Protein and RNA elements directing YBX1 association with mt tRNAGly and mt tRNALeu(UUR). (A) YBX1 binding is directed by the D and T stem–loop of mt tRNALeu(UUR) and mt tRNAGly, respectively. About 2 fmol of internally labeled wild-type and mutant mt tRNAs carrying internal truncations (shaded), sequence alterations (boxed) or other mt tRNA elements (italics) were mixed with 2 µg of cold E. coli tRNA and transfected into HeLa cells. After 24 h of incubation, endogenous YBX1 was immunoprecipitated and co-IP of labeled tRNAs was tested. (B) Gel shift analyses. In vitro transcribed labeled mt tRNAPhe (1 pmol) was incubated without or with 0.2, 0.4, 1.5, and 4 pmol of recombinant mutant (YBX1CSDm) and 4 pmol of wild-type YBX1 in the absence or presence of cold mt tRNAPhe (50 pmol) before analysis on a 5% native gel. (C) Intact CSD is required for in vivo association of YBX1 with mt tRNALys and mt tRNAPhe. Transiently expressed Xpress-YBX1 and Xpress-YBX1CSDm were immunoprecipitated from transfected or nontransfected (NT) cell extracts and analyzed by western blotting. Co-IP of mt tRNAs Lys and Phe was monitored by northern blotting.
