The Nucleus-Encoded HCF107 Gene of Arabidopsis Provides a Link between Intercistronic RNA Processing and the Accumulation of Translation-Competent psbH Transcripts in Chloroplasts
The Nucleus-Encoded HCF107 Gene of Arabidopsis Provides a Link between Intercistronic RNA Processing and the Accumulation of Translation-Competent psbH Transcripts in Chloroplasts
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DOI：
10.1105/tpc.010090
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发表时间：
2001-09
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通讯作者：
Susanne Felder;K. Meierhoff;A. P. Sane;J. Meurer;C. Driemel;H. Plücken;P. Klaff;B. Stein;N. Bechtold;P. Westhoff
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作者：
Susanne Felder;K. Meierhoff;A. P. Sane;J. Meurer;C. Driemel;H. Plücken;P. Klaff;B. Stein;N. Bechtold;P. Westhoff
为了了解RNA加工对质体编码的光合作用基因表达的功能意义，我们研究了拟南芥核突变hcf 107。该突变由两个等位基因表示，这两个等位基因都导致缺陷的光系统II（PSII）。在体内蛋白质标记，体外磷酸化，和免疫印迹实验表明，psbB基因产物（CP 47）和8 kD磷蛋白，psbH基因产物（PsbH），是不存在的突变体植物。PsbH和PsbB是光合真核生物中PSII组装的基本要求，它们在hcf 107中的缺失与PSII较少的突变表型一致。RNA凝胶印迹杂交显示，hcf 107突变特异性地损害了通过基因间核酸内切裂解从五顺反子psbB-psbT-psbH-petB-petD前体RNA释放的一些但不是全部的寡顺反子psbH转录物的积累。相反，无论是水平还是大小的psbB含有RNA的影响。S1核酸酶保护分析显示，psbH RNA仅在psbH是前导顺反子的地方缺失，并且它们在psbH的5′前导片段的−45位被加工。这些数据和对细胞色素b6 f复合物突变体hcf 152（3′ psbH加工缺陷）的额外实验表明，只有那些在−45 5 5′端加工的含有psbH的转录物才能被翻译。对5′ psbH前导序列的二级结构分析预测，在未加工的转录本中会形成稳定的茎环，这些茎环通过在−45位点加工而展开。我们建议，这种展开的psbH前导片段作为RNA加工的结果是必不可少的psbH阅读框架的翻译。我们进一步认为HCF 107具有双重功能：它参与psbH 5′非翻译区的顺反子间加工或5′加工psbH RNA的稳定化，同时，它也是合成CP 47所必需的。
To understand the functional significance of RNA processing for the expression of plastome-encoded photosynthesis genes, we investigated the nuclear mutation hcf107 of Arabidopsis. The mutation is represented by two alleles, both of which lead to a defective photosystem II (PSII). In vivo protein labeling, in vitro phosphorylation, and immunoblot experiments revealed that the psbB gene product (CP47) and an 8-kD phosphoprotein, the psbH gene product (PsbH), are absent in mutant plants. PsbH and PsbB are essential requirements for PSII assembly in photosynthetic eukaryotes, and their absence in hcf107 is consistent with the PSII-less mutant phenotype. RNA gel blot hybridizations showed that the hcf107 mutation specifically impairs the accumulation of some but not all oligocistronic psbH transcripts that are released from the pentacistronic psbB-psbT-psbH-petB-petD precursor RNA by intergenic endonucleolytic cleavage. In contrast, neither the levels nor the sizes of psbB-containing RNAs are affected. S1 nuclease protection analyses revealed that psbH RNAs are lacking only where psbH is the leading cistron and that they are processed at position −45 in the 5′ leader segment of psbH. These data and additional experiments with the cytochrome b6f complex mutant hcf152, which is defective in 3′ psbH processing, suggest that only those psbH-containing transcripts that are processed at their −45 5′ ends can be translated. Secondary structure analysis of the 5′ psbH leader predicted the formation of stable stem loops in the nonprocessed transcripts, which are unfolded by processing at the −45 site. We propose that this unfolding of the psbH leader segment as a result of RNA processing is essential for the translation of the psbH reading frame. We suggest further that HCF107 has dual functions: it is involved in intercistronic processing of the psbH 5′ untranslated region or the stabilization of 5′ processed psbH RNAs, and concomitantly, it is required for the synthesis of CP47.