UNK’s RNA binding domain is required for PLK4-induced centriole overduplication. (A) Exogenous expression of WTsiR and Mut.siR UNK localizes to centrosomes. Confocal images of RPE-1-Tet-PLK4 Cetn2-GFP p53 WT cells showing expression of mCherry WTsiR and Mut.siR UNK at the centrosomes with PLK4 overexpression in S phase. UNK, magenta, and centrioles (centrin), green. Scale bar, 5 μm. Insets scale bar, 1 μm. (B) Knockdown of PCM1 and CEP131 reduces centriolar satellites. Left panels: Centrosomal PCM1 and CEP131 mean normalized fluorescence intensities based on binned central 2 μm of RPE-1-Tet-PLK4 Cetn2-GFP p53 WT cells depleted with PCM1, CEP131, or double-depleted cells with overexpressed PLK4 in S phase. Graph values are expressed as the means of three replicates of 25–30 cells per replicate and SD. P values were determined using one-way ANOVA with the Dunnett post hoc test. Right panel: Frequency of centriole under duplication in RPE-1-Tet-PLK4 Cetn2-GFP p53 WT depleted with PCM1, CEP131, or double-depleted cells with endogenous PLK4 in S phase. Graph values are expressed as the means of three replicates of 100 cells per replicate and SD. P values were determined using one-way ANOVA with the Dunnett post hoc test. (C) High expression of WTsiR but not Mut.siR UNK blocks centriole overduplication. Frequency of RPE-1-Tet-PLK4 Cetn2-GFP p53 WT cells with PLK4 overexpression in S phase. Graph values are expressed as the mean of one biological replicate of 100 cells. (D) UNK’s RNA-binding domain is dispensable for centriolar satellite localization. 8-μm radial fluorescence intensity and corresponding ratio quantification of CEP131 using centrin as the centrosome centroid of RPE-1-Tet-PLK4 Cetn2-GFP p53 WT cells with PLK4 overexpression in S phase. (E) OPP labels newly synthesized peptides and is blocked by cycloheximide. Confocal images of RPE-1-Tet-PLK4 Cetn2-GFP p53 WT cells with controls for OPP labeling: OPP with click chemistry fluor reaction, click chemistry fluor reaction without OPP, OPP with click chemistry fluor reaction with 50 μM of cycloheximide, OPP only, and DMSO only, all with PLK4 overexpression in S phase. OPP, magenta, and centrioles (centrin), green. Scale bar, 5 μm. Insets scale bar, 1 μm. (F) 6 and 12 h of PLK4 overexpression causes reduced translation at the centrosome. Mean normalized fluorescence quantification of centrosomal OPP levels 6 and 12 h of PLK4 overexpression in RPE-1-Tet-PLK4 Cetn2-GFP p53 WT cells in S phase. Graph values are expressed as means of three biological replicates of 25–30 cells per replicate and SD. P values were determined using one-way ANOVA with the Dunnett post hoc test. (G) 6, 12, and 16 h of GFP overexpression causes no effect of translation at the centrosome. Left panels: Mean normalized fluorescence quantification of centrosomal OPP levels 6 and 12 h of GFP overexpression in RPE-1-Tet-GFP p53 WT cells in S phase. Right panel: Mean normalized fluorescence quantification of centrosomal OPP levels with 16 h of GFP overexpression in RPE-1-Tet-GFP p53 WT cells in S phase. Graph values are expressed as means of three biological replicates of 25–30 cells per replicate and SD. P value was determined using a one-way ANOVA with the Dunnett post hoc test and unpaired two-tailed t test. (H) Further suppressing translation blocks centriole overduplication. RPE-1-Tet-PLK4 Cetn2-GFP p53 WT cells with PLK4 overexpression, followed by cycloheximide treatment, and frequency of centriole overduplication 16 h of PLK4 overexpression in S phase. Graph values are expressed as the mean of one biological replicate of 50 cells. (I) Centriolar satellites interact with proteins involved in RNA regulation. Panther analysis of BioID data from PCM1 and CEP131 interacting proteins (Gheiratmand et al., 2019) and their functional classification. (J) UNK does not impact translation on a whole-cell level 16 h of PLK4 overexpression. Mean normalized fluorescence quantification of whole-cell OPP levels with 16 h of PLK4 overexpression in RPE-1-Tet-PLK4 Cetn2-GFP p53 WT cells in S phase. Graph values are expressed as the means of three biological replicates of 25–30 cells per replicate and SD. P values were determined using an unpaired two-tailed t test. (K) UNK does not impact translation at the centrosome 6 or 12 h of PLK4 overexpression. Mean normalized fluorescence quantification of centrosomal OPP levels 6 and 12 h of PLK4 overexpression and depleted UNK in RPE-1-Tet-GFP p53 WT cells in S phase. Graph values are expressed as means of three biological replicates of 25–30 cells per replicate and SD. P values were determined using one-way ANOVA with the Šídák post hoc test. (L) CEP131 does not impact translation at the centrosome 6 or 12 h of PLK4 overexpression. Mean normalized fluorescence quantification of centrosomal OPP levels 6 and 12 h of PLK4 overexpression and depleted CEP131 in RPE-1-Tet-GFP p53 WT cells in S phase. Graph values are expressed as means of four biological replicates of 25–30 cells per replicate and SD. P values were determined using one-way ANOVA with the Šídák post hoc test.