THA8 is found in large ribonucleoprotein particles. (A) Sucrose gradient fractionation of untreated (−RNAse) and RNAse A-treated chloroplast stroma. An equivalent aliquot of each fraction was used for Western blot analysis with THA8 antibody. The underlined fractions in the untreated sample contain the two major THA8 peaks. Rubisco (∼550 kDa) serves as a size marker. This sedimentation pattern was observed in two independent experiments (data not shown). RNA extracted from fractions of the control gradient (−RNAse) was analyzed by RNA gel-blot hybridization with probes specific for the ycf3-2 and trnA introns. The RNAs detected have the size expected for excised intron RNAs. (B) THA8 coimmunoprecipitates with the trnA splicing factors WTF1 and RNC1. Chloroplast stroma was used for coimmunoprecipitations with antibodies against various chloroplast splicing factors, as indicated at top. An immunoprecipitation with an antibody to OE16 served as a negative control. The immunoprecipitation pellets were analyzed by probing immunoblots with antibodies to THA8.
