Institut für Biologie, Humboldt-Universität Berlin, Philippstr. 13, Rhoda-Erdmann-Haus, Berlin, 10115, Germany.
Max-Planck-Institut für Molekulare Pflanzenphysiologie (MPI-MP), Am Mühlenberg 1, Potsdam-Golm, 14476, Germany.
Plant J. 2018 Apr;94(1):8-21. doi: 10.1111/tpj.13863.
The chloroplast is a prime target for genetic engineering in plants, offering various advantages over nuclear transformation. For example, chloroplasts allow the expression of polycistronic transcripts and thus to engineer complex metabolic pathways. Each cistron within such a longer transcript needs its own expression elements. Within the 5'-UTR, such expression elements are needed for stabilizing mRNAs and for translation of the downstream reading frame. One of the few effective expression elements used so far in transplastomic approaches is the intercistronic expression element (IEE). The IEE is derived from the psbT-psbH intergenic region and includes a target sequence of the RNA binding protein HCF107. We here show that excessive expression of the IEE can lead to specific defects of endogenous chloroplast mRNA stabilization, likely via depletion of HCF107. Key players in chloroplast transcript stabilization and translation are pentatricopeptide repeat (PPR) proteins, which are structurally related to HCF107. PPR proteins that stabilize mRNAs leave behind short RNA footprints that are indicators of their activity. We identified such sRNAs in tobacco, and demonstrate that they are sufficient to stabilize and stimulate translation of mRNAs from synthetic dicistronic transgenes in chloroplasts. Thus, minimal sequence elements are generally adequate to support key steps in chloroplast gene expression, i.e. RNA stability and translation. Furthermore, our analysis expands the repertoire of available expression elements to facilitate the assembly and expression of multi-gene ensembles in the chloroplast.
叶绿体是植物基因工程的主要目标，相对于核转化具有多种优势。例如，叶绿体允许多顺反子转录物的表达，从而能够构建复杂的代谢途径。这种较长转录物中的每个顺反子都需要自己的表达元件。在 5'UTR 内，这种表达元件对于稳定 mRNA 和翻译下游阅读框是必需的。迄今为止，在质体转化方法中使用的少数有效表达元件之一是内含子间表达元件（IEE）。IEE 源自 psbT-psbH 基因间区，包含 RNA 结合蛋白 HCF107 的靶序列。我们在这里表明，IEE 的过度表达可能导致内源叶绿体 mRNA 稳定性的特定缺陷，可能是通过耗尽 HCF107 引起的。叶绿体转录物稳定和翻译的关键参与者是五肽重复（PPR）蛋白，它与 HCF107 在结构上相关。稳定 mRNA 的 PPR 蛋白会留下短的 RNA 足迹，这是其活性的指标。我们在烟草中鉴定了这些 sRNA，并证明它们足以稳定和刺激叶绿体中合成的双顺反子转基因的 mRNA 翻译。因此，一般来说，最小的序列元件足以支持叶绿体基因表达的关键步骤，即 RNA 稳定性和翻译。此外，我们的分析扩展了可用表达元件的范围，以促进多基因组件在叶绿体中的组装和表达。
