WTF1 is associated with intron RNAs in chloroplast extract. RNA purified from the pellets and supernatants of immunoprecipitations with antisera to WTF1 or OE16 was applied to slot blots and hybridized with the indicated probes. All probes were intron-specific, except that for trnR, which lacks introns. One tenth of the RNA from each immunoprecipitation supernatant (Sup) and one fifth of the RNA from the corresponding pellet (Pel) was analyzed with each probe.

Mutant alleles of wtf1. (A) Mu transposon insertions in the wtf1 gene. The wtf1 ORF lacks introns and is indicated by a rectangle. (B) Phenotypes of wtf1 mutants. Plants indicated by 2 alleles are the heteroallelic progeny of complementation crosses. (C) Loss of WTF1 protein in wtf1 mutant chloroplasts. Chloroplasts purified from seedlings of the indicated genotypes were analyzed on immunoblots with anti-WTF1 antiserum. Cpn60 was used as a loading control. Strong wtf1 alleles could not be analyzed in this manner because plastids cannot be purified in sufficient quantity from albino plants.

WTF1 forms a stable complex with RNC1. (A) Amylose pull-down assay. Recombinant RNC1, MBP, and/or MBP-WTF1 were combined as indicated (Input), and incubated with amylose affinity beads. Material retained on the beads after washing (Pulldown) was assayed by SDS/PAGE. (B) Gel filtration interaction assays. Elution of proteins from a Superdex 200 column was monitored by SDS/PAGE and Coomassie staining. Shown from top to bottom are the elution of: TEV protease-cleaved MBP-WTF1; TEV-protease cleaved MBP-RNC1; a preincubated mixture of MBP-WTF1 and RNC1; the pooled fractions indicated in the box, cleaved with TEV-protease and then applied again to the column. Fraction numbers and the positions of size standards are indicated at top.

Gel-mobility shift assays demonstrating RNA-binding activity of recombinant WTF1 and DUF860. The proteins indicated at top were incubated with (A) a 141-nt RNA from the petB intron, or (B) a 31-nt RNA from the petA gene. The latter RNA was either heated and snap-cooled to maintain it in single-stranded form (at left, SS), or annealed to its complement (at right, DS). Proteins were used in serial 2-fold dilutions (maximum concentrations of 1 μM for MBP and MBP-WTF1, and 2 μM for MBP-DUF860). RNAs were present at 20 pM. Bound (B) and unbound (U) RNAs were resolved on native polyacrylamide gels. (C) Purity of MBP-DUF860 used for RNA-binding assays. Consecutive fractions from the gel filtration column used as the final purification were analyzed by SDS/PAGE and staining with Coomassie Blue. The purity of MBP-WTF1 is shown in Fig. 6.

Nucleus-encoded proteins that promote chloroplast group II intron splicing in angiosperms. Introns are designated as subgroup IIA or IIB, according to ref. 35. Introns found in Arabidopsis but not in maize are marked with asterisks. Splicing factors are shown to the outside, annotated with their conserved domains. Where analyzed (9, 10, 28), functions are conserved between monocots and dicots. Results are summarized from this work and from refs. 7–12, 14, 16, 17, and 36. Not shown are WHY1, which associates with and stimulates splicing of the atpF intron (15), and HCF152, which is required for the accumulation of spliced petB RNA but not its excised intron (37). In addition to its primary role in ycf3-2 splicing, OTP51 stimulates the splicing of the atpF, trnV, and trnK introns (8).

Splicing defects in wtf1 mutants. Assays used seedling leaf RNA from plants of the indicated genotypes. Introns are designated as subgroup IIA or IIB, according to ref. 35. (A) Poisoned-primer extension assays. Oligonucleotides complementary to exon sequences near the 3′-splice junction of the indicated introns were used to prime reverse transcription in the presence of a dideoxynucleotide that terminates cDNA synthesis after different distances on spliced and unspliced templates. The asterisk marks a product terminating at the branchpoint adenosine formed during the first splicing step. (B) RNA gel blots probed with exon sequences from the tRNA gene indicated at bottom. Asterisks identify unspliced precursors. (C) Ribonuclease-protection assay. The probe was body-labeled and spanned the splice junction diagrammed below. The lengths in nucleotides of probe segments corresponding to intron and exon are indicated in the diagram. S, spliced; U, unspliced; pg, pale green seedlings; iv, ivory seedlings.

WTF1 is associated with splicing factors in chloroplast stroma. (A) Chloroplasts (Cp) and chloroplast subfractions (Thy, thylakoid; Env, envelope) are from the fractionated chloroplast preparation described previously (30); material in each lane is derived from the same quantity of chloroplasts. The blot was reprobed to detect mitochondrial MDH and the thylakoid protein D1. The Ponceau S-stained blot below illustrates the distribution of the stromal protein RbcL and the loading of the leaf and mitochondrial (Mito) samples. (B) Coimmunoprecipitation of WTF1 with chloroplast splicing factors. Stromal extract was used for immunoprecipitations with the antibodies listed at top. Immunoprecipitates were analyzed by immunoblot analysis with the antibodies listed at left. (C) Cosedimentation of WTF1 with intron RNPs. Stromal extract was fractionated by sucrose gradient sedimentation. An equal proportion of each fraction and of the pelleted material (P) was analyzed by probing immunoblots with the antibodies indicated at left. The ribosomal protein Rpl2 marks the position of ribosomes. RbcL marks the position of Rubisco (≈550 kDa). The WTF1, RNC1, CAF1, and CAF2 peaks coincide with those of CRS1, CFM3, and group II intron RNAs in analogous assays (9, 10, 12, 13).