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.
Highlights
Centrosomal proteins function in tango with cell cycle proteins and the loss-of-function of these proteins leads to cell cycle misregulation [1]
To find novel interacting partners for the C-terminal region (CTR) of ASPM, we cloned this region corresponding to amino acids 3,276–3,477 in the Y2H DNA binding domain vector pGBKT7 (Figure 1A)
The clone was subsequently used as a bait to screen a human fetal brain cDNA library cloned in the Y2H activation domain vector pACT2
Summary
Centrosomal proteins function in tango with cell cycle proteins and the loss-of-function of these proteins leads to cell cycle misregulation [1]. Autosomal recessive primary microcephaly (MCPH; OMIM 251200) is a subtype defined by congenital microcephaly and associated mental retardation (MR). It is genetically heterogeneous with seven known loci and the genes for all seven loci have been identified: MCPH1-MCPH1, MCPH2-WDR62, MCPH3-CDK5RAP2, MCPH4-CEP152, MCPH5-ASPM, MCPH6-CENPJ, and MCPH7STIL [2,3,4,5,6,7,8]. ASPM is the human orthologue of the Drosophila melanogaster asp (abnormal spindles) gene It encodes for a 3,477 amino acids protein and is widely expressed in many human fetal and adult organs including brain, kidney, muscle and lung [13]. Using RNAi knockdown approach, Fish and colleagues demonstrated that the loss of Aspm in mouse neuroepithelium alters the orientation of cleavage plane in the progenitor cells, resulting in an increase in asymmetric divisions and reduction in the neuronal progenitor pool [14]
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