Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients
2010; Springer Nature; Volume: 29; Issue: 7 Linguagem: Inglês
10.1038/emboj.2010.21
ISSN1460-2075
AutoresChantal Sellier, Fredérique Rau, Yilei Liu, Flora Tassone, Renate K. Hukema, Renata Gattoni, Anne Schneider, Stéphane Richard, Rob Willemsen, David J. Elliott, Paul J. Hagerman, Nicolas Charlet‐Berguerand,
Tópico(s)RNA Research and Splicing
ResumoArticle25 February 2010free access Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients Chantal Sellier Chantal Sellier Department of Neurobiology and Genetics, IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France Search for more papers by this author Frédérique Rau Frédérique Rau Department of Neurobiology and Genetics, IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France Search for more papers by this author Yilei Liu Yilei Liu Institute of Human Genetics, Newcastle University, Newcastle, UK Search for more papers by this author Flora Tassone Flora Tassone M.I.N.D. Institute, University of California, Davis, Health System, Sacramento, CA, USA Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, CA, USA Search for more papers by this author Renate K Hukema Renate K Hukema CBG-Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Renata Gattoni Renata Gattoni Department of Neurobiology and Genetics, IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France Search for more papers by this author Anne Schneider Anne Schneider Department of Neurobiology and Genetics, IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France Search for more papers by this author Stéphane Richard Stéphane Richard Lady Davis Institute for Medical Research, Departments of Medicine and Oncology, McGill University, Montreal, Quebec, Canada Search for more papers by this author Rob Willemsen Rob Willemsen CBG-Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author David J Elliott David J Elliott Institute of Human Genetics, Newcastle University, Newcastle, UK Search for more papers by this author Paul J Hagerman Paul J Hagerman M.I.N.D. Institute, University of California, Davis, Health System, Sacramento, CA, USA Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, CA, USA Search for more papers by this author Nicolas Charlet-Berguerand Corresponding Author Nicolas Charlet-Berguerand Department of Neurobiology and Genetics, IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France Search for more papers by this author Chantal Sellier Chantal Sellier Department of Neurobiology and Genetics, IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France Search for more papers by this author Frédérique Rau Frédérique Rau Department of Neurobiology and Genetics, IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France Search for more papers by this author Yilei Liu Yilei Liu Institute of Human Genetics, Newcastle University, Newcastle, UK Search for more papers by this author Flora Tassone Flora Tassone M.I.N.D. Institute, University of California, Davis, Health System, Sacramento, CA, USA Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, CA, USA Search for more papers by this author Renate K Hukema Renate K Hukema CBG-Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Renata Gattoni Renata Gattoni Department of Neurobiology and Genetics, IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France Search for more papers by this author Anne Schneider Anne Schneider Department of Neurobiology and Genetics, IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France Search for more papers by this author Stéphane Richard Stéphane Richard Lady Davis Institute for Medical Research, Departments of Medicine and Oncology, McGill University, Montreal, Quebec, Canada Search for more papers by this author Rob Willemsen Rob Willemsen CBG-Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author David J Elliott David J Elliott Institute of Human Genetics, Newcastle University, Newcastle, UK Search for more papers by this author Paul J Hagerman Paul J Hagerman M.I.N.D. Institute, University of California, Davis, Health System, Sacramento, CA, USA Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, CA, USA