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Angelman syndrome protein UBE3A interacts with primary microcephaly protein ASPM, localizes to centrosomes and regulates chromosome segregation.

Singhmar P, Kumar A - PLoS ONE (2011)

Bottom Line: Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis.The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis.Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, Karnataka, India.

ABSTRACT
Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH) proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly) protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.

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Localization of ASPM and interaction of ASPM with UBE3A in vivo.(A) Indirect immunofluorescence of HEK293 cells stained with anti-γ-tubulin and raised anti-ASPM antibodies. Upper panel: Note ASPM (red) colocalizes with the centrosomal marker γ-tubulin (green). Lower panel: Pre-immune serum (PIS/normal IgG) does not show any signal. Scale bar = 2 µm. (B) Immunofluorescence images of HEK293 cells stained with anti-α-tubulin and raised anti-ASPM antibodies. Upper panel: note ASPM localizes at midbody during cytokinesis (arrow heads). Lower panel: note ASPM localizes at spindle poles during metaphase (arrow heads). Scale bar = 2 µm. (C) Expression profile of ASPM and UBE3A by Western blot analysis using lysates from human fetal brain, human fetal kidney, HEK293, A549, HeLa and HepG2. Anti-ASPM and anti-UBE3A (anti-UBE3A-sc-8926) antibodies recognize 410 kDa and 100 kDa bands, respectively. Note the ubiquitous expression of both proteins. (D) Co-immunoprecipitation of ASPM and UBE3A in human fetal kidney tissue. Upper panel: immunoprecipitation of ASPM co-precipitates UBE3A (100 kDa). Lower panel: immunoprecipitation of UBE3A co-precipitates ASPM (410 kDa). Input lane represents tissue lysate used in pulldown. None of these proteins coprecipitated with either normal IgG or PA/G beads. Abbreviations: IB, immunoblotting; and, IP, immunoprecipitation.
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pone-0020397-g002: Localization of ASPM and interaction of ASPM with UBE3A in vivo.(A) Indirect immunofluorescence of HEK293 cells stained with anti-γ-tubulin and raised anti-ASPM antibodies. Upper panel: Note ASPM (red) colocalizes with the centrosomal marker γ-tubulin (green). Lower panel: Pre-immune serum (PIS/normal IgG) does not show any signal. Scale bar = 2 µm. (B) Immunofluorescence images of HEK293 cells stained with anti-α-tubulin and raised anti-ASPM antibodies. Upper panel: note ASPM localizes at midbody during cytokinesis (arrow heads). Lower panel: note ASPM localizes at spindle poles during metaphase (arrow heads). Scale bar = 2 µm. (C) Expression profile of ASPM and UBE3A by Western blot analysis using lysates from human fetal brain, human fetal kidney, HEK293, A549, HeLa and HepG2. Anti-ASPM and anti-UBE3A (anti-UBE3A-sc-8926) antibodies recognize 410 kDa and 100 kDa bands, respectively. Note the ubiquitous expression of both proteins. (D) Co-immunoprecipitation of ASPM and UBE3A in human fetal kidney tissue. Upper panel: immunoprecipitation of ASPM co-precipitates UBE3A (100 kDa). Lower panel: immunoprecipitation of UBE3A co-precipitates ASPM (410 kDa). Input lane represents tissue lysate used in pulldown. None of these proteins coprecipitated with either normal IgG or PA/G beads. Abbreviations: IB, immunoblotting; and, IP, immunoprecipitation.

Mentions: To confirm the interaction of ASPM with UBE3A using co-immunoprecipitation in vivo, we first raised a rabbit polyclonal antibody against the N-terminal region of ASPM (Figure 1A), as a commercial antibody was unavailable when we started this experiment. The specificity of the ASPM antibody was verified by Western blotting (Figure S1) and immunofluorescence (Figure 2A and B). The raised anti-ASPM antibody recognized the 56 kDa immunogen (Figure S1A). As expected Western blot analysis with human fetal kidney lysate showed a 410 kDa band corresponding to a full-length ASPM protein (Figure S1B). The same result was obtained with a commercial ASPM antibody when it became available (Figure S1B). Here after, we have used the anti-ASPM antibody raised in our laboratory for all the experiments.


