Fig 2: Depletion of SON splicing targets partially phenocopy SON depletion. (A) CEP131 knockdown partially inhibits centriole assembly. SIM images of CEP131 and centrobin (CNTROB) stained cells depleted of SON, CEP131, centrobin, or both CEP131 and centrobin. (B) Fluorescence quantification of widefield images of CEP131 (5-µm radius—cell) and centrobin (1-µm radius—centrosome). N = siControl: 165 centrosomes, 85 cells; siSON: 158 centrosomes, 78 cells; siCEP131: 151 centrosomes, 71 cells; siCNTROB: 161 centrosomes, 81 cells; siCEP131 and siCNTROB: 173 centrosomes, 86 cells. (C) Centriole counts after S phase arrest and PLK4 induction in indicated siRNA conditions. N = 180 cells for each condition. Quantifications are from two biological replicates. (D) Depletion of CEP131 results in a reduction in PCM1 around the centrosome. Widefield images of cells stained for PCM1 after SON or CEP131 depletion and S phase arrest and PLK4 induction. (E) PCM1 fluorescence quantification of a 5-µm radius circle around centrosomes in images from D. N = siControl: 67 cells; siSON: 68 cells; siCEP131: 75 cells. Quantifications are from two biological replicates. (F) PCNT depletion reduces centriole assembly and CEP131 localization. SIM images of S phase-arrested and PLK4-induced cells stained for CEP131 and PCNT. SON, CEP131, and PCNT were depleted as indicated. (G) Fluorescence quantification of widefield images of CEP131 and PCNT within a 5-µm radius around the centrosomes. N = siControl: 57 cells; siSON: 56 cells; siCEP131: 49 cells; siPCNT: 45 cells from two biological replicates. (H) Centriole counts after S phase arrest and PLK4 induction in indicated siRNA conditions. N = 186 cells for each condition. Quantifications are from two biological replicates. (I) Mean radial fluorescence intensity measurements from the centrosome of CEP131 and PCNT. The SD is plotted as thin lines in the corresponding colors. N = siControl: 63 centrosomes; siSON: 61 centrosomes; siCEP131: 46 centrosomes; siPCNT: 44 centrosomes. Quantifications are from two biological replicates. (J) KATNB1 depletion does not alter gross MT and PCM1 distributions. SIM images of S phase-arrested and PLK4-induced cells stained for MTs and PCM1. (K) Centriole counts after S phase arrest and PLK4 induction in indicated siRNA conditions. N = 183 cells for each condition. Quantifications are from two biological replicates. (L) Mean radial fluorescence intensity measurements from the centrosome of MTs and PCM1. The SD is plotted as thin lines in the corresponding colors. N = siControl: 70 centrosomes; siSON: 76 centrosomes; siKATNB1: 77 centrosomes. Quantifications are from two biological replicates. Bar and error bars: mean and SD. Scale bars = 1 and 10 µm for whole-scell images in J.

Fig 3: SON depletion disrupts splicing of genes responsible for centriole assembly, centriolar satellites, and the MT cytoskeleton. (A) The centrosome fraction of the CCDB, centrosome DB (Gupta et al., 2015), and genes determined by proximity mapping of centriolar satellites (satellite proximity) (Gheiratmand et al., 2019) was included with GO terms in opaque colors. The top 20 terms for reduced gene expression (DESeq2) and alternative splicing (as determined by either the MAJIQ or rMATS software packages) are ordered by p values. The dashed lines indicate a p value of 0.05. (B) Venn diagrams displaying the overlap of the CD (Centrosome), the centriolar satellite proximity list (Satellite) and reduced expression or spliced genes. Areas of each circle are proportionate with the gene set. (C) SON depletion results in improper splicing of CNTROB and CEP131. Top panels show the location of introns and exons and the primers used for reverse transcriptase PCR (RT-PCR), middle panel. Bottom panel: quantification of the RT-PCR bands normalized first to the amount of RNA as determined by ACT B RT-PCR and second to the amount of the properly spliced fragment in the control condition. RNA-seq and RT-PCR were performed and quantified on three biological replicates. Error bars: SD.

Fig 4: SON splicing targets disrupt centrobin, centriolar satellites, and trafficking granules. (A) SON targets are diminished at the centrosome. SIM images of siRNA-treated, S phase-arrested cells induced for PLK4 expression and stained for the identified SON targets. Scale bar = 1 µm. (B) ?-Tsubulin and PCNT are reduced most in the region 1.0 to 2.5 µm from the centrosome center. Normalized mean radial quantification of fluorescence intensities from widefield images (Supplemental Figure S5A) (y-axis on the left, black = siControl, red = siSON). The ratio of the intensity of siSON/siControl is also plotted (y-axis on the right, purple); p values from the Student’s t test are plotted beneath each graph on a semilog scale. The mean value is plotted, and standard deviations are plotted as thin lines in the corresponding color. N = ?-tubulin: siControl, 76 centrosomes; siSON, 73 centrosomes; PCNT: siControl, 72 centrosomes; siSON 76 centrosomes; centrobin (CNTROB): siControl, 101 centrosomes; siSON, 106 centrosomes; CEP131: siControl, 70 centrosomes; siSON, 96 centrosomes. Data are compiled from two biological replicates. (C) SIM images of siRNA-treated, S phase-arrested cells induced for PLK4 expression and stained for PCM1. Scale bar = 1 µm. (D) Radial quantification of PCM1 fluorescence intensity as in B. N = siControl, 78 centrosomes; siSON, 70 centrosomes. Data are compiled from two biological replicates. (E) Fluorescence quantification of the indicated proteins in a 5-µm radius circle around the centrosomes. N = ?-tubulin: siControl, 65 cells; siSON, 63 cells; centrobin (CNTROB): siControl, 90 cells; siSON 100 cells; PCNT: siControl, 65 cells; siSON, 66 cells; PCM1: siControl, 74 cells; siSON, 67 cells; Cep131: siControl, 66 cells; siSON, 73 cells; MTs: siControl, 55 cells; siSON 68 cells. Bar and error bars: mean and SD. Data are compiled from two biological replicates. (F) Total mean in cell fluorescence intensities. Fluorescence was measured simultaneously for all cells in each image, and 10 images total were used for each marker except for CEP131, where nine images were used, and centrobin (CNTROB), where 16 images were used. Data are compiled from two biological replicates. Bars and error bars: mean and SD; ns, not significant.