Site-recognition of RNA editing in chloroplasts

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C to U RNA editing occurs in transcripts from higher plant chloroplast transcripts at specific sites. This editing is observed one C out of about 1,000 Cs in the transcripts.1. Improvement of chloroplast preparationTo isolate a sizable amount of trans-factors, rapid and economic procedure for preparing chloroplasts was devised using sucrose-gradients. Useful advices were come from our German colleagues.2. Improvement of the in vitro RNA editing systemOur previous in vitro system requires ^<32>P-labeled mRNA substrates. As the synthesis of ^<32>P-labeled mRNAs was tedious and time-consuming, we developed a non-RI in vitro system with fluorescent nucleotides using a primer extension method. Using the new system, we assayed editing activities for tobacco chloroplast ndh mRNAs. The activity varies from ca. 30% (ndh2) to not detectable. Cis-sequences for ndh-2 and ndhF mRNA editing were then identified using mutated mRNAs. The cis-sequence for ndh-2 resides 10 to 6 nt upstream from AUG and that for ndhF does 40 to 36 nt and 15 to 6 nt upstream from AUG. As ndhF mRNA has two cis-sequences, we proposed a new model for RNA editing.3. Isolation of a recognition factor for editingAs the highest editing activity was observed for psbE mRNA, the corresponding 56 kDa trans-factor was isolated from 12 kg tobacco green leaves using ammonium sulfate fractionation, heparin column, gel filtration and SDS gel electrophoresis. The 56 kDa band was excised and subjected to MS/MS analysis. Several amino acid sequences were obtained. This protein is likely to be a new protein unique to tobacco since no similar sequence was found by BLAST search.4. Identification of additional trans-factorsUsing UV crosslinking, trans-factors for ndhF and rpoB mRNA editing were identified to be 82 kDa and 60 kDa, respectively.