Fig 2: Knockdown of PAF49 results in the rapid degradation of PAF53, but not vice versa.A, cells were treated with 1 mM IAA or vehicle for 3 h to knock down AID-PAF49 levels. Cells were then harvested and lysed in HEPES lysis buffer. The expression of AID-PAF49 and PAF53 was determined by Western blot analysis. B, three independent repeats of the Western blot experiment presented in A were performed. The levels of PAF53 were corrected for ß-actin. The data were analyzed by a two-tail t test, p < 0.0001. C, cells with PAF53-AID were treated with 1 mM IAA or vehicle for 3 h and were harvested and lysed in HEPES lysis buffer. The expression of PAF53-AID and PAF49 was determined via Western blot analysis. D, three independent repeats of the Western blot experiment presented in C were performed. The levels of PAF49 were corrected with ß-actin. A two-tail t test was performed to test for significance, p = 0.3007. E, cells expressing AID- PAF49 were treated with 1 mM IAA for 3 h and were harvested and lysed at the indicated time points: 0, 15, 30, 60, 120, and 180 min after IAA treatment. Western blot analysis was performed to determine the protein levels of AID-PAF49 and PAF53 at each time point. F, three independent repeats of the experiment presented in E were performed. The levels of AID-PAF49 and PAF53 were corrected for the ß-actin content of the sample. The change in AID-PAF49 and PAF53 levels with time were plotted with a nonlinear fit with GraphPad Prism. The dotted lines indicate the approximate half-life of each protein in these experiments.

Fig 3: RPA12 knockdown affects the expression and localization of RNA Polymerase I subunits RPA194 and RPA135.(A) qPCR analysis of RPA12 transcript in A375 melanoma cells with transient RPA12 knockdown using siRNA. Mean fold change ± SD of n = 4 biological replicates is shown. Mann-Whitney two-tailed t-test *, p < 0.05. (B) Immunofluorescence staining of RPA12. Representative biological replicate of n = 4 is shown. (C) Quantification of the images for RPA12. Mean fold change ±SD of n = 3 biological replicates is shown. Analysis of N = 50 cells per treatment. Student’s two-tailed t-test **, p = 0.0018. (D) Immunofluorescence staining of siCtrl and siRPA12 knockdown cells. A375 melanoma cells were stained for the indicated Pol I subunits and fibrillarin (FBL) following siRNA knockdown of RPA12. DNA was counterstained using Hoechst. Representative biological replicates of n = 4 are shown. (E) Quantification of the images for RPA194. Mean fold change ±SD of n = 3 biological replicates is shown. N = 50–100 cells per treatment. Student’s two-tailed t-test *, p = 0.0306. (F) Immunofluorescence staining of siCtrl and siRPA12 knockdown cells for PAF53 and CAST. (G) Quantification of the images for PAF53 and CAST. N = over 100 cells per treatment. Scale bars, 10 µm.

Fig 4: Inhibition of rDNA transcription by depletion of PAF49 or PAF53 does not affect the steady-state levels of other Pol I subunits.A, cells expressing AID-PAF49 were treated with 1 mM IAA or vehicle for 3 h and were harvested and lysed in HEPES lysis buffer. The levels of multiple subunits of RNA Polymerase I (A194, A127, and A43) were determined by Western blot analysis. B, cells with PAF53-AID were treated with 1 mM IAA or vehicle for 3 h and were harvested and lysed in HEPES lysis buffer. The expression of the same Pol I subunits as in A were visualized by Western blot analysis. C, cells with AID-PAF49 were treated with IAA for 3 days. On each day, including day 0, cells were harvested and lysed as described previously. The expression of A194, PAF53, and A43 was determined by Western blot analysis. D, cells with PAF53-AID were treated with IAA for 3 days. On each day, including dy 0, cells were harvested and lysed as described previously. The expression of A194, PAF49, and A43 was determined by Western blot analysis.

Fig 5: The functionality of PAF49 requires the dimerization domain and adjacent sequences.A, diagrams of the mPAF49 constructs used in the following experiments. B, 24 h prior to IAA treatment, cells were transfected with the mPAF49 mutants indicated in (A). Cells were then treated with 1 mM IAA or vehicle for 3 h to knock down endogenous PAF49-AID. After 3 h, the cells were pulsed with 5-EU for 15 min and de novo synthesized RNA was visualized as described (red) against a DAPI background (94). Scale bar = 50 µm. C, to assess cell proliferation via active DNA replication, cells were treated with 1 mM IAA or vehicle for 3 days instead of 3 h as done in (B). Cells were then pulsed with EdU for 1 h and newly synthesized DNA was visualized as described (red) against a DAPI background. Scale bar = 50 µm. D, cells were transfected with either WT mPAF49 or one of the mPAF49 mutants described in A 24 h prior to IAA treatment. After 3 h of 1 mM IAA treatment, cells were harvested and lysed as described. The levels of hPAF53 and the mPAF49 mutants were determined via Western blot analysis. All mPAF49 constructs were tagged with the FLAG epitope. mPAF491-100 is also tagged with the GST epitope in order to make the protein large enough to detect on a Western blot. This explains why mPAF491-100 has a larger molecular weight than mPAF491-200. E, three independent repeats of the WB data presented in (D) were performed. The relative levels of PAF53 were quantified and corrected with ß-actin. The data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test; ns indicates p > 0.05. F, for the WB data presented in (D), the relative expression levels of the ectopically expressed mPAF49 mutants (and WT). They were quantitated and normalized to ß-actin. Significance was determined via a one-way ANOVA with Tukey’s multiple comparisons test. The error bars represent mean ± SD; * = 0.05 to 0.01 and ** <0.01. G, to assess how well each mPAF49 mutant was able to rescue the level of PAF53 in the absence of endogenous PAF49, relative rescue of PAF53 was calculated for each construct (% control/relative density * 1E4). This calculation resulted in an integer that could be used to compare the activity of each mutant. Significance was determined via a one-way ANOVA with Tukey’s multiple comparisons test. The error bars represent mean ± S.D; * = 0.05 to 0.01 and ** <0.01. H, HEK293 cells were transfected with the indicated FLAG-PAF49 mutant. 48 h later, the cells were lysed, and the FLAG-tagged proteins recovered with immobilized anti-FLAG antibodies and analyzed by western blotting for FLAG and A127. PAF49 1 to 100 did not blot well (lane 2).