Gott Jonatha M, Parimi Neeta, Bundschuh Ralf
Center for RNA Molecular Biology, Case Western Reserve University Cleveland, OH 44106, USA.
Nucleic Acids Res. 2005 Sep 7;33(16):5063-72. doi: 10.1093/nar/gki820. Print 2005.
Gene finding is complicated in organisms that exhibit insertional RNA editing. Here, we demonstrate how our new algorithm Predictor of Insertional Editing (PIE) can be used to locate genes whose mRNAs are subjected to multiple frameshifting events, and extend the algorithm to include probabilistic predictions for sites of nucleotide insertion; this feature is particularly useful when designing primers for sequencing edited RNAs. Applying this algorithm, we successfully identified the nad2, nad4L, nad6 and atp8 genes within the mitochondrial genome of Physarum polycephalum, which had gone undetected by existing programs. Characterization of their mRNA products led to the unanticipated discovery of nucleotide deletion editing in Physarum. The deletion event, which results in the removal of three adjacent A residues, was confirmed by primer extension sequencing of total RNA. This finding is remarkable in that it comprises the first known instance of nucleotide deletion in this organelle, to be contrasted with nearly 500 sites of single and dinucleotide addition in characterized mitochondrial RNAs. Statistical analysis of this larger pool of editing sites indicates that there are significant biases in the 2 nt immediately upstream of editing sites, including a reduced incidence of nucleotide repeats, in addition to the previously identified purine-U bias.
在表现出插入式RNA编辑的生物体中，基因发现过程较为复杂。在此，我们展示了我们的新算法——插入编辑预测器（PIE），它可用于定位其mRNA经历多次移码事件的基因，并将该算法扩展到包括对核苷酸插入位点的概率预测；在设计用于测序编辑后RNA的引物时，这一特性特别有用。应用此算法，我们成功鉴定了多头绒泡菌线粒体基因组中的nad2、nad4L、nad6和atp8基因，这些基因在现有程序中未被检测到。对其mRNA产物的表征意外发现了多头绒泡菌中的核苷酸缺失编辑。通过对总RNA进行引物延伸测序，证实了导致三个相邻A残基缺失的缺失事件。这一发现意义非凡，因为它是该细胞器中已知的首例核苷酸缺失，与之形成对比的是，在已表征的线粒体RNA中有近500个单核苷酸和双核苷酸添加位点。对这一更大编辑位点库的统计分析表明，除了先前确定的嘌呤-U偏向之外，编辑位点上游紧邻的2个核苷酸存在显著偏向，包括核苷酸重复发生率降低。
