Limits...
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.

Show MeSH

Related in: MedlinePlus

UBE3A is a cell cycle dependent protein and it fails to degrade ASPM.(A) Flow cytometric analysis of cells to confirm synchronization in different cell cycle phases. Note the cells in G1 phase show a 2n peak, cells in M phase with a 4n peak and cells in S phase fall in between 2n-4n peaks, suggesting cell cycle arrest at these phases. UN: unsynchronized cells. (B) HEK293 cells synchronized at different cell cycle phases were analyzed for UBE3A and ASPM expression by Western blot analysis. Note the expression of UBE3A was found to peak in M phase as compared to its levels in G1 and S phases. The level of ASPM peaks during G1 and M phases. Erk1/2 was used a loading control. The level of Erk1/2 is known to remain unaffected during different phases of cell cycle. (C) Western blot analysis of lysates from HEK293 cells transfected with an increasing concentration of the pCMV4-HA-UBE3A construct. Note UBE3A overexpression fails to degrade ASPM. Overexpression of UBE3A was determined by probing the blot with anti-HA antibody. β-Actin was used as a loading control. (D) Immunofluorescence analysis of HEK293 cells overexpressing the pCMV4-HA-UBE3A construct. UBE3A and ASPM staining were examined by anti-HA and anti-ASPM antibodies respectively. Note overexpression of UBE3A does not alter ASPM localization or protein level (compare ASPM signals in upper and lower panels). Also note colocalization of UBE3A with ASPM at the centrosome (upper panel). Cells transfected with the pCMV-HA vector were also stained with anti-HA antibody as a control (lower panel).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3102111&req=5

pone-0020397-g005: UBE3A is a cell cycle dependent protein and it fails to degrade ASPM.(A) Flow cytometric analysis of cells to confirm synchronization in different cell cycle phases. Note the cells in G1 phase show a 2n peak, cells in M phase with a 4n peak and cells in S phase fall in between 2n-4n peaks, suggesting cell cycle arrest at these phases. UN: unsynchronized cells. (B) HEK293 cells synchronized at different cell cycle phases were analyzed for UBE3A and ASPM expression by Western blot analysis. Note the expression of UBE3A was found to peak in M phase as compared to its levels in G1 and S phases. The level of ASPM peaks during G1 and M phases. Erk1/2 was used a loading control. The level of Erk1/2 is known to remain unaffected during different phases of cell cycle. (C) Western blot analysis of lysates from HEK293 cells transfected with an increasing concentration of the pCMV4-HA-UBE3A construct. Note UBE3A overexpression fails to degrade ASPM. Overexpression of UBE3A was determined by probing the blot with anti-HA antibody. β-Actin was used as a loading control. (D) Immunofluorescence analysis of HEK293 cells overexpressing the pCMV4-HA-UBE3A construct. UBE3A and ASPM staining were examined by anti-HA and anti-ASPM antibodies respectively. Note overexpression of UBE3A does not alter ASPM localization or protein level (compare ASPM signals in upper and lower panels). Also note colocalization of UBE3A with ASPM at the centrosome (upper panel). Cells transfected with the pCMV-HA vector were also stained with anti-HA antibody as a control (lower panel).

Mentions: We investigated the role of UBE3A in cell cycle further by studying its cell cycle dependence. For this, we synchronized HEK293 cells at three different stages- G1/S boundary, M phase and S phase. The cells were stained with propidium iodide (PI) for DNA content and analyzed by Flow cytometry to check for the efficiency of synchronization (Figure 5A). Western blot analysis of synchronous cells revealed that UBE3A levels fluctuate across the cell cycle phases (Figure 5B). UBE3A was found to be maximally expressed in M phase (Figure 5B). Whereas a minimum expression of UBE3A was seen in S phase (Figure 5B). We observed that the expression level of ASPM in S phase corroborates with UBE3A (Figure 5B). Taken together, above observations imply that UBE3A is under cell cycle regulation and its high expression level in mitosis may be playing an important function.