Search for more papers by this author Nicolas Charlet-Berguerand Corresponding Author Nicolas Charlet-Berguerand Department of Neurobiology and Genetics, IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France Search for more papers by this author Author Information Chantal Sellier1, Frédérique Rau1, Yilei Liu2, Flora Tassone3,6, Renate K Hukema4, Renata Gattoni1, Anne Schneider1, Stéphane Richard5, Rob Willemsen4, David J Elliott2, Paul J Hagerman3,6 and Nicolas Charlet-Berguerand 1 1Department of Neurobiology and Genetics, IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France 2Institute of Human Genetics, Newcastle University, Newcastle, UK 3M.I.N.D. Institute, University of California, Davis, Health System, Sacramento, CA, USA 4CBG-Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands 5Lady Davis Institute for Medical Research, Departments of Medicine and Oncology, McGill University, Montreal, Quebec, Canada 6Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, CA, USA *Corresponding author. Department of Neurobiology and Genetics, IGBMC, 1 rue Laurent Fries, Strasbourg, Illkirch 67400, France. Tel.: +33 388 653 309; Fax: +33 388 653 201; E-mail: [email protected] The EMBO Journal (2010)29:1248-1261https://doi.org/10.1038/emboj.2010.21 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by expansion of 55–200 CGG repeats in the 5′-UTR of the FMR1 gene. FXTAS is characterized by action tremor, gait ataxia and impaired executive cognitive functioning. It has been proposed that FXTAS is caused by titration of RNA-binding proteins by the expanded CGG repeats. Sam68 is an RNA-binding protein involved in alternative splicing regulation and its ablation in mouse leads to motor coordination defects. Here, we report that mRNAs containing expanded CGG repeats form large and dynamic intranuclear RNA aggregates that recruit several RNA-binding proteins sequentially, first Sam68, then hnRNP-G and MBNL1. Importantly, Sam68 is sequestered by expanded CGG repeats and thereby loses its splicing-regulatory function. Consequently, Sam68-responsive splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 tyrosine phosphorylation modulates its localization within CGG aggregates and that tautomycin prevents both Sam68 and CGG RNA aggregate formation. Overall, these data support an RNA gain-of-function mechanism for FXTAS neuropathology, and suggest possible target routes for treatment options. Introduction Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a recently identified neurodegenerative disorder that affects principally older adult males who are carriers of pre-mutation expansions (55–200 CGG repeats) in the 5′-untranslated region (UTR) of the Fragile X Mental Retardation-1 (FMR1) gene (Hagerman et al, 2001; Hagerman and Hagerman, 2004; Ostra and Willemesen, 2009). The major clinical features of FXTAS are progressive intention tremor and gait ataxia, with more variable associated features, including parkinsonism, dysautonomia, anxiety, peripheral neuropathy and cognitive decline (Jacquemont et al, 2003). The neuropathological hallmark of FXTAS is the presence of ubiquitin-positive intranuclear inclusions in both astrocytes and neurons throughout the brain (Greco et al, 2002). It is estimated that nearly 1 in 3000 males has a lifetime risk of developing FXTAS, which would make FXTAS one of the most common single gene causes of tremor, ataxia and cognitive decline among older adults (Jacquemont et al, 2004). In Fragile X syndrome full mutations (>200 CGG repeats) result in hypermethylation and silencing of the FMR1 gene. In contrast, carriers of the pre-mutation alleles (55–200 CGG repeats) have increased FMR1 mRNA levels but normal, or moderately low, FMR1 protein expression, especially in the upper pre-mutation range (Tassone et al, 2000a, 2000b; Kenneson et al, 2001; Primerano et al, 2002). These observations, coupled with the fact that mRNAs containing expanded CGG repeats accumulate in intranuclear aggregates, suggest a toxic RNA gain-of-function model for FXTAS (Tassone et al, 2004). In support of this hypothesis, a