Site-recognition of RNA editing in chloroplasts

从C到U的RNA编辑发生在高等植物叶绿体转录本的特定位置。在转录本中观察到这种编辑是大约1,000个C中的一个C。叶绿体制备的改进为了分离大量的反式因子，利用蔗糖梯度设计了快速而经济的叶绿体制备方法。我们的德国同事提供了一些有用的建议。体外RNA编辑系统的改进我们以前的体外系统需要^&lt；32&gt；P标记的mRNA底物。针对~&lt；32&gt；P标记的mRNAs合成过程繁琐耗时的问题，我们采用了一种引物延伸的方法，建立了一种非RI的体外荧光核苷酸检测系统。使用新的系统，我们检测了烟草叶绿体NDH mRNAs的编辑活性。活性从大约30%(Ndh2)到检测不到。然后使用突变的mRNAs鉴定用于NDH-2和ndhF mRNA编辑的顺式序列。NDH-2的顺式序列自8月起位于上游10至6个核苷酸，而ndhF的顺式序列自8月起位于上游40至36个核苷酸和15至6个核苷酸。由于ndhF基因有两个顺式序列，我们提出了一种新的RNA编辑模型。经硫酸铵分级、肝素柱层析、凝胶过滤和十二烷基硫酸钠凝胶电泳法，从12公斤烟叶中分离得到56 kDa的反式转录因子。提取56 kDa条带，进行MS/MS分析。获得了几个氨基酸序列。该蛋白很可能是烟草特有的一种新蛋白，因为BLAST搜索没有发现类似的序列。额外反式因子的鉴定利用紫外光交联，ndhF和rpoB mRNA编辑的反式因子分别为82 kDa和60 kDa。

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.