Fig. 3.

MatK specifically coimmunoprecipitates with seven group IIA introns. (A) Immunological analysis of aliquots from pellet and supernatant fractions of tagged MatK immunoprecipitations from transplastomic plants using a peroxidase-linked HA antiserum (Sigma). A 35th of the total supernatant and a 10th of the pellet fraction were loaded on the gel. P, pellet; S, supernatant; RbcL, large subunit of RuBisCo. (B) Identification of RNAs associated with HA-tagged MatK in chloroplast stroma by RNA coimmunoprecipitation and microarray analysis (RIP-Chip). The enrichment ratios (F635:F532) were normalized between four assays with C+ lines, three assays with N+ lines, three control experiments with C− lines, and three control experiments with N− lines. The background-corrected, median-normalized values for replicate spots from the tagged MatK assays were divided by those from the control assays and plotted according to their chromosomal position. Scale for y-axis is logarithmic. Corresponding data can be found in Table S1. (C) Validation of RIP-Chip data by dot-blot hybridization of radiolabeled RNAs that coimmunoprecipitated with HA-tagged MatK. RNA that coimmunoprecipitated with HA-tagged MatK was reverse transcribed in the presence of labeled deoxynucleotides and random primers, and hybridized to DNA probes on a nylon filter. For each experiment, signals were normalized to the psbE signal. (Normalization to other probes including psbA, rrn16, and rrn23 yielded similar results.) Normalized values from two C+ replicates were compared to two C− replicates to yield the relative enrichment ratios shown here (with standard deviations). The same analysis was done for one pair of N+/N− experiments. Scale for y-axis is logarithmic.