knock-in (KI) mouse model, in which the endogenous eight CGG repeats in the Fmr1 gene has been replaced with an expansion containing ∼100 CGG repeats of human origin, shows ubiquitin-positive intranuclear inclusions and mild neuromotor and behavioural disturbance (Willemsen et al, 2003; Van Dam et al, 2005; Brouwer et al, 2008). Furthermore, sole expression of mRNAs containing 90 CGG repeats outside the context of Fmr1 in a transgenic mouse model is sufficient to recapitulate the neuropathological and molecular features of FXTAS (Hashem et al, 2009). Similarly, heterologous expression of 90 CGG repeats in Drosophila is sufficient to cause neurodegeneration along with formation of neuronal inclusions (Jin et al, 2003). These models show that sole expression of expanded CGG repeats is necessary and sufficient to cause a pathology similar to human FXTAS, and thus indicate that the expanded CGG repeats in RNA are the likely cause of the neurodegeneration in FXTAS. The FXTAS toxic RNA gain-of-function model show similarities with Myotonic Dystrophies (DM), where expanded CUG or CCUG repeats accumulate in nuclear RNA aggregates that sequester the Muscleblind-like (MBNL1) splicing factor. In DM, partial depletion of the free pool of MBNL1 leads to specific alternative splicing changes, which ultimately result in the symptoms of DM (Ranum and Cooper, 2006; Wheeler and Thornton, 2007). Extending this model to FXTAS, expanded CGG repeats are predicted to sequester specific proteins resulting in loss of their normal molecular functions. Several proteins, including a number of heat-shock proteins, Purα, hnRNP-A2/B1, CUGBP1, MBNL1, lamin-A/C and MBP were found to localize with ubiquitin-positive inclusions in CGG-expressing Drosophila, KI mouse model and FXTAS patients (Iwahashi et al, 2006; Jin et al, 2007; Sofola et al, 2007). However, these proteins were not found to be sequestered by expanded CGG repeats and consequently they are not expected to lose their functions in FXTAS patients. In this study, we found that in contrast to CUG repeats, expanded CGG repeats accumulate in dynamic intranuclear RNA structures that expand over time. Formation of giant CGG RNA aggregates ultimately results in disorganization of the nuclear lamin structure and cell death. MBNL1 and hnRNP-G proteins were found localized within CGG aggregates but only in the larger inclusions and at late time points after transfection, suggesting these are not the primary defects. In contrast, we identified the Src-Associated substrate during mitosis of 68-kDa (Sam68) protein as the only protein that colocalizes with CGG RNA aggregates at each time point. Sam68 is a nuclear RNA-binding protein involved in alternative splicing regulation (Stoss et al, 2001; Paronetto et al, 2007; Chawla et al, 2009), and its ablation in a mouse knock-out model leads to motor coordination defects (Lukong and Richard, 2008). Sam68 splicing activity, RNA-binding ability and localization are regulated by phosphorylation (Haegebarth et al, 2004; Lukong et al, 2005), and Sam68 interacts with various RNA-binding proteins through several protein–protein interaction domains (Lukong and Richard, 2003). We found that Sam68 is required for subsequent aggregation of MBNL1 and hnRNP G proteins within CGG aggregates. Most importantly, Sam68 is sequestered by expanded CGG-repeat aggregates and thereby loses its splicing-regulatory function. As a consequence, Sam68-regulated splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 phosphorylation modulates its localization within CGG aggregates. Strikingly, among the various kinase and phosphatase inhibitors tested, we found one, tautomycin, which not only prevents Sam68 colocalization within CGG aggregates, but also abolishes CGG RNA aggregate formation. Results Expanded CGG repeats form dynamic nuclear RNA aggregates that expand over time We first questioned whether pre-mutation-length CGG repeats can form nuclear RNA aggregates in cultured cells. We transfected plasmids expressing either 20, 40, 60 or 100 CGG repeats under the control of a cytomegalovirus (CMV) promoter in various cell lines, and tested