Identification of an RNA-protein complex involved in chloroplast group II intron trans-splicing in Chlamydomonas reinhardtii

Abstract

In Chlamydomonas reinhardtii, the psaA mRNA is assembled by a process involving trans-splicing of separate transcripts, encoded at three separate loci of the chloroplast genome. At least 14 nuclear loci and one chloroplast gene, tscA, are needed for this process. We have cloned Raa3, the first nuclear gene implicated in the splicing of intron 1. The predicted sequence of Raa3 consists of 1783 amino acids and shares a small region of homology with pyridoxamine 5'-phosphate oxidases. Raa3 is present in the soluble fraction of the chloroplast and is part of a large 1700 kDa complex, which also contains tscA RNA and the first psaA exon transcript. These partners, in association with other factors, form a chloroplast RNP particle that is required for the splicing of the first intron of psaA and which may be the counterpart of eukaryotic snRNPs involved in nuclear splicing.

Fig. 1. Genomic complementation of M18. (A) Mapping of Raa3. Cosmid represents the entire cosmid of ∼40 kb isolated from the indexed cosmid library that is able to rescue the M18 mutant. HSl9 is a HindIII–SalI subfragment of the cosmid. HSl9SA is the longer SacI subfragment of HSl9. ΔNco is an NcoI subclone of HSl9SA. cDNAs-B and -A are two independently isolated cDNAs (see Materials and methods). (B) Photoautotrophic growth and photosensitivity of M18 and the rescued strains obtained by transformation of M18 with the constructs described in (A). GHA3-6 is the M18 strain transformed with a HSl9SA fragment containing the entire sequence of Raa3 and an HA tag at the 3′-terminal end of the coding sequence. + and – indicate normal and absent growth, respectively. DL, dim light (6 µE/m²s); ML, medium light (60 µE/m²s); HL, high light (600 µE/m²s); TAP, Tris–acetate–phosphate medium; HSM, high salt minimal medium. (C) Immunoblot analysis. Total cell proteins (10 µg) from the indicated strains were separated by SDS–PAGE, blotted and decorated with PsaA and RbcL antibodies. (D) RNA blot analysis. The blots were hybridized with the three different psaA exon probes and RbcS as a loading control. The sizes of the major transcripts are indicated: the 2.7, 3.8, 2.4 and 0.4 kb bands correspond to the mature psaA mRNA, the exon 2–exon 3 precursor (containing the 3′ end of intron 1), the exon 3 precursor and the exon 1 precursor, respectively.

Fig. 2. Raa3 sequence. (A) Deduced amino acid sequence of Raa3. The putative transit peptide is framed and the domain related to pyridoxamine 5′ phosphate oxidases is shaded. The asterisk indicates the site of the mutation of M18. The black arrow and the black inverted triangle correspond to the 5′ ends of the ΔNco fragment and of cDNA-A, respectively. The sites corresponding to the four introns are indicated by open inverted triangles. (B) Amino acid comparison of the homologous domains of pyridoxamine 5′ phosphate oxidases and Raa3. Sequences were aligned using ClustalW (gap open 10; gap ext 0.2; Blosum matrix). * = conserved residues (shaded), : = conservative substitutions, ⋅ = semi-conservative substitution (Thompson et al., 1994). Accession Nos for PDXs are Zymomonas mobilis: AF179611; yeast, P38075; Mycobacterium leprae, O33065; E.coli, P28225; Myxococcus xanthus, P21159; Synechocystis PCC 6803, P74211; O74250.

Fig. 3. Accumulation and subcellular localization of Raa3. (A) Immuno blot analysis of Raa3 in wild-type and M18 rescued with the genomic (cosmid and ΔNcoI) and cDNA (cDNA-A and -B) clones. The antibody used was directed against recombinant Raa3. Dots indicate Raa3 and its truncated versions. The arrowhead indicates a non-specific band that is also present in M18. (B) Immunoblot analysis of Raa3 in whole-cell extracts from wild-type and M18 and from cell fractions of M18 transformed with the Raa3-HA construct (total cell, intact chloroplasts, soluble and insoluble chloroplast fractions) prepared as described in Materials and methods. To detect Raa3, 100 µg of proteins were loaded on a 6% polyacrylamide gel and decorated with αHA. For PsaA, RbcL and eIF4A detection, 10 µg of proteins were loaded on a 10% polyacrylamide gel and decorated with the corresponding antibodies.

Fig. 4. Raa3 is part of a multimolecular complex. (A) Immunoblot analysis of cell extracts of GHA3-6 fractionated by size exclusion chromatography. A soluble fraction (see Materials and methods) was prepared in the presence of 0.5 mg/ml heparin (+heparin), or incubated on ice without heparin and with 100 µg/ml RNase (+ RNase), and subjected to size exclusion chromatography. Part of each fraction (90%) was electrophoresed on a 6% polyacrylamide gel, blotted and reacted with HA antiserum. Molecular weight size standards are included. (B) The remaining part of the fractions (10%) was electrophoresed on a 10% polyacrylamide gel, blotted and decorated with antibodies directed against RB60 and RbcL. (C) Immunoblot analysis of total cell extracts from wild type (with dilution series) and representatives from the different class C and class B psaA complementation groups probed with Raa3 and RbcL antibodies.

Fig. 5. The Raa3 complex includes tscA RNA and the psaA exon 1 transcript. (A) RNA blot analysis of cell extracts from GHA3-6 and M18 fractionated by size exclusion chromatography. Soluble extracts were prepared in the presence of heparin (0.5 mg/ml). The RNA was separated on a 1.25% formamide–agarose gel, blotted and hybridized with labelled probes of tscA, psaA exon1 and atpH. The absence of an RNA signal in fraction 12 of GHA3-6 ΔtscA is due to the accidental loss or degradation of the RNA of this strain during the lengthy preparation procedure. (B and C) Immunoblot analysis of cell extracts of the GHA3-6 strain and its derivative lacking tscA (GHA3-6 ΔtscA). Samples were handled as described in Figure 4. Blots were decorated with antibodies against HA and RbcL.