A DYW-protein knockout in Physcomitrella affects two closely spaced mitochondrial editing sites and causes a severe developmental phenotype
A DYW-protein knockout in Physcomitrella affects two closely spaced mitochondrial editing sites and causes a severe developmental phenotype
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DOI：
10.1111/tpj.12304
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发表时间：
2013-11-01
期刊：
影响因子：
7.2
通讯作者：
Knoop, Volker
中科院分区：
文献类型：
--
作者：
Schallenberg-Ruedinger, Mareike;Kindgren, Peter;Knoop, Volker
RNA结合五肽重复序列(PPR)携带类似胞苷脱氨酶的羧基末端dyw结构域，是植物细胞器中C-to-U RNA编辑的位点特异性因子。在本研究中，我们报道了Patens中Dyw-PPR_65的敲除导致苔藓严重的发育表型，并特异性地影响线粒体ccmFC mRNA上相距18个核苷酸的两个编辑位点。有趣的是，PPR_71，另一种dyw类型的PPR，以前被确定为仅影响下游编辑站点ccmFCeU122SF的编辑因子。现在表征的PPR_65仅与上游靶点ccmFCeU103PS特异性结合，与最近用于PPR阵列的RNA识别代码完全一致。这两个编辑事件之间的功能干扰可能是由三个因素共同引起的：(I)PPR_65先前结合干扰PPR_71结合的RNA二级结构的不稳定；(Ii)由此产生的上游C-U转换；或(Iii)两个dyw蛋白之间的直接相互作用。事实上，我们发现Physcomitrella dyw-Pprs在酵母双杂交试验中相互作用。苔藓DYW-PPRS还与拟南芥的Morf(多器官RNA编辑因子)/RIP(RNA编辑因子相互作用蛋白)蛋白质相互作用更强，这些蛋白质是开花植物中的通用编辑因子，尽管MOSS中完全没有Morf同源基因。最后，我们展示了Physcomitrella dyw-PPR_98与其编辑位点atp9eU92SL上游的预测靶序列的结合，该序列不能产生KO线。结合dyw-PPR_65的功能鉴定，完成了对Physcomitrella线粒体转录组中所有编辑位点的RNA编辑因子的分配。
RNA-binding pentatricopeptide repeat (PPR) proteins carrying a carboxy-terminal DYW domain similar to cytidine deaminases have been characterized as site-specific factors for C-to-U RNA editing in plant organelles. Here we report that knockout of DYW-PPR_65 in Physcomitrella patens causes a severe developmental phenotype in the moss and specifically affects two editing sites located 18 nucleotides apart on the mitochondrial ccmFC mRNA. Intriguingly, PPR_71, another DYW-type PPR, had been identified previously as an editing factor specifically affecting only the downstream editing site, ccmFCeU122SF. The now characterized PPR_65 binds specifically only to the upstream target site, ccmFCeU103PS, in full agreement with a recent RNA-recognition code for PPR arrays. The functional interference between the two editing events may be caused by a combination of three factors: (i) the destabilization of an RNA secondary structure interfering with PPR_71 binding by prior binding of PPR_65; (ii) the resulting upstream C-U conversion; or (iii) a direct interaction between the two DYW proteins. Indeed, we find the Physcomitrella DYW-PPRs to interact in yeast-two-hybrid assays. The moss DYW-PPRs also interact yet more strongly with MORF (Multiple Organellar RNA editing Factor)/RIP (RNA editing factor interacting proteins) proteins of Arabidopsis known to be general editing factors in flowering plants, although MORF homologues are entirely absent in the moss. Finally, we demonstrate binding of Physcomitrella DYW-PPR_98, for which no KO lines could be raised, to its predicted target sequence upstream of editing site atp9eU92SL. Together with the functional characterization of DYW-PPR_65, this completes the assignment of RNA editing factors to all editing sites in the Physcomitrella mitochondrial transcriptome.