the formation of CGG aggregates by RNA fluorescence in situ hybridation (FISH) analysis. We confirmed the specificity of the FISH conditions, and the RNA composition of CGG aggregates, as they were sensitive to RNAse treatment (Supplementary Figure S1). Consistent with an RNA gain-of-function model, expression of 60 or 100 CGG repeats within COS7 cells generated numerous intranuclear CGG aggregates, whereas expression of 20 CGG repeats did not (Figure 1A). Expression of 40 CGG repeats resulted in an intermediate condition with formation of rare small intranuclear aggregates. This is consistent with observations in FXTAS patients in whom it is estimated that 'normal' CGG polymorphic repeat lengths are 5–45 repeats long, 'gray zone' alleles contain 45 to 55 repeats and FXTAS patients are defined by pre-mutation allele containing 55–200 CGG repeats (Tassone et al, 2007; Leehey et al, 2008). Figure 1.Expanded CGG repeats form intranuclear RNA aggregates. (A) COS7 cells were transfected with a plasmid expressing either no (a), 20 (b), 40 (c), 60 (d) or 100 (e) CGG repeats, transferred to 0.1% serum to block cell divisions and analysed 24 h after transfection by RNA FISH using a (CCG)8x-Cy3 DNA probe. (B) Primary cultures of hippocampal embryonic mouse neurons (a), differentiated PC12 (b), SKOV3 (c) and COS7 (d) cells were transfected with a plasmid expressing 60 CGG repeats and analysed 24 h after transfection by FISH. (C) COS7 cells were co-transfected with a plasmid expressing 60 CGG repeats and a plasmid expressing GFP-tagged lamin-A, and analysed by RNA FISH either 24 (a), 48 (b) or 72 (c) hours after transfection. In all the figures, the magnification is × 630. The scale bars represent 10 μm; nuclei were counterstained with DAPI and one representative experiment from at least three separate experiments is shown. Download figure Download PowerPoint Next, expression of CGG aggregates was investigated in various cell types. Transfection of constructs containing 60 CGG repeats led to intranuclear CGG RNA aggregate formation in primary cultures of hippocampal embryonic mouse neurons, as well as in various immortalized cell lines such as neuronal (differentiated PC12), ovarian (SKOV3 and SW626) and kidney (COS7)-derived cell lines (Figure 1B). However, no aggregates were observed in A172, U-937, THP1, HeLa, HEK293, NG108-15, IMR-32, Neuro-2a, SH-SY5Y, SK-N-MC or SK-N-SH cell lines (data not shown), confirming a previous report that not all cell lines can support CGG-repeat aggregate formation (Arocena et al, 2005). Tests of colocalization with various nuclear structures indicated that in transfected cells most of the CGG aggregates are associated with nuclear speckles, but not with other structures such as lamin, nucleoli, PML, Gems or Cajal bodies (Supplementary Figure S2A). Finally, kinetic formation of CGG aggregates was investigated. COS7 cells were transfected with a plasmid expressing 60 CGG repeats and analysed by RNA FISH either 24, 48 or 72 h after transfection. Surprisingly, expanded CGG repeats formed dynamic nuclear structures that expanded over time, resulting in giant inclusions, nuclear lamin architecture disruption and cell death 72–96 h after transfection (Figure 1C). In contrast, expanded CUG (DM1 mutation) or AUUCU (SCA10 mutation) repeats were less toxic and formed stable nuclear aggregates, whose size did not evolve over time (Supplementary Figure S2B). Annexin-V–PE apoptosis tests were negative indicating that the cytotoxicity of CGG repeats is not linked to apoptotic cell death (data not shown), and in agreement with a previous report (Arocena et al, 2005) no ubiquitin-positive aggregates were observed in transfected cells. CGG aggregates recruit Sam68, then MBNL1 and hnRNP G To identify which proteins are associated with expanded CGG repeats, we first adopted an in vitro approach. Proteins extracted from mouse brain or COS7-cell nuclei were captured on streptavidin resin coupled to biotinylated in vitro-transcribed RNA composed of 60 CGG repeats, eluted, separated on SDS–PAGE gels and identified by MALDI-TOF analysis. More than 20 proteins were identified (Supplementary Table 1), including a heat-shock protein and several RNA-binding proteins, including MBNL1 and hnRNP-G. To discard non-specific binding proteins, we tested for colocalization of these candidates with RNA aggregates in COS7 cells transfected with 60 CGG repeats. CGG aggregates expand over time, suggesting that these repeats may recruit different proteins at different time points. Thus, we tested our candidates at 24 and 72 h after transfection (Figure 2A and B). Colocalization of MBNL1 within CGG aggregates increased from 14% at 24 h to 41% at 72 h after transfection. Similarly, colocalization of hnRNP-G increased from 26 to 73% (Figure 2D), suggesting that CGG aggregates form dynamic structures, which constantly recruit proteins. Figure 2.Sam68 colocalizes with CGG RNA aggregates. (A) COS7 cells were transfected with a plasmid expressing 60 CGG repeats and analysed by FISH/IF using an antibody against MBNL1 (a) 24 h after transfection. None of the antibodies against hnRNP-G that we tested supported FISH conditions. Consequently, endogenous hnRNP-G (b) was analysed by co-transfection of COS7 cells with a plasmid expressing 60 CGG repeats fused to three MS2 tags and a plasmid expressing the GFP-MS2 Coat Protein. The MS2 Coat Protein (MS2CP) possesses a very high and specific affinity for MS2 RNA tags. Endogenous hnRNP-G (b) was detected by IF and the CGG aggregates by localization of the GFP-MS2CP protein, which is bound to the MS2-(CGG)60x RNA. In the absence of MS2-(CGG)60x, GFP-MS2CP was diffuse in the nucleoplasm (data not shown). (B) Similar to panel A but analysed 72 h after transfection. (C) COS7 cells (a, b) or differentiated PC12 neuronal cells (c) were transfected with a plasmid expressing 60 CGG repeats and analysed by FISH/IF using an antibody against Sam68, 24 h (a and c) or 72 h after transfection (b). (D) The percentage of endogenous Sam68, MBNL1 and hnRNP-G colocalized within CGG RNA aggregates in transfected COS7 cells 24 or 72 h after transfection. In all the experiments, three independent transfections totalling a hundred cells were counted, and results are presented as mean±s.d. Download figure Download PowerPoint By contrast, other in vitro-identified candidates such as SPNR, hnRNP-A1, hnRNP-A2/B, hnRNP-C, hnRNP-D, hnRNP-E and hnRNP-H showed no or very little colocalization with RNA aggregates, and any colocalization observed was only within the giant CGG aggregates that form just before cell death (data not shown). We also observed that neither CUGBP1, nor Purα, colocalized with CGG aggregates in COS7-transfected cells. These results suggest that MBNL1 and hnRNP-G are not initially recruited by CGG repeats, but join the larger aggregates later on, probably through protein–protein interactions. On the basis of that hypothesis, we searched for proteins that would colocalize with expanded CGG repeats early after transfection, and may capture other RNA-binding proteins through RNA or protein interactions. We screened ∼50 candidates known to bind to RNA or RNA-binding proteins (Supplementary Table 2 and data not shown) and found one, Sam68, that consistently co-localized with CGG RNA aggregates at each time point, including the earliest (Figure 2C and D). Next, we confirmed by FISH/IF that endogenous Sam68 colocalized with CGG aggregates in neuronal-differentiated PC12 cells (Figure 2C). Similar to COS7 cells, endogenous MBNL1 and hnRNP-G were not recruited within CGG aggregates in PC12 cells 24 h after transfection. We were not able to test Sam68, MBNL1 or hnRNP-G colocalization within CGG aggregates at later transfection time points as PC12 cells are very sensitive to CGG toxicity and die after less than 48 h of CGG expression. Sam68 is a nuclear protein involved in various aspects of mRNA metabolism and interacts with several RNA-binding proteins, raising question its specificity towards CGG aggregates versus other expanded RNA repeats. COS7 cells were transfected by plasmids expressing either expanded CGG, CUG, CCUG or AUUCU repeats, which are the cause of FXTAS, DM1, DM2 and SCA10 diseases, respectively. We found that 24 or 72 h after