Journal: Cell

Article Title: Pumilio1 Haploinsufficiency Leads to SCA1-like Neurodegeneration by Increasing Wild-Type Ataxin1 Levels

doi: 10.1016/j.cell.2015.02.012

Figure Lengend Snippet: (A) Schematic representation of Human ATXN1-3′UTR showing three putative PUM1 binding motifs (gray and red boxes) and their conservation in different species. The numbers indicate positions of PUM1 motifs in the human ATXN1-3′UTR. (B) ATXN1 mRNA quantification by qRT-PCR in HEK293T cells upon overexpression (left panel) or knockdown (siPUM1-81 and -82) (right panel) of PUM1. The destination-cloning vector (Control), scrambled siRNAs (siScr.) and cel-miR-67 were used as negative controls. The housekeeping gene GAPDH was used to normalize the expression of genes in all the qRT-PCR experiments. (C) Luciferase assay in HEK293 cells overexpressing the reporter construct harboring the full length ATXN1-3′UTR. In these conditions, we overexpressed (left panel) or decreased expression (right panel) of PUM1. (D) Luciferase assay in HEK293T cells transfected with single WT and mutant (MUT) putative PUM1 binding site on the ATXN1-3′UTR. The position of cloned regions for each PUM1 binding motif are indicated. The mutagenized nucleotides are highlighted in blue. In panels C and D the destination-vector (control), RNAi scramble (siScramble) and cel-miR-67 were used as negative controls; miR-101a was used as positive control. (RL) Renilla, (FL) Firefly luciferase. All the experiments were performed in triplicate (data represent mean ± S.E.M.). P values were calculated by Student’s t-test. Statistical significance is indicated as follows: *P<0.05, **P<0.01, ***P<0.0001. See also Figure S1.

Article Snippet: HEK293T cells were transfected with 30ng of psiCHECK-2 vectors (Promega) containing ATXN1 -3′UTR WT- or Mut binding sitess using Lipofectamine 2000 (Invitrogen).

Techniques: Binding Assay, Quantitative RT-PCR, Over Expression, Clone Assay, Plasmid Preparation, Expressing, Luciferase, Construct, Transfection, Mutagenesis, Positive Control

Journal: Cell

Article Title: Pumilio1 Haploinsufficiency Leads to SCA1-like Neurodegeneration by Increasing Wild-Type Ataxin1 Levels

doi: 10.1016/j.cell.2015.02.012

Figure Lengend Snippet: (A) RNA-Clip for the conserved Pum1 binding site in mouse cerebrum and cerebellum. PCRu and PCRd highlight the PCR fragments upstream and downstream of the conserved Atxn1-3′UTR Pum1 binding site (BS). IP with IgG as well Pum1 null mice were used as negative controls. Isolated RNA from a fraction (10%) of pre-cleared lysate was used as input. The experiment was performed in triplicate. Quantification of Atxn1 protein (B) and mRNA levels (C) in WT and Pum1−/− mice in cerebrum and cerebellum (n=8 per genotype). Data represent mean ± S.E.M. and normalized to Gapdh. See Experimental Procedures for more details. P values were calculated by Student’s t-test; *P<0.05, **P<0.01, ***P<0.0001. See also Figure S2.

Article Snippet: HEK293T cells were transfected with 30ng of psiCHECK-2 vectors (Promega) containing ATXN1 -3′UTR WT- or Mut binding sitess using Lipofectamine 2000 (Invitrogen).

Techniques: Binding Assay, Isolation

Journal: Cell

Article Title: Pumilio1 Haploinsufficiency Leads to SCA1-like Neurodegeneration by Increasing Wild-Type Ataxin1 Levels

doi: 10.1016/j.cell.2015.02.012

Figure Lengend Snippet: Representative western blot (upper panel) of protein lysates 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 negative controls were destination-cloning vector (Control), RNAi scramble (siScramble) and cel-miR-67. All data were normalized to α-Tubulin (TUBA). (E) mRNA half-life quantification of wild-type (WT) and Mutated (Mut) PUM1 ATXN1-3′UTR binding sites in HEK293T cells at different time points upon DRB treatment (time zero). The numeric values within the panel given the extrapolated half-life for WT and Mut RNA, P=3.6×10−06. Firefly (FL) RNA levels were quantified and normalized to Renilla (RL). Bottom panel: representative western blot of PUM1 in HEK293T cells at different time points. Data were normalized to GAPDH. (F) ATXN1 mRNA half-life quantification in HEK293T cells at different time points, from zero (DRB treatment) to 8 hours total upon RNAi of PUM1 (siPUM1) or RNAi of scramble (siScramble) transfection. The numeric values within the panel given the extrapolated half-life for siPUM1 and siScramble RNA, P=0.012. Bottom panel: representative western blot of PUM1 in HEK293T cells at different time points. Data were normalized to GAPDH mRNA (top panel) or protein (bottom panel). All experiments were performed in triplicate (data represent mean ± S.E.M.); ***P<0.0001. See also Figure S3.

Article Snippet: HEK293T cells were transfected with 30ng of psiCHECK-2 vectors (Promega) containing ATXN1 -3′UTR WT- or Mut binding sitess using Lipofectamine 2000 (Invitrogen).

Techniques: Western Blot, Over Expression, Clone Assay, Plasmid Preparation, Binding Assay, Transfection

Journal: Cell

Article Title: Pumilio1 Haploinsufficiency Leads to SCA1-like Neurodegeneration by Increasing Wild-Type Ataxin1 Levels

doi: 10.1016/j.cell.2015.02.012

Figure Lengend Snippet: (A) Representative western blot showing that haploinsufficiency of Pum1 restores physiological Atxn1 protein levels. All experiments were performed in triplicate in cerebrum and cerebellum from mice at 5 weeks of age (data represent mean ± SD). All data were normalized to Gapdh. Pum1+/−;Atxn1+/− mice showed (B) significant improvement in motor performance on the accelerating rotarod (n=12 per genotype), (C) reduced hind-paw clasping (n>12 per genotype), and (D) reduced kyphosis (curvature of the spine; photo taken at 25 weeks). Purkinje cells loss (E–F) and loss of dendritic arborization (G) were rescued in Pum1+/−;Atxn1+/− mice (n=6 per genotype). Staining was performed with Calbindin/IP3R1 in 3D-deepth-coding images. Data represent mean ± S.E.M. See Experimental Procedures. P values was calculated by Student’s t-test. ns=not significant; *P<0.05, **P<0.01, ***P<0.0001. See also Figure S6.

Article Snippet: HEK293T cells were transfected with 30ng of psiCHECK-2 vectors (Promega) containing ATXN1 -3′UTR WT- or Mut binding sitess using Lipofectamine 2000 (Invitrogen).

Techniques: Western Blot, Staining