A site-specific factor interacts directly with its cognate RNA editing site in chloroplast transcripts

Abstract

RNA editing involves a variety of genetic systems and occurs by different mechanisms. In higher plant chloroplasts, specific sites of some transcripts are subject to C-to-U conversion. We have previously shown that site-specific trans-acting factors for psbE and petB mRNA editing bind corresponding cis-elements, which are located 5 nucleotides upstream from the editing site. Here we report that, by using mRNAs labeled either at the center of the upstream cis-element or at the editing site, the site-specific factors can be cross-linked with nucleotides at both positions. Mutations of nucleotides in the proximal region of the editing site revealed a correlation between editing activity and cross-linking efficiency of factors with the editing site, even though cross-linking with the upstream cis-element was unaffected. These observations suggest that the site-specific factor binds stably to the upstream ciselement, whereas it interacts weakly with the editing site. This finding raises the intriguing possibility that the site-specific factor is involved in both site-determination and C-to-U conversion in chloroplast RNA editing.

Fig. 1.

Cis-analysis in the proximal region of psbE and petB mRNAs. (A) Schematic representation of altered regions in mRNA substrates (Left, psbE mRNA; Right, petB mRNA). The top sequence represents a portion of the wild-type mRNAs (19), and mutated residues are shown below. (B) Editing activity of mutated mRNA substrates. One femtomole of each mutated mRNA was incubated with a chloroplast extract as described in Materials and Methods, and resulting ³²P-mononucleotides were separated by TLC. (C) Competition analysis using the mutated mRNAs as competitors. One femtomole of each ³²P-labeled wild-type mRNA substrate was incubated with 1 pmol of each unlabeled mutated mRNA as competitor. U, marker pU; W, wild-type mRNA; E, psbE wild-type mRNA as competitor; B, petB wild-type mRNA as competitor; –Ex, without chloroplast extract; 0, without competitor.

Fig. 2.

Preference for the 5′ neighboring residue for psbE mRNA (Left) and petB mRNA (Right) editing. The –1 residue of psbE and petB mRNAs was changed to three other nucleotides. Editing activities of mutated RNA substrates were assayed as in Fig. 1.

Fig. 3.

UV cross-linking with the upstream cis-element or the editing site of psbE and petB mRNAs. (A) Schematic representation of mRNA substrates with ³²P-labels (marked by asterisks) at the center of the upstream cis-element (underlined) or at the editing site (C). (B) Gel patterns of proteins bound to the upstream cis-element and the editing site. (Left) psbE mRNA. (Right) perB mRNA. Each mRNA was incubated for 1 h at 28°C in the editing reaction mixture and then UV-irradiated (254 nm). The mixture was treated with a mixture of RNase A, nuclease P1, and C. adamanteus venom phosphodiesterase I followed by SDS/PAGE. Protein size markers (Rainbow, Amersham Pharmacia) are shown between the two gel patterns.

Fig. 4.

Determination of the sequence involved in p56 binding to psbE mRNA. Schematic representation of altered regions of psbE mRNAs used as competitors are shown above. One femtomole of the wild-type psbE RNA labeled at the +1 residue was used as a substrate, and 1 pmol of each competitor was added. Cross-linking assays were as described in Fig. 3. 0, without competitor.

Fig. 5.

Effect of mutations in neighboring residues of the editing site in psbE mRNAs on the cross-linking of p56 with the editing site or the upstream cis-element. UV cross-linking experiments were performed by using psbE mRNAs with mutations in 5′ and 3′ neighboring residues (–1 and +2). (A) Assays with mRNAs labeled at the editing site (+1). (B) Assays with mRNAs labeled at the center of the upstream cis-element (–10).

Fig. 6.

Model of chloroplast RNA editing. First, a site-specific trans-acting factor binds an upstream cis-element in a sequence-specific manner. Based on this binding, the factor then interacts with an editing site also in a sequence-specific manner. This interaction is most likely necessary for C-to-U conversion, possibly by allowing contact of the putative catalytic domain of the site-specific factor with the target C residue.