Fig. 4. Co-immunoprecipitation of CRS2 with CAF1 and CAF2 from chloroplast extract. Chloroplast stroma was incubated with the affinity-purified antiserum indicated above each lane. Immunoprecipitates were analyzed on immunoblots to detect CAF1 (top panel), CAF2 (middle panel) or CRS2 (bottom panel). Antiserum to OE33, which is a component of the photosynthetic apparatus that does not bind RNA, was used as a negative control. The αCRS1 immunoprecipitation successfully precipitated CRS1 (not shown) and the CRS1-dependent atpF intron (see Figure 6).

Fig. 3. CRM domains in group II intron splicing factors. (A) Schematic illustrating regions of similarity between CRS1, CAF1 and CAF2. Similar shading indicates amino acid sequence similarity. (B) Alignment of CRM domains in CAF1, CAF2 and CRS1 with E.coli YhbY. Residue numbers indicate the position of each CRM domain within the context of the full-length protein. The complete YhbY sequence is shown.

Fig. 2. Alignment of CAF1 and CAF2 with predicted orthologs in rice and Arabidopsis. Identical residues are shaded in black and similar residues in gray. The predicted cleavage sites of the chloroplast targeting sequences are indicated by diamonds. Double arrowheads show the starting points of truncated CAFs identified in the yeast two-hybrid screen. The positions of the Mu insertions in the caf mutants analyzed in Figure 5 are shown by inverted triangles. Alignments were calculated with ClustalW (Higgins et al., 1994) and shaded with BOXSHADE. Accession Nos: rice CAF1, BAC05662; rice CAF2, BAB21243; Arabidopsis CAF1, At2g20020; Arabidopsis CAF2, At1g23400. The CAF1 and CAF2 sequences have been deposited in GenBank under accession Nos AY264368 and AY264369, respectively.

Fig. 6. Co-immunoprecipitation of group II intron RNA with CAF1 and CAF2 from chloroplast extract. Chloroplast stroma was subject to immunoprecipitation with the indicated affinity-purified antisera. RNA was extracted from the immunoprecipitate pellet and supernatant (Sup) and applied to a nylon membrane with a slot-blot manifold. Duplicate slot-blots were hybridized with probes specific for the indicated intron. Negative controls included a mock precipitation (no Ab), and precipitation with antisera to OE23, which is a component of the photosynthetic apparatus that does not bind RNA. RNA extracted from total stroma was applied to one slot on each blot as a positive hybridization control.

Fig. 7. Summary of intron targets of four host-encoded group II intron splicing factors in maize chloroplasts. (A) The maize chloroplast group II introns and their requirements for CRS1, CRS2, CAF1 and CAF2. Requirements were deduced from the splicing defects detected in crs1, crs2, caf1 and caf2 mutants. The crs1 and crs2 mutant data are from Jenkins et al. (1997) and Vogel et al. (1999). The caf mutant data are from this work. Although crs2-1 and caf2-1 mutants exhibit defects in subgroup IIA splicing, these are thought to be indirect effects of their plastid ribosome deficiency and are therefore designated as (NO) in this table. (B) Diagram summarizing both the genetic and biochemical intron specificities of CRS2–CAF1 and CRS2–CAF2 complexes. Arrows point to the set of introns that require either CAF1 or CAF2 to splice in vivo. Superscripts indicate whether CAF1 antiserum (superscript 1) or CAF2 antiserum (superscript 2) strongly co-immunoprecipitated the indicated intron. The hash sign indicates that the rpl16 intron was poorly precipitated by both antisera.

Fig. 1. Identification of CAF1 and CAF2. (A) CRS2 interacts with CAF1 and CAF2 in a yeast two-hybrid assay. Yeast strains were streaked on plates containing (left) and lacking histidine (right). Growth in the absence of histidine indicates a positive two-hybrid interaction. Cells contained plasmids encoding fusion proteins to the Gal4 DNA-binding domain (BD) and transcription activation domain (AD), as follows: (1) BD and AD fused to a λcI fragment that homodimerizes (positive interaction control); (2) BD–CRS2 and AD–cI (negative control); (3) BD–cI and AD–CAF1 (negative control); (4) BD–CRS2 and AD–CAF1; (5) BD–cI and AD–CAF2 (negative control); and (6) BD–CRS2 and AD–CAF2. (B) RNA gel blot hybridization showing the size of the caf1 and caf2 mRNAs. Lanes contain 0.5 µg of poly(A)-enriched RNA from wild-type seedling leaf (wt poly-A), or 20 µg of total seedling leaf RNA from wild-type, caf1 mutant or caf2 mutant. The upper panel was probed with a caf1 gene-specific probe; the lower panel was probed with a caf2 gene-specific probe. The position of a 2.37 kb RNA marker is indicated.

Fig. 5. RNase protection analysis of group II intron splicing defects in caf1 and caf2 mutants. The RNase protection probes used here have been described previously (Jenkins et al., 1997). They spanned either the 5′- or the 3′-splice junction. Total leaf RNA from wild-type (wt) or the indicated mutant line was analyzed. The crs2-1 allele analyzed here conditions defects in both subgroup IIA and subgroup IIB splicing (Jenkins et al., 1997). tRNA indicates reactions in which tRNA was substituted for leaf RNA. Unspliced RNA protects both intron and exon probe sequence (U), whereas spliced RNA protects exon sequence (S) and, in some cases, excised intron sequence (S-intron). (A) Schematic of a 3′-splice site probe. (B) Representative CAF1-dependent introns. (C) Representative CAF2-dependent introns. (D) The ycf3 introns. ycf3-1 requires both CAF1 and CAF2 for splicing. The ycf3-2 intron, which is the only subgroup IIB intron that does not require CRS2, likewise does not require CAF1 or CAF2. (E) Representative subgroup IIA introns. These introns are not spliced in mutants such as iojap, with severe plastid ribosome deficiencies. Evidence presented in Jenkins et al. (1997) suggested that the subgroup IIA splicing defect in the crs2-1 allele is a pleiotropic effect of its plastid ribosome loss. Intron co-immunoprecipitation data presented in Figure 6 suggest the same is true for caf2 mutants.