Figure 1.
ATXN1 interacts with FOX-2 splice variants.
(A) Schematic view of ATXN1 and the regions used in the Y2H analyses (left) and FOX-2 variants (right). Prey plasmids pACT-ATXN1-NTQ30 or pACT-ATXN1-NTQ82 cover amino acids 1–576, pACT-ATXN1-AXH amino acids 559–701, and pACT-ATXN1-CT amino acids 530–816. Bait plasmid pBTM-FOX-2V1 covers amino acids 1–380 of variant 1, including the putative NLS in the C-terminal region, and pBTM-FOX-2cyt covers amino acids 1–391 of the cytoplasmic FOX-2 variant. Different C-terminal regions of both FOX-2 variants are highlighted in blue and green. (B; C) For directed Y2H analyses yeast strain L40ccua was co-transformed with the relevant bait and prey plasmids, and transformants were selected and spotted onto selective media or membrane to analyze activity of the reporter genes. (D) For Co-IP experiments, HEK293T cell lysates derived from cells overexpressing FLAG-ATXN1-Q30 (left panel) or HEK293T cell lysates (right panel) were incubated with an antibody directed against FOX-2 (Bethyl or Abnova, respectively). Cell lysates incubated without primary antibody served as controls. Then, membranes were treated with an antibody directed against the FLAG tag or ATXN1 to detect precipitated protein.
Figure 2.
The FOX-2 splice variants FOX-2V1 and FOX-2cyt co-localize with nuclear ATXN1 inclusions.
Confocal microscopy of HeLa cells transfected with (A) pCMV-HA-FOX-2V1 and pcDNA1-FLAG-SCA1-Q30 or pcDNA1-FLAG-SCA1-Q82, or (B) pCMV-MYC-FOX-2cyt and pcDNA1-FLAG-SCA1-Q30 or pcDNA1-FLAG-SCA1-Q82, and (C) pCMV-MYC-TIAR and pcDNA1-FLAG-SCA1-Q30 or pcDNA1-FLAG-SCA1-Q82, respectively. Forty-eight hours post transfection cells were fixed and prepared for microscopic analyses. Proteins were visualized using the respective antibodies against the tag as described in Material and Methods. Nuclei were stained using Hoechst. Bars represent 20 µm.
Figure 3.
FOX-2 co-localizes with nuclear ATXN1 inclusions.
(A) Endogenous localization of FOX-2 in HeLa cells visualized with an antibody directed against FOX-2 (Bethyl). (B; C) HeLa cells expressing normal and mutant ATXN1 were fixed forty-eight hours post transfection. Endogenous proteins were visualized using respective antibodies against (B) FOX-2 (Bethyl) and FLAG or (C) TIAR and FLAG as described. Nuclei were stained using Hoechst. Bars represent 20 µm.
Figure 4.
Co-localization of FOX-2 with nuclear ATXN1 inclusions is independent of the polyglutamine region.
Confocal microscopy of HeLa cells transfected with CFP-ATXN1-Q0, CFP-ATXN1-Q30 or CFP-ATXN1-Q82, respectively. Forty-eight hours post transfection cells were fixed and prepared for microscopic analyses. FOX-2 protein was visualized using a specific antibody (Abnova). CFP fluorescence was pseudo-coloured green. Nuclei were stained using Hoechst. Bars represent 20 µm.
Figure 5.
ATXN1 overexpression affects FOX-2 splicing activity.
(A) Schematic view of a partial genomic region of the MAP3K7 gene. Exons are represented by white boxes, the intronic regions by a black line. The FOX-binding sites are shown as black rectangles. (B; C) Total RNA was isolated from HEK293T cells transiently overexpressing ATXN1 with 30 or 82 glutamines. (B) Splicing of the MAP3K7 exon 12 was analyzed by RT-qPCR. Inclusion/exclusion ratio of MAP3K7 exon 12 is illustrated. (C) ATXN1 overexpression was analyzed by RT-qPCR. ATXN1 transcript levels detected in the vector control were set as value one. (D; E) Total RNA was isolated from HEK293T and HeLa cells that were either transfected with FOX-2-specific siRNA or with control siRNA and splicing of the MAP3K7 exon 12 was analyzed by RT-qPCR. (D) Inclusion/exclusion ratio of MAP3K7 exon 12 is demonstrated. (E) FOX-2 reduction was analyzed by RT-qPCR in HeLa and HEK293T cells. Error bars indicate standard error of the mean.
Figure 6.
ATXN2 interacts with FOX-2 splice variants and co-localizes with nuclear ATXN1 inclusions upon overexpression.
(A) Schematic illustration of ATXN1 interactions. Black lines represent interactions reported by Lim and co-workers [16], the blue line represents the investigated interaction in this study. (B) Schematic view of ATXN2 regions used in Y2H studies as described earlier [44], [51], [66]. (C; D) L40ccua yeast cells expressing the corresponding LexA-ATXN2 and AD-FOX-2 fusion proteins were spotted onto selective media or membrane as indicated and the activity of the reporter genes was monitored. (E) HeLa cell lysates were incubated with an antibody directed against FOX-2 (Bethyl) and membranes were treated with an anti-ATXN2 antibody (BD-Biosciences) to detect precipitated protein. (F) HeLa cells were transfected with pCMV-MYC-ATXN2-Q22 or co-transfected with pCMV-MYC-ATXN2-Q22 and pcDNA1-FLAG-SCA1-Q30 or pcDNA1-FLAG-SCA1-Q82, respectively. Forty-eight hours post transfection cells were fixed and prepared for microscopic analyses. Nuclei were stained using Hoechst. Bars represent 20 µm.
Figure 7.
Splicing of ATXN2 transcripts is affected by both, reduced FOX-2 levels and ATXN1 overexpression.
(A) Schematic view of a partial genomic region of the SCA2 gene. Exons are represented by white boxes, the respective intronic regions by a black line. The two potential FOX-binding sites 40 bp downstream of exon 18 are shown as black rectangles. (B) HEK293T and HeLa cells were either transfected with FOX-2-specific siRNA or with control siRNA. Subsequently, total RNA was isolated and RT-qPCR experiments were performed as described in Material and Methods. Inclusion/exclusion ratio of ATXN2 exon 18 is shown. Error bars indicate standard error of the mean. (C) Schematic view of a region of ATXN2 transcripts lacking exon 18 (upper panel) and the primers used for its detection in various cell lines as indicated (lower panel). (D) HEK293T cells were transfected with pcDNA1-FLAG-SCA1-Q30 or pcDNA1-FLAG-SCA1-Q82 and processed as described in (B).
Figure 8.
Schematic model of FOX-2 and ATXN1 effects on ATXN2 transcript.
The SCA2 gene bears two putative FOX-binding sites downstream of exon 18 in the ATXN2 transcript as illustrated. Under normal conditions, FOX-2 binding resulted in inclusion of exon 18, whereas depletion of FOX-2 or overexpression of ATXN1 resulted in increased levels of ATXN2 transcripts lacking exon 18.
Table 1.
: Oligonucleotides used in this study.