Fig. 4
Pbp1 polyQ expansions inhibit Ty1 retromobility at the post-translational level. a Effects of Pbp1-polyQ expansion on Ty1his3AI retromobility (Mean ± SD; n = 5 independent cultures; Mann–Whitney U-test). b Semi-quantitative PCR products reflecting Ty1 integration upstream of 12 tRNAGLY loci. TEL1 amplification serves as control. c ChIP examining the localization of RNA Pol II at TyB using an α-RNA Pol II (phosphorylated serine 5) antibody (One-way ANOVA and Dunnett’s post hoc). d Effects of Pbp1-polyQ expansion on the levels of Ty1 mRNA in RT-qPCR (One-way ANOVA and Dunnett’s post hoc). e Confirmation of ATXN2-polyQ expression in transfected HEK293T cells by western blotting. f Effects of ATXN2 polyQ expansion on the levels of retroelement RNAs as detected by RT-qPCR. Values are normalized to GAPDH and statistics are relative to 23Q (Student’s t-test). g Pbp1 polyQ forms exhibit an increased interaction with Trf4 (n = 2). TAP-tagged Pbp1 proteins with indicated glutamine features were subjected to anti-TAP pulldowns followed by immunoblotting for endogenous Trf4, TAP tag or Actin control. h, i Interactions between Pbp1 forms or Trf4 with Ty1 mRNA. Presented are levels of Ty1 mRNA immunoprecipitated relative to input, as detected by RT-qPCR. Values are normalized to pull-down in untagged (h) or trf4∆ cells (i). j Effects of Pbp1-polyQ expansion on Ty1 mRNA levels in the presence/absence of TRF4 as detected by RT-qPCR (One-way ANOVA and Dunnett’s post hoc). k Ty1 mRNA stability assay in Pbp1 polyQ cells. Following RNA Pol II inhibition, Ty1 mRNA levels were assessed over time by RT-qPCR and the line of best fit was plotted (n = 2). l Quantification of slopes in k. m Western blot examining in Pbp1-polyQ-expanded cells (n = 2), the levels of Ty1 Gag forms p49 (unprocessed), p45 (processed), and p22/18 (truncated). c, d, f, h–j Mean ± SD; n = 3. c, d, j Statistics are presented relative to levels detected in Pbp1-1Q cells. a–m *p < 0.05; **p < 0.01; ****p < 0.0001
