YBX1 and YBX3 associate with mt tRNAs in the cytosol. (A) Cell fractionation. YBX1 and YBX3 were immunoprecipitated from HeLa cytoplasmic and nuclear soluble extracts (lower panels). Co-IP of mt tRNAs was monitored by northern blot analyses (upper panels). (Lanes no Ab) Control IPs without antibody. Distribution of the U2 nuclear and the Y5 cytoplasmic RNAs was also tested. (B) YBX1 and YBX3 are not detectable in HeLa mitochondria. Crude mitochondrial fraction (lane 1) was digested with proteinase K in the absence (lane 2) and presence (lane 3) of Triton X-100 before western blot analyses of YBX1, YBX3, and the mitochondrial marker proteins TOM20 and ATP5A1. (C) HeLa YBX1- and YBX3-mt tRNA complexes are present in a cytosolic extract devoid of mitochondrial contamination. A HeLa cytosolic extract was prepared by gentle digitonin extraction (lane 2) and its protein (YBX1, YBX3, ATP5A1, Cyt c, and TOM20), mRNA (COXIII), and tRNA (mt tRNAPhe, mt tRNALys, cyt tRNAGly and cyt tRNAThr) contents were compared to those of a diluted (10 times) total cell sonic extract (lane 1). Co-IP of mt tRNAs Phe and Lys with YBX3 and YBX1 from the digitonin extract was confirmed.
