Dynamic association of YBX1 with mt tRNAs. (A) Transcription inhibition increases YBX1 association with mt tRNAs. Human HeLa cells were nontreated (NT) or treated with α-amanitin (a-ama), 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), or actinomycin D (ActD). After extract preparation, YBX1 was immunoprecipitated and coprecipitation of mt tRNAs Leu(UUR), Lys, Phe, and Val was measured by slot blot analyses and PhosphorImager quantification. For each transcription inhibitor, 12 independent inhibition, IP, and slot blot experiments were performed and the obtained relative hybridization intensity values were normalized to the means of values obtained for nontreated control cells. The means and standard deviations (sd) for each condition are indicated. The obtained values of treated cells were compared to the values obtained for NT control cells in a statistical analysis using Student's t-test. For conditions statistically different from the corresponding control condition, the significance levels are indicated. (B) DRB treatment of HeLa cells increases YBX1 association with mt tRNAs in a reversible manner. YBX1 association with mt tRNAs Leu(UUR), Lys, Phe, and Val was measured in nontreated HeLa cells (0 h), in cells grown for 1 h in the presence (1 h) and for another hour in the absence of DRB (2 h). Other details are identical to panel A. (C) YBX1 and YBX3 association with mt tRNAs is decreased in apoptotic cells. YBX3 and YBX1 were immunoprecipitated from extracts prepared from HeLa cells nontreated (NT) or treated with staurosporine (STS) for 4 h. Co-IP of mt tRNAs was measured as described in panel A.
