simple in vitro RNA editing assay for chloroplast transcripts using fluorescent dideoxynucleotides: distinct types of sequence elements required for editing of ndh transcripts

• Published in: The Plant journal : for cell and molecular biology 2006-09, Vol.47 (5), p.802-810
• Main Authors: Sasaki, Tadamasa, Yukawa, Yasushi, Wakasugi, Tatsuya, Yamada, Kyoji, Sugiura, Masahiro
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; chloroplast; Chloroplasts - genetics; Chloroplasts - metabolism; cis‐element; Deoxyadenine Nucleotides - chemistry; Deoxyguanine Nucleotides - chemistry; Deoxyribonucleotides - chemistry; Dideoxynucleotides; Fluorescent Dyes - chemistry; Fundamental and applied biological sciences. Psychology; Gene Components; Gene expression; in vitro; Molecular and cellular biology; Molecular genetics; NADH Dehydrogenase - genetics; NADH Dehydrogenase - metabolism; ndh mRNA; Nicotiana - genetics; Nicotiana - metabolism; Plant Proteins - genetics; Plant Proteins - metabolism; Regulatory Sequences, Ribonucleic Acid; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA Editing; RNA, Messenger - chemistry; RNA, Messenger - metabolism; RNA, Plant - chemistry; RNA, Plant - metabolism; Sequence Analysis, RNA; tobacco
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313x.2006.02825.x

RNA editing is found in various transcripts from land plant chloroplasts. In tobacco chloroplasts, C-to-U conversion occurs at 36 specific sites including two sites identified in this work. Our RNA editing assay system using chloroplast extracts facilitated biochemical analyses of editing reactions but required mRNAs labeled with ³²P at specific sites. Here, we have improved the in vitro system using fluorescence-labeled chain terminators, ddGTP and ddATP, and have measured the editing activity at 19 sites in ndh transcripts. Editing activities varied from site to site. It has been reported that one editing site in ndhA mRNAs is present in spinach but absent in tobacco, but a corresponding editing capacity had been found in vivo in tobacco using biolistic transformation. We confirmed biochemically the existence of this activity in tobacco extracts. Using the non-radioactive assay, we examined sequences essential for editing within a 50-nt mRNA region encompassing an editing site. Editing of the ndhB-2 site requires a short sequence in front of the editing site, while that of the ndhF mRNA requires two separate regions, a sequence surrounding the editing site and a 5' distal sequence. These results suggest that distinct editing mechanisms are present in chloroplasts.