was statisti" @default.
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