transfection, endogenous Sam68 colocalized only with CGG RNA aggregates, but not with other expanded RNA repeats (Figure 3). These results suggest that Sam68 is a specific and early component of CGG aggregates in FXTAS. Figure 3.Sam68 colocalization within nuclear RNA aggregates is specific to expanded CGG repeats. COS7 cells were transfected with a plasmid expressing either 60 CGG repeats (A), 160 AUUCU repeats (B), 960 CUG repeats (C) or 300 CCUG repeats (D). Endogenous Sam68 and CGG repeats were analysed 24 h after transfection by FISH/IF. Download figure Download PowerPoint Sam68 is essential for recruitment of MBNL1 and hnRNP-G within CGG aggregates Sam68 is recruited earlier than MBNL1 and hnRNP-G, suggesting sequential recruitment of proteins within the CGG aggregates. Furthermore, Sam68 protein contains several protein–protein interaction domains, raising the question whether Sam68 directly recruits hnRNP-G and MBNL1. In agreement with a previous report (Venables et al, 1999), we found by co-immunoprecipitation that Sam68 interacts with hnRNP-G. However, we found no robust interactions between MBNL1 and Sam68 or between MBNL1 and hnRNP-G (Supplementary Figure S3A). Next, we mapped the domain required for Sam68 colocalization with CGG aggregates. Sam68 protein contains a central KH RNA-binding domain, and N- and C-terminal protein–protein interaction domains. Deletion of the Sam68 N-terminal domain abolished colocalization with CGG aggregates, whereas deletion of the Sam68 RNA-binding domain did not (Supplementary Figure S3B). Similarly, Sam68 paralog proteins, Slm1 and Slm2 (T-Star), which are devoid of the N-terminal extended region of the Sam68 protein, did not colocalize with expanded CGG aggregates 24 h after transfection. The N-terminal part of the Sam68 protein consists of a number of potential protein–protein interaction domains, suggesting that association of Sam68 with CGG repeats might not be direct but mediated through protein–protein interactions. Finally, we tested whether Sam68 is required for colocalization of MBNL1 and hnRNP-G within CGG aggregates. Transfection of COS7 cells with an shRNA directed against Sam68 greatly reduced (>80%) the expression of endogenous Sam68 (Figure 4C). Importantly, depletion of endogenous Sam68 abolished the colocalization of MBNL1 and hnRNP-G within CGG aggregates (Figure 4 and Supplementary Table 3). These data show that Sam68 protein is required for subsequent recruitment of MBNL1 and hnRNP-G proteins within CGG aggregates. Figure 4.Sam68 is essential for recruitment of MBNL1 and hnRNP-G within CGG aggregates. (A) COS7 cells were co-transfected with a plasmid expressing 60 CGG repeats and a plasmid expressing either a control LacZ shRNA (a), or a Sam68 shRNA (b), and analysed 72 h after transfection by FISH/IF. Endogenous MBNL1 was detected by IF using an Alexa-488-labelled secondary antibody. Simultaneous detection of Sam68 by IF using a Cy5-labelled secondary antibody confirmed shRNA-mediated depletion of endogenous Sam68. (B) Endogenous hnRNP-G cannot be detected by FISH/IF. Thus, COS7 cells were co-transfected with a plasmid expressing 60 CGG repeats, a plasmid expressing GFP-hnRNP-G and either control (a) or Sam68 (b) shRNA and analysed 72 h after transfection by FISH. (C) shRNA-mediated depletion of endogenous Sam68 was confirmed by western blotting against Sam68. (D) The percentage of endogenous Sam68, MBNL1 and GFP-hnRNP-G colocalized within CGG RNA aggregates 72 h after transfection in COS7 cells transfected with a plasmid expressing either a control or a Sam68 shRNA. Download figure Download PowerPoint Sam68 colocalizes with CGG aggregates in FXTAS patients Next, we tested whether Sam68 protein colocalizes with endogenous CGG aggregates. We first analysed the localization of endogenous Sam68 and CGG repeats in the brain sections of a KI mouse model, in which endogenous CGG repeats had been replaced with an expansion of 98 CGG repeats (Willemsen et al, 2003). FISH/IF experiments showed the presence of intranuclear CGG aggregates that colocalized with Sam68 in mice expressing 98 CGG repeats (Figure 5A). By