Phloem and RNA: Identification and Characterization of Distinct Phloem-Allocated Non-coding RNA Molecules

该项目的总体目标是研究通过开花植物脉管系统分配的tRNA一半的生物发生和功能。在植物中，已经确定韧皮部是营养物质和激素的全系统传递途径。然而，在过去的十年中，这种观点受到了韧皮部也分配特定RNA分子的发现的挑战。这些韧皮部传递的RNA包括信使RNA （mRNA）、沉默诱导RNA （siRNA）和微RNA （miRNA）。此外，这些韧皮部分配的RNA分子被证明是影响生长的重要长距离信号。在我们的研究过程中，我们在韧皮部中发现了相当数量的全长和片段的小非编码RNA (ncRNA)，如核糖体RNA （rRNA）、转移RNA （tRNA）、核小RNA （snoRNA）和其他未知的小ncRNA。虽然我们对PS ncrna的研究在分析和鉴定转录本的数量方面并不全面，但我们在韧皮部分泌物中发现的tRNA池中发现了一个有趣的偏差。一些独特的和必要的trna缺失，这表明有选择性地传递到韧皮部流。此外，我们还在韧皮部检测到大量特定的tRNA片段（tRNA半）。为了强调我们的发现的重要性，我们证明，与天然叶片RNA库相比，天然韧皮部RNA库以非特异性的方式抑制翻译。根据后来在人类细胞上进行的tRNA一半研究，我们可以证明tRNA一半有助于这种抑制。在酵母和人类细胞等其他生物体中也观察到这种tRNA的一半。许多研究表明，tRNA的一半出现在发育开关和应激条件下。除此之外，tRNA一半的作用在所有生物体中仍有待证实。在酵母中，一种特殊的RNase T2和哺乳动物中，一种与RNase a相关的酶，称为血管生成素，在它们的反密码子环上切割特定的tRNA，并产生tRNA的一半。植物中不存在与血管生成素相关的RNase酶，但编码RNase T2相关酶的基因丰富（如拟南芥中有5个）