Angelman syndrome protein UBE3A interacts with primary microcephaly protein ASPM, localizes to centrosomes and regulates chromosome segregation.

Singhmar P, Kumar A - PLoS ONE (2011)

Localization of ASPM and interaction of ASPM with UBE3A in vivo.(A) Indirect immunofluorescence of HEK293 cells stained with anti-γ-tubulin and raised anti-ASPM antibodies. Upper panel: Note ASPM (red) colocalizes with the centrosomal marker γ-tubulin (green). Lower panel: Pre-immune serum (PIS/normal IgG) does not show any signal. Scale bar = 2 µm. (B) Immunofluorescence images of HEK293 cells stained with anti-α-tubulin and raised anti-ASPM antibodies. Upper panel: note ASPM localizes at midbody during cytokinesis (arrow heads). Lower panel: note ASPM localizes at spindle poles during metaphase (arrow heads). Scale bar = 2 µm. (C) Expression profile of ASPM and UBE3A by Western blot analysis using lysates from human fetal brain, human fetal kidney, HEK293, A549, HeLa and HepG2. Anti-ASPM and anti-UBE3A (anti-UBE3A-sc-8926) antibodies recognize 410 kDa and 100 kDa bands, respectively. Note the ubiquitous expression of both proteins. (D) Co-immunoprecipitation of ASPM and UBE3A in human fetal kidney tissue. Upper panel: immunoprecipitation of ASPM co-precipitates UBE3A (100 kDa). Lower panel: immunoprecipitation of UBE3A co-precipitates ASPM (410 kDa). Input lane represents tissue lysate used in pulldown. None of these proteins coprecipitated with either normal IgG or PA/G beads. Abbreviations: IB, immunoblotting; and, IP, immunoprecipitation.
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pone-0020397-g002: Localization of ASPM and interaction of ASPM with UBE3A in vivo.(A) Indirect immunofluorescence of HEK293 cells stained with anti-γ-tubulin and raised anti-ASPM antibodies. Upper panel: Note ASPM (red) colocalizes with the centrosomal marker γ-tubulin (green). Lower panel: Pre-immune serum (PIS/normal IgG) does not show any signal. Scale bar = 2 µm. (B) Immunofluorescence images of HEK293 cells stained with anti-α-tubulin and raised anti-ASPM antibodies. Upper panel: note ASPM localizes at midbody during cytokinesis (arrow heads). Lower panel: note ASPM localizes at spindle poles during metaphase (arrow heads). Scale bar = 2 µm. (C) Expression profile of ASPM and UBE3A by Western blot analysis using lysates from human fetal brain, human fetal kidney, HEK293, A549, HeLa and HepG2. Anti-ASPM and anti-UBE3A (anti-UBE3A-sc-8926) antibodies recognize 410 kDa and 100 kDa bands, respectively. Note the ubiquitous expression of both proteins. (D) Co-immunoprecipitation of ASPM and UBE3A in human fetal kidney tissue. Upper panel: immunoprecipitation of ASPM co-precipitates UBE3A (100 kDa). Lower panel: immunoprecipitation of UBE3A co-precipitates ASPM (410 kDa). Input lane represents tissue lysate used in pulldown. None of these proteins coprecipitated with either normal IgG or PA/G beads. Abbreviations: IB, immunoblotting; and, IP, immunoprecipitation.
Mentions: To confirm the interaction of ASPM with UBE3A using co-immunoprecipitation in vivo, we first raised a rabbit polyclonal antibody against the N-terminal region of ASPM (Figure 1A), as a commercial antibody was unavailable when we started this experiment. The specificity of the ASPM antibody was verified by Western blotting (Figure S1) and immunofluorescence (Figure 2A and B). The raised anti-ASPM antibody recognized the 56 kDa immunogen (Figure S1A). As expected Western blot analysis with human fetal kidney lysate showed a 410 kDa band corresponding to a full-length ASPM protein (Figure S1B). The same result was obtained with a commercial ASPM antibody when it became available (Figure S1B). Here after, we have used the anti-ASPM antibody raised in our laboratory for all the experiments.

Bottom Line: Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis.The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis.Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, Karnataka, India.

ABSTRACT
Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH) proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly) protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.

Show MeSH
Related in: MedlinePlus