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

Singhmar P, Kumar A - PLoS ONE (2011)

UBE3A is a cell cycle dependent protein and it fails to degrade ASPM.(A) Flow cytometric analysis of cells to confirm synchronization in different cell cycle phases. Note the cells in G1 phase show a 2n peak, cells in M phase with a 4n peak and cells in S phase fall in between 2n-4n peaks, suggesting cell cycle arrest at these phases. UN: unsynchronized cells. (B) HEK293 cells synchronized at different cell cycle phases were analyzed for UBE3A and ASPM expression by Western blot analysis. Note the expression of UBE3A was found to peak in M phase as compared to its levels in G1 and S phases. The level of ASPM peaks during G1 and M phases. Erk1/2 was used a loading control. The level of Erk1/2 is known to remain unaffected during different phases of cell cycle. (C) Western blot analysis of lysates from HEK293 cells transfected with an increasing concentration of the pCMV4-HA-UBE3A construct. Note UBE3A overexpression fails to degrade ASPM. Overexpression of UBE3A was determined by probing the blot with anti-HA antibody. β-Actin was used as a loading control. (D) Immunofluorescence analysis of HEK293 cells overexpressing the pCMV4-HA-UBE3A construct. UBE3A and ASPM staining were examined by anti-HA and anti-ASPM antibodies respectively. Note overexpression of UBE3A does not alter ASPM localization or protein level (compare ASPM signals in upper and lower panels). Also note colocalization of UBE3A with ASPM at the centrosome (upper panel). Cells transfected with the pCMV-HA vector were also stained with anti-HA antibody as a control (lower panel).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102111&req=5

pone-0020397-g005: UBE3A is a cell cycle dependent protein and it fails to degrade ASPM.(A) Flow cytometric analysis of cells to confirm synchronization in different cell cycle phases. Note the cells in G1 phase show a 2n peak, cells in M phase with a 4n peak and cells in S phase fall in between 2n-4n peaks, suggesting cell cycle arrest at these phases. UN: unsynchronized cells. (B) HEK293 cells synchronized at different cell cycle phases were analyzed for UBE3A and ASPM expression by Western blot analysis. Note the expression of UBE3A was found to peak in M phase as compared to its levels in G1 and S phases. The level of ASPM peaks during G1 and M phases. Erk1/2 was used a loading control. The level of Erk1/2 is known to remain unaffected during different phases of cell cycle. (C) Western blot analysis of lysates from HEK293 cells transfected with an increasing concentration of the pCMV4-HA-UBE3A construct. Note UBE3A overexpression fails to degrade ASPM. Overexpression of UBE3A was determined by probing the blot with anti-HA antibody. β-Actin was used as a loading control. (D) Immunofluorescence analysis of HEK293 cells overexpressing the pCMV4-HA-UBE3A construct. UBE3A and ASPM staining were examined by anti-HA and anti-ASPM antibodies respectively. Note overexpression of UBE3A does not alter ASPM localization or protein level (compare ASPM signals in upper and lower panels). Also note colocalization of UBE3A with ASPM at the centrosome (upper panel). Cells transfected with the pCMV-HA vector were also stained with anti-HA antibody as a control (lower panel).
Mentions: We investigated the role of UBE3A in cell cycle further by studying its cell cycle dependence. For this, we synchronized HEK293 cells at three different stages- G1/S boundary, M phase and S phase. The cells were stained with propidium iodide (PI) for DNA content and analyzed by Flow cytometry to check for the efficiency of synchronization (Figure 5A). Western blot analysis of synchronous cells revealed that UBE3A levels fluctuate across the cell cycle phases (Figure 5B). UBE3A was found to be maximally expressed in M phase (Figure 5B). Whereas a minimum expression of UBE3A was seen in S phase (Figure 5B). We observed that the expression level of ASPM in S phase corroborates with UBE3A (Figure 5B). Taken together, above observations imply that UBE3A is under cell cycle regulation and its high expression level in mitosis may be playing an important function.

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