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A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function.

Okray Z, de Esch CE, Van Esch H, Devriendt K, Claeys A, Yan J, Verbeeck J, Froyen G, Willemsen R, de Vrij FM, Hassan BA - EMBO Mol Med (2015)

Bottom Line: We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells.We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP.In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes.

View Article: PubMed Central - PubMed

Affiliation: VIB Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, University of Leuven School of Medicine and University Hospitals Leuven, Leuven, Belgium Program in Molecular and Developmental Genetics, Doctoral School of Biomedical Sciences, University of Leuven, Leuven, Belgium.

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Novel G-insertion mutation identified in male with FXS symptomsPredicted effects of the G-insertion mutation on FMRP are schematized. The insertion site corresponds to the beginning of the RNA-binding RGG Box, where it alters the open reading frame and thus disrupts the domain entirely. The frameshift creates a novel peptide sequence [RKRTRRKRTWRRLQRKRRSLPNR] followed by a premature stop codon, causing the truncation of the FMRP C-terminus.Photograph of male patient with FMR1G-ins. allele. Typical physical and behavioral features of FXS were noted in the patient, who shows moderate to severe intellectual disability.FMR1 mRNA levels were analyzed via RT–qPCR in EBV-transformed lymphocyte cells derived from the patient's blood samples. Patient cells showed a ˜60% decrease in FMR1 mRNA compared to control cells (*P = 0.010, 0.383 ± 0.110 SD, n = 3). Treatment with translational blocker puromycin restored FMR1 mRNA levels in patient cells (*P = 0.048, 0.383 ± 0.110 SD versus 1.25 ± 0.166 SD, n = 3), suggesting that the reduction of FMR1 mRNA in these cells is primarily due to nonsense-mediated decay. RT-qPCR reactions were run in triplicate in three independent experiments. Fold changes in FMR1 expression, normalized to HRPT expression, were calculated using the ΔΔCT method, and analyzed statistically with a two-tailed t-test (GraphPad). Error bars represent mean values with SD.Western blot analysis shows a significant decrease (> 90%) in FMRP protein levels in patient-derived cells (***P = 0.008, 0.13 ± 0.031 SD, n = 2), and the patient FMRP migrates slightly lower than the wild-type protein. Band intensities for the different cell lines were quantified, normalized for actin and analyzed statistically with a twot-tailed t-test (GraphPad). Error bars represent mean values with SD.
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fig01: Novel G-insertion mutation identified in male with FXS symptomsPredicted effects of the G-insertion mutation on FMRP are schematized. The insertion site corresponds to the beginning of the RNA-binding RGG Box, where it alters the open reading frame and thus disrupts the domain entirely. The frameshift creates a novel peptide sequence [RKRTRRKRTWRRLQRKRRSLPNR] followed by a premature stop codon, causing the truncation of the FMRP C-terminus.Photograph of male patient with FMR1G-ins. allele. Typical physical and behavioral features of FXS were noted in the patient, who shows moderate to severe intellectual disability.FMR1 mRNA levels were analyzed via RT–qPCR in EBV-transformed lymphocyte cells derived from the patient's blood samples. Patient cells showed a ˜60% decrease in FMR1 mRNA compared to control cells (*P = 0.010, 0.383 ± 0.110 SD, n = 3). Treatment with translational blocker puromycin restored FMR1 mRNA levels in patient cells (*P = 0.048, 0.383 ± 0.110 SD versus 1.25 ± 0.166 SD, n = 3), suggesting that the reduction of FMR1 mRNA in these cells is primarily due to nonsense-mediated decay. RT-qPCR reactions were run in triplicate in three independent experiments. Fold changes in FMR1 expression, normalized to HRPT expression, were calculated using the ΔΔCT method, and analyzed statistically with a two-tailed t-test (GraphPad). Error bars represent mean values with SD.Western blot analysis shows a significant decrease (> 90%) in FMRP protein levels in patient-derived cells (***P = 0.008, 0.13 ± 0.031 SD, n = 2), and the patient FMRP migrates slightly lower than the wild-type protein. Band intensities for the different cell lines were quantified, normalized for actin and analyzed statistically with a twot-tailed t-test (GraphPad). Error bars represent mean values with SD.

Mentions: Among screened individuals, we found seven with previously unreported variations in the FMR1 (Supplementary Fig S1A). In 5 of these individuals, single base polymorphisms were identified in intronic regions. In the other 2, single mutations were present in coding exons. In one of these individuals, we recovered a silent base substitution in exon 15. In the other, we identified a guanine insertion in exon 15 [1457insG] (Supplementary Fig S1A and B). This G-insertion mutation alters the open reading frame to one that is not used by any of the alternative FMR1 isoforms (Ensembl Genome Browser), and is predicted to create a novel peptide sequence followed by a premature stop codon, which results in the truncation of the C-terminus of FMRP (Fig1A). This modification also leads to the disruption of the RGG box, one of the three RNA-binding domains characterized in FMRP (Darnell et al, 2001; Ramos et al, 2003; Bagni & Greenough, 2005; Blackwell et al, 2010).


A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function.

