Primary microcephaly (MCPH)(MIM 251200) is a neurodevelopmental disorder. 1 Microcephaly at birth and non-progressive mental retardation are the two principle features of this disease. The microcephaly is a normal brain with architectural reduction in size. This size reduction is thought to be due to reduced neuron production during fetal life rather than defective neuron migration or increased apoptosis. 2 Six loci (MCPH1-6) have been mapped that, when mutated, account for this autosomal recessive disease. 1 The genes at four of those loci have been identified so far. MCPH3 and MCPH6 encode CDK5RAP2 (cyclin dependent kinase 5 regulatory associated protein 2) and CENPJ (centromere-associated protein J), also known as CPAP (centrosomal protein 4.1-associated protein), respectively, both of which encode centrosomal proteins. 3, 4 MCPH5 encodes ASPM (abnormal spindle-like, microcephaly-associated). 5, 6 Homozygous mutations in the ASPM gene appear to be the most common cause of MCPH. 5, 6 ASPM is the putative human ortholog of the Drosophila melanogaster abnormal spindles gene (asp), which is essential for organization and bundling of microtubules at the spindle poles and necessary for the proper formation of mitotic spindles in mitosis and meiosis. 1 It has been recently demonstrated that ASPM is a centrosomal protein, too. 7, 8 Centrosomal localization of MCPH proteins suggests a novel centrosomal mechanism in controlling neuron production. 3 MCPH1 encodes Microcephalin, 9 a member of the PTCB (proteins with twin carboxylterminal BRCT domains) family that includes BRCA1, 53BP1 (p53 binding protein 1), and NFBD1/MDC1. 10 We have shown that MCPH1 is involved in regulation of BRCA1 and CHK1, and cellular responses to DNA damage. 10 These observations were confirmed by another independent study. 11 MCPH1 also has a role in the initiation of chromosome condensation during mitosis. 12 In this report, we examine if MCPH1 is a centrosomal protein.

To detect endogenous MCPH1 in cultured cells, we tested 4 different affinity purified rabbit polyclonal anti-peptide antibodies against human MCPH1 produced by Bethyl Laboratories. Both BL1609 and BL1610 recognized two major bands, one with a slower electrophoretic mobility than that of the 100-kDa protein marker (about 105 kDa), and the other band near the 100-kDa protein marker, on immunoblots of HEK293T and U2OS cell extracts (Fig. 1A and data not shown). To determine specificity of the MCPH1 antibodies, we inhibited MCPH1 expression with a siRNA oligonucleotide duplex (MCPH1si). 10, 11 The full-length MCPH1 protein level (the band that migrated slower than the 100-kDa protein marker) was significantly decreased in U2OS cells 60 hours post transfection with MCPH1si, while it was not affected by a control siRNA oligonucleotide duplex (LacZsi) that targets the LacZ gene (Fig. 1A). The band near the 100-kDa protein marker recognized by both antibodies represented a non-specific cross-reaction or a spliced variant missing the sequence targeted by MCPH1si since this signal was not changed after transfection with a control or a MCPH1-specific siRNA duplex. We reported previously that MCPH1 knockdown decreases BRCA1 protein level and this regulation is at the transcriptional and/or post-transcriptional level. 10 It has been reported that both BRCA1 regulates centrosome duplication13 and CHK1 localizes to interphase, but not mitotic, centrosomes. 14 Furthermore, all the MCPH genes, except MCPH1, have been shown to encode a centrosomal protein. 3, 7, 8 Therefore, we would expect that human MCPH1 may have …