Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
Plant Cell. 2013 Jun;25(6):2265-75. doi: 10.1105/tpc.113.111567. Epub 2013 Jun 4.
The profiling of ribosome footprints by deep sequencing has revolutionized the analysis of translation by mapping ribosomes with high resolution on a genome-wide scale. We present a variation on this approach that offers a rapid and cost-effective alternative for the genome-wide profiling of chloroplast ribosomes. Ribosome footprints from leaf tissue are hybridized to oligonucleotide tiling microarrays of the plastid ORFeome and report the abundance and translational status of every chloroplast mRNA. Each assay replaces several time-consuming traditional methods while also providing information that was previously inaccessible. To illustrate the utility of the approach, we show that it detects known defects in chloroplast gene expression in several nuclear mutants of maize (Zea mays) and that it reveals previously unsuspected defects. Furthermore, it provided firm answers to several lingering questions in chloroplast gene expression: (1) the overlapping atpB/atpE open reading frames, whose translation had been proposed to be coupled, are translated independently in vivo; (2) splicing is not a prerequisite for translation initiation on an intron-containing chloroplast RNA; and (3) a feedback control mechanism that links the synthesis of ATP synthase subunits in Chlamydomonas reinhardtii does not exist in maize. An analogous approach is likely to be useful for studies of mitochondrial gene expression.
通过深度测序对核糖体足迹进行分析，通过在全基因组范围内对核糖体进行高分辨率作图，彻底改变了对翻译的分析。我们提出了这种方法的一种变体，它为叶绿体核糖体的全基因组分析提供了一种快速且具有成本效益的替代方法。来自叶片组织的核糖体与叶绿体 ORFeome 的寡核苷酸平铺微阵列杂交，并报告每个叶绿体 mRNA 的丰度和翻译状态。每个测定都替代了几个耗时的传统方法，同时还提供了以前无法获得的信息。为了说明该方法的实用性，我们表明它可以检测出几种玉米（Zea mays）核突变体中叶绿体基因表达的已知缺陷，并且还揭示了以前未被怀疑的缺陷。此外，它为叶绿体基因表达中的几个悬而未决的问题提供了明确的答案：（1）atpB/atpE 开放阅读框重叠，其翻译曾被提议为偶联的，但在体内独立翻译；（2）剪接不是内含子含有叶绿体 RNA 翻译起始的先决条件；（3）在莱茵衣藻（Chlamydomonas reinhardtii）中不存在一种反馈控制机制，该机制将 ATP 合酶亚基的合成联系起来。类似的方法可能对线粒体基因表达的研究有用。