contrast, no RNA aggregates were detected and Sam68 was diffuse throughout the nucleoplasm in control mice (Figure 5A). Figure 5.Sam68 colocalizes with endogenous CGG aggregates. (A) Brain sections of mouse expressing either control eight CGG repeats (a) or expanded 98 CGG repeats (b) were analysed by FISH/IF. (B) Similar to panel A. Brain sections (hippocampal area) of age-matched control (a) or FXTAS (b) patients were analysed by FISH/IF. Magnification: × 630. Scale bar, 10 μm. (C) Brain (hippocampal area) sections of age-matched control (a) or FXTAS (b) patients were analysed by immunohistochemistry directed against Sam68. Magnification: × 350. Download figure Download PowerPoint Then, we investigated the presence of Sam68 and CGG aggregates in FXTAS patients. FISH/IF experiments show that Sam68 consistently colocalized with CGG intranuclear RNA aggregates in the brain sections of FXTAS patients (Figure 5B), but neither Sam68 nor CGG aggregates were found in control brain tissue (Figure 5B). We noted that in FXTAS patients, Sam68 and CGG aggregates were larger and more frequent than in KI mice. This is consistent with the milder neuromotor and behavioural disturbances observed in KI mice as compared with that in FXTAS patients (Willemsen et al, 2003; Van Dam et al, 2005). Finally, presence of Sam68 aggregates in brain sections of FXTAS patients was confirmed by immunohistochemistry. In brain sections of control patients, Sam68 was diffuse within the nucleoplasm and no detectable Sam68 inclusions were found. In contrast, large intranuclear aggregates of Sam68 were observed in FXTAS patients (Figure 5C), thus confirming that CGG expanded repeats alter Sam68 localization. Sam68 protein is partially immobilized within CGG expanded repeats An RNA gain-of-function model for FXTAS predicts that CGG expanded repeats should immobilize Sam68 protein and deplete its molecular activity. To test Sam68 sequestration by CGG repeats, we first analysed its mobility by FRAP (Fluorescence Recovery After Photobleaching) experiments. FRAP of transfected GFP-Sam68 was measured in nuclear regions containing Sam68 aggregates and compared to nuclear regions containing diffuse Sam68 either located within the same nucleus or located in the nuclei of cells not transfected with CGG repeats (Figure 6A). In both cases, nucleoplasmic areas without Sam68 aggregates recovered ∼95% of their initial fluorescence after photobleaching, whereas areas containing Sam68 aggregates recovered only ∼60% of their initial fluorescence (Figure 6B). This shows that a fraction of Sam68 is less mobile in CGG-transfected cells. Figure 6.FRAP analysis uncovers an immobile fraction of GFP-Sam68 within CGG aggregates. (A) Photobleaching was performed 24 h after transfection of COS7 cells with GFP-Sam68 and a plasmid expressing either no CGG repeats (a) or 60 CGG repeats (b). The white circles denote the photobleached regions in the aggregates and the nucleoplasm. Representative images show a single z-section obtained before photobleaching (pre-bleach) and at the indicated time points after photobleaching. (B) Recovery curves of photobleached aggregates and nucleoplasm in cells expressing 0 or 60 CGG repeats are shown as relative fluorescence intensity versus time. In CGG-expressing cells, recovery reached a plateau at ∼60% around 300 s. Each data point is the average of 10 nuclei. The error bars indicate the s.e.m.'s. Download figure Download PowerPoint Sam68 depletion by CGG repeats affects alternative splicing Sam68 is a nuclear RNA-binding protein with roles in alternative splicing regulation (Stoss et al, 2001; Paronetto et al, 2007; Chawla et al, 2009). According to the RNA gain-of-function model, titration of free nuclear Sam68 into CGG nuclear aggregates should deplete its functional activity and result in detectable pre-mRNA splicing alterations. To test this hypothesis, we co-transfected constructs encoding expanded CGG repeats with minigenes that recapitulate splicing events directly regulated by Sam68. In agreement with a previous report (Paronetto et al, 2007), o
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