ATAXIN2 is crucial for megakaryocyte protein homeostasis and proper platelet functionality

Expression of the RNA-binding protein is increased upon megakaryocyte commitment, and may coordinate with mRNA stability and translation during megakaryopoiesis. Reduced expression of ATXN2 in human megakaryocytic cells decreased protein synthesis and total protein content despite equal mRNA expression. Genome-wide comparision of subpolysomal versus polysomal mRNA showed that both protein synthesis and protein degradation are derailed in absence of ATXN2. Furthermore, ATXN2 was associated with PABP and DDX6, proteins that control mRNA stability through the polyA-tail. These findings indicate that ATXN2 is involved in protein metabolism in megakaryocytes and platelet function. The human megakaryoblastic cell line Meg01 was transduced with either a short hairpin against ATXN2 (sh95) or a short hairpin control (shc002). 48 hours after transduction, cells were sorted for successful transduction by GFP-expression, lysed and subsequently loaded on a sucrose gradient to separate mRNA based on the amount of ribosomes attached to it. The first eight fractions of the gradient containing subpolysomal, untranslated mRNA were pooled as were the remaining eight fractions containing polyribosomal, translated mRNA. Subpolysomal and polysomal mRNA from three independent experiments were then hybridized to an Illumina beadchip array.