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. Author manuscript; available in PMC: 2015 Dec 11.
Published in final edited form as: Nat Genet. 2014 Oct 26;46(12):1283–1292. doi: 10.1038/ng.3122

Figure 2. Transcriptional and protein consequences of PLK4 mutations.

Figure 2

a) Schematic of the human PLK4 gene. Coding exons (black), UTRs (white), alternatively spliced region in exon 5 (grey), arrow heads, RT-PCR primer positions.

b) PLK4 protein domain structure. Kinase domain (KD)( grey), PEST sequences (1-3) (black), polo-box domains (PBD1-3) (blue/turquoise/green). Middle panel, the c.2811-5G>C mutation creates a new splice acceptor site that leads to retention of 4 bp of intron 15 sequence in the PLK4 mRNA, resulting in premature truncation of the protein at its C-terminus, disrupting the terminal polo-box domain (PB3). Lower panel depicts domain structure for the alternative isoform (ALT) resulting from use of an internal exon 5 splice donor site.

c) Sequence electropherograms of the exon 15-16 junction of PLK4 amplified by RT-PCR from patient and control RNA.

d) Alternative splicing of an internal exon 5 splice donor site is not detected in other vertebrates by RT-PCR.

e) Levels of functional PLK4 are reduced to 25% of normal levels, in patients with the c.1299_1309delTAAAG mutation. Transcript levels plotted from quantitative RT-PCR on RNA extracted from patient primary fibroblast cell lines (n=3 experiments (exp), performed in triplicate; error bars, s.e.m). P value, two-tailed t-test *p≤0.05. Further details and characterization of PLK4 ALT and FL transcript levels, Fig. S5.

f) Immunoblotting demonstrates reduced endogenous PLK4 protein levels in patient fibroblasts. Cell lysates of asynchronous cells. Left two lanes, RNAi of PLK4 in RPE1 cells demonstrating specificity of PLK4 antibody, with two PLK4-specific protein bands visualized. Remaining lanes, patient and control primary fibroblasts, upper panel standard exposure PLK4 immunoblot with middle panel overexposed to demonstrate residual detectable protein in P1 patient. Note also loss of the upper PLK4 band in P6 and P7. Lower panel, loading control, blot probed with anti-Actin antibody.

g) PLK4 protein levels at the centrosome are reduced in patient fibroblasts. Top panel, representative immunofluorescence images of primary fibroblasts treated with 10μM MG132 for 5 hrs prior to fixation. (Specificity of the anti-PLK4 antibody confirmed by performing RNAi-mediated PLK4 depletion, Fig. S6). Scale bar = 10 μm. Bottom panel, quantitation of PLK4 protein levels at the centrosome by immunofluorescence analysis. Exp=3, n=50 cells/exp; error bars, s.e.m of n=150 cells. P value, two-tailed t-test ****p≤0.0001.