Distributions of Enrichment Ratios in CAF1 RIP-Chip Experiments.
(A) Histograms showing the distribution of enrichment ratios in CAF1 RIP-chip assays involving wild-type or crp1 stroma. F635/F532 is the ratio of fluorescence deriving from the pellet and supernatant RNAs, respectively. Gray bars show the distribution of ratios for all array elements; black bars show the distribution for the subset of elements diagrammed in (B).
(B) Map positions of array fragments corresponding to the most highly enriched sequences in CAF1 RIP-chip assays. Fragment numbers are shown below each fragment. Residue numbers within the chloroplast genome (Maier et al., 1995) are indicated.

Overview of the RIP-Chip Experiments.
(A) Experimental design. Chloroplast stroma from either wild-type or crp1 mutants was used for immunoprecipitation (IP) with the indicated antisera. RNA purified from immunoprecipitation pellets or supernatants (sup) was labeled with Cy5 or Cy3, respectively, combined, and used to probe the maize chloroplast (Zm Cp) microarray. Replicate data sets for the wild-type extract derive from two independent immunoprecipitations, one of which was used for two labeling/hybridization reactions. Replicate data sets for the crp1 mutant extract derive from two independent immunoprecipitations.
(B) Excerpts of merged F635:F532 images from a CRP1 and a CAF1 experiment involving wild-type stroma. Fragment names are indicated, and fragment numbers are in parentheses. Each DNA fragment is represented five times on the array, in clusters of two and three spots. These excerpts show three-spot clusters.

CRP1 RIP-Chip Data Plotted According to Genomic Position.
The log₂-transformed enrichment ratios (F635:F532) were normalized between the three CRP1 RIP-chip assays involving wild-type stroma and the two control assays with crp1 mutant stroma. The difference between the median normalized values for replicate spots in the experimental and control data sets is plotted as a function of fragment number. Fragments for which fewer than two spots per array yielded an F532 signal above background were excluded and appear as gaps in the curve. The data used to generate this curve are provided in Supplemental Table 3 online. Supplemental Figure 3 online shows analogous plots for the five individual experiments without data normalization. The valley within the psaC peak region is artifactual: the F532 signal/base pair for fragment 236 (the low point) is substantially lower than that for the fragments that overlap it, indicating that the DNA in this spot is defective.

Representative CRP1 RIP-Chip Data.
(A) Immunoblots probed with the affinity-purified CRP1 antibody used in the RIP-chip assays. The left panel shows an immunoblot of chloroplast stroma from wild-type and crp1 mutant seedlings. Cross-reacting proteins are indicated with asterisks. The right panel shows an immunoblot of an α-CRP1 immunoprecipitation involving wild-type stroma; an immunoprecipitation with a different antibody (α-OE23) is shown as a negative control. IgGs in the immunoprecipitates are detected by the secondary antibody used to probe the immunoblot.
(B) Histograms showing the distribution of enrichment ratios in representative experimental and control CRP1 RIP-chip assays. Gray bars show the distribution of ratios for all array elements and are drawn to the frequency scale at left. Solid lines show the distribution for replicate spots overlapping the petA 5′ UTR (fragments 113 and 114) and the psaC 5′ UTR (fragments 233 and 235); the positions of these fragments are diagrammed in Figure 6C. Dashed lines show the distribution for spots corresponding to fragments 139, 142, 169, and 194, which derive from the petB, rpl2, and rps12 genes. The line plots are drawn to the frequency scale at right.

CAF1 RIP-Chip Data Plotted According to Genomic Position.
Log₂-transformed F635/F532 ratios were normalized between replicate CAF1 RIP-chip data sets (see Methods). The median normalized values for replicate spots across the two replicate experiments are plotted according to fragment number. Fragments for which fewer than two spots per array yielded an F532 signal above background were excluded and appear as gaps in the curve. The normalized data used to generate this curve are provided in Supplemental Table 2 online. Supplemental Figure 2 online shows analogous plots for the two individual experiments without normalization and illustrates the high degree of reproducibility. The enrichment values in the petD intron region show a bimodal peak in which F635/F532 correlates with the proportion of the fragment containing intron sequence (see Supplemental Table 1 online). Enrichment of the petD, rps16, trnG, and ycf3 introns was previously shown by slot-blot analysis of RNAs that coimmunoprecipitate with CAF1 (Ostheimer et al., 2003). The slight enrichment of rpl2 and ndhB intron sequences was confirmed by slot-blot hybridizations of pellet and supernatant RNAs derived from an independent CAF1 immunoprecipitation (data not shown).

Fine-Mapping of RNAs That Coimmunoprecipitate with CRP1.
(A) Verification that petA and psaC RNAs are enriched in CRP1 coimmunoprecipitates. Coimmunoprecipitations and RNA extractions were performed as for the RIP-chip assays; the RNAs were then analyzed by slot-blot hybridization with the indicated probes. The top panels, showing CRP1-dependent enrichment of petA 5′ sequences, are duplicate blots of RNAs from parallel CRP1 and CAF2 immunoprecipitations. The CAF2 immunoprecipitation was included as a negative control; the ndhB intron is a known CAF2 substrate (Ostheimer et al., 2003) and was analyzed to demonstrate the integrity of the CAF2 pellet RNA. The bottom panels, showing CRP1-dependent enrichment of psaC 5′ sequences, are duplicate blots of RNAs from parallel CRP1 and CRS1 immunoprecipitations. The CRS1 immunoprecipitation was included as a negative control; the atpF intron is a known CRS1 substrate (Ostheimer et al., 2003) and was analyzed to demonstrate the integrity of the CRS1 pellet RNA. The psaC/ndhE (fragment 235) and cemA/petA (fragment 114) probes correspond to the two highest ranking fragments in the CRP1 RIP-chip experiments and are diagrammed in (C). Sup, supernatant.
(B) Fine-mapping of CRP1-associated RNAs from the psaC and petA 5′ UTRs. Immunoprecipitation with a different antibody (α-OE23) served as a negative control. Duplicate slot blots were prepared as in (A) except that RNase inhibitor was omitted from the immunoprecipitation reactions. Blots were probed with 5′ end-labeled 70-mer oligonucleotides as diagrammed in (C). Results were quantified with a phosphorimager and plotted at right.
(C) Summary of CRP1 RIP-chip and slot-blot data for the psaC and petA regions. The PCR fragments on the microarray (identified by name and number) and the 70-mer oligonucleotides used for slot-blot probing are indicated with lines whose thickness reflects the degree to which the sequences were enriched in CRP1 immunoprecipitates. The nucleotide (nt) sequences shown span the pair of 70-mers from each region that detected the most highly enriched sequences; they are aligned to highlight the 7-mer and 11-mer motifs (large boldface letters), with start codons shown in large lightface letters.
(D) Model for RNA recognition by CRP1. The cartoon illustrates how a CRP1 monomer might bind the RNA encompassed by the shared 7-mer and 11-mer motifs in the psaC and petA 5′ UTRs. The 14 repeating units in CRP1 represent its 14 tandem PPR motifs. The RNA is shown bound to the concave face of the predicted PPR superhelix; this predicted surface of CRP1 appears well-suited for RNA binding (Williams and Barkan, 2003).