2004 Fiscal Year Final Research Report Summary
Site-recognition of RNA editing in chloroplasts
| Project/Area Number |
15370025
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
植物生理・分子
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| Research Institution | Nagoya City University |
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Principal Investigator |
SUGIURA Masahiro Nagoya City Univ., Graduate School of Natural Sciences, Prof., 大学院・システム自然科学研究科, 教授 (80027044)
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| Co-Investigator(Kenkyū-buntansha) |
SAKURAI Norihiko Nagoya City Univ., Graduate School of Natural Sciences, Assistant Prof., 大学院・システム自然科学研究科, 講師 (00255233)
OBOKATA Junichi Nagoya Univ., Gene Research Center, Associate Prof., 遺伝子実験施設, 助教授 (50185667)
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| Project Period (FY) |
2003 – 2004
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| Keywords | RNA editing / mRNA / in vitro / cis-sequence / trans-factor / fluorescent-label / tobacco |
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| Research Abstract |
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 preparation
To 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 system
Our 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 editing
As 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-factors
Using UV crosslinking, trans-factors for ndhF and rpoB mRNA editing were identified to be 82 kDa and 60 kDa, respectively.
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