Okray Z, de Esch CE, Van Esch H, Devriendt K, Claeys A, Yan J, Verbeeck J, Froyen G, Willemsen R, de Vrij FM, Hassan BA - EMBO Mol Med (2015)

Novel G-insertion mutation identified in male with FXS symptomsPredicted effects of the G-insertion mutation on FMRP are schematized. The insertion site corresponds to the beginning of the RNA-binding RGG Box, where it alters the open reading frame and thus disrupts the domain entirely. The frameshift creates a novel peptide sequence [RKRTRRKRTWRRLQRKRRSLPNR] followed by a premature stop codon, causing the truncation of the FMRP C-terminus.Photograph of male patient with FMR1G-ins. allele. Typical physical and behavioral features of FXS were noted in the patient, who shows moderate to severe intellectual disability.FMR1 mRNA levels were analyzed via RT–qPCR in EBV-transformed lymphocyte cells derived from the patient's blood samples. Patient cells showed a ˜60% decrease in FMR1 mRNA compared to control cells (*P = 0.010, 0.383 ± 0.110 SD, n = 3). Treatment with translational blocker puromycin restored FMR1 mRNA levels in patient cells (*P = 0.048, 0.383 ± 0.110 SD versus 1.25 ± 0.166 SD, n = 3), suggesting that the reduction of FMR1 mRNA in these cells is primarily due to nonsense-mediated decay. RT-qPCR reactions were run in triplicate in three independent experiments. Fold changes in FMR1 expression, normalized to HRPT expression, were calculated using the ΔΔCT method, and analyzed statistically with a two-tailed t-test (GraphPad). Error bars represent mean values with SD.Western blot analysis shows a significant decrease (> 90%) in FMRP protein levels in patient-derived cells (***P = 0.008, 0.13 ± 0.031 SD, n = 2), and the patient FMRP migrates slightly lower than the wild-type protein. Band intensities for the different cell lines were quantified, normalized for actin and analyzed statistically with a twot-tailed t-test (GraphPad). Error bars represent mean values with SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4403044&req=5

fig01: Novel G-insertion mutation identified in male with FXS symptomsPredicted effects of the G-insertion mutation on FMRP are schematized. The insertion site corresponds to the beginning of the RNA-binding RGG Box, where it alters the open reading frame and thus disrupts the domain entirely. The frameshift creates a novel peptide sequence [RKRTRRKRTWRRLQRKRRSLPNR] followed by a premature stop codon, causing the truncation of the FMRP C-terminus.Photograph of male patient with FMR1G-ins. allele. Typical physical and behavioral features of FXS were noted in the patient, who shows moderate to severe intellectual disability.FMR1 mRNA levels were analyzed via RT–qPCR in EBV-transformed lymphocyte cells derived from the patient's blood samples. Patient cells showed a ˜60% decrease in FMR1 mRNA compared to control cells (*P = 0.010, 0.383 ± 0.110 SD, n = 3). Treatment with translational blocker puromycin restored FMR1 mRNA levels in patient cells (*P = 0.048, 0.383 ± 0.110 SD versus 1.25 ± 0.166 SD, n = 3), suggesting that the reduction of FMR1 mRNA in these cells is primarily due to nonsense-mediated decay. RT-qPCR reactions were run in triplicate in three independent experiments. Fold changes in FMR1 expression, normalized to HRPT expression, were calculated using the ΔΔCT method, and analyzed statistically with a two-tailed t-test (GraphPad). Error bars represent mean values with SD.Western blot analysis shows a significant decrease (> 90%) in FMRP protein levels in patient-derived cells (***P = 0.008, 0.13 ± 0.031 SD, n = 2), and the patient FMRP migrates slightly lower than the wild-type protein. Band intensities for the different cell lines were quantified, normalized for actin and analyzed statistically with a twot-tailed t-test (GraphPad). Error bars represent mean values with SD.
Mentions: Among screened individuals, we found seven with previously unreported variations in the FMR1 (Supplementary Fig S1A). In 5 of these individuals, single base polymorphisms were identified in intronic regions. In the other 2, single mutations were present in coding exons. In one of these individuals, we recovered a silent base substitution in exon 15. In the other, we identified a guanine insertion in exon 15 [1457insG] (Supplementary Fig S1A and B). This G-insertion mutation alters the open reading frame to one that is not used by any of the alternative FMR1 isoforms (Ensembl Genome Browser), and is predicted to create a novel peptide sequence followed by a premature stop codon, which results in the truncation of the C-terminus of FMRP (Fig1A). This modification also leads to the disruption of the RGG box, one of the three RNA-binding domains characterized in FMRP (Darnell et al, 2001; Ramos et al, 2003; Bagni & Greenough, 2005; Blackwell et al, 2010).

Bottom Line: We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells.We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP.In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes.

View Article: PubMed Central - PubMed

Affiliation: VIB Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, University of Leuven School of Medicine and University Hospitals Leuven, Leuven, Belgium Program in Molecular and Developmental Genetics, Doctoral School of Biomedical Sciences, University of Leuven, Leuven, Belgium.

Show MeSH
Related in: MedlinePlus