A simple in vitro RNA editing assay for chloroplast transcripts using fluorescent dideoxynucleotides: distinct types of sequence elements required for editing of ndh transcripts.
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 (32)P 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.
Sasaki
Tadamasa
T
Graduate School of Natural Sciences, Nagoya City University, Yamanohata, Mizuho, Nagoya 467-8501, Japan.
Yukawa
Yasushi
Y
Wakasugi
Tatsuya
T
Yamada
Kyoji
K
Sugiura
Masahiro
M
D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000737:chemistry; D003848:Deoxyguanine Nucleotides / Q000737:chemistry; D003854:Deoxyribonucleotides / Q000737:chemistry*; D054306:Dideoxynucleotides; D005456:Fluorescent Dyes / Q000737:chemistry; D040461:Gene Components; D009245:NADH Dehydrogenase / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D017393:RNA Editing; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D018749:RNA, Plant / Q000737:chemistry / Q000378:metabolism*; D038621:Regulatory Sequences, Ribonucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017423:Sequence Analysis, RNA; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D054306:Dideoxynucleotides; D005456:Fluorescent Dyes; D010940:Plant Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D038621:Regulatory Sequences, Ribonucleic Acid; C037773:2',3'-dideoxyadenosine triphosphate; C031709:2',3'-dideoxyguanosine 5'-triphosphate; D009245:NADH Dehydrogenase