Fig 1: SerRS directly interacts with Shelterin POT1. a Immunofluorescent staining to show the colocalization of SerRS (green) and POT1 (red) in the nucleus of HeLa VST cells. Scale bars represent 5 µm. b Flag-tagged SerRS (Flag-SerRS) and V5-tagged POT1 (V5-POT1) vectors were transfected into HeLa cells, and immunoprecipitation (IP) was performed with the indicated antibodies. Cell lysates and immunoprecipitated proteins were analyzed by immunoblotting (IB) with the indicated antibodies. c HeLa VST cell lysates were immunoprecipitated with a POT1 antibody or a nonspecific IgG and analyzed by immunoblotting with the indicated antibodies. d A GST tag was fused with full length (FL) SerRS or the tRNA-binding domain (TBD), catalytic domain (CD) and unique to SerRS domain (UNE-S) of SerRS (upper panel). The GST-fused SerRS proteins were used to pull down purified His6-tagged POT1, and they were analyzed by immunoblot with anti-His6 antibody and anti-GST antibody (lower panel). e GST pull-down assays showed the direct interaction between SerRS and different domains of POT1. Full-length (FL) POT1 and its domains were fused with GST at the N-terminus to pull down purified His6-tagged SerRS. OB1, OB2 and OB3 stand for oligonucleotide/oligosaccharide DNA-binding domains 1, 2, and 3; HJR stands for Holliday junction resolvase-like domains
Fig 2: SerRS promotes the recruitment of POT1 to telomeres. a Flag-tagged POT1 was transfected into HeLa 1.2.11 cells with V5-tagged SerRS or empty vectors (right panel). POT1-associated telomeric DNA fragments were immunoprecipitated with an anti-Flag antibody or a control IgG and analyzed by dot blot using DIG-labeled (CCCTAA)6 probes (left panel). b Quantification of the data shown in a. Data are represented as the means ± SEM (n = 3). **P < 0.005, two-tailed Student’s t-test. c The levels of POT1 expression in SerRS-overexpressing HeLa 1.2.11 cells as determined by immunoblot analysis. d IF-FISH showing the localization of POT1 at telomeres in HeLa 1.2.11 cells transfected with SerRS or empty vector. Cells were hybridized with Cy3-labeled (CCCTAA)3 PNA probe (red) and immunostained with POT1 (green). Scale bars represent 5 µm. e Quantification of the data in d to show the average percentages of POT1-associated telomere foci per cell (means ± SEM, n = 50, ***P<0.001, two-tailed Student’s t-test). f Quantification of the data shown in d to show the percentages of cells with more than ten POT1-associated telomeric foci (mean ± SEM, n = 3, **P < 0.01, two-tailed Student’s t-test)
Fig 3: SerRS prevents the recruitment of telomerase to telomeric DNA. a Telomerase RNA (TERC; red) and telomeres were visualized by RNA FISH and immunofluorescent staining with anti-TRF1 antibody (green), respectively, in HeLa 1.2.11 cells transfected with the SerRS expression vector or an empty vector. Arrows indicate telomerase foci colocalizing with telomeres. Scale bars represent 5 µm. b Data from a were quantified and plotted as a bar graph (means ± SEM, n = 3, *P < 0.05, two-tailed Student’s t-test). Cells with =2 telomerase-associated foci were counted as positive, and more than 80 cells were examined for each cell line. c Model proposing a role for SerRS as a regulator of telomere length. The SerRS dimer may bind to telomeric DNA repeats, and it tethers two POT1 molecules onto a telomeric single-stranded 3’-overhang, preventing the access of telomerase and the elongation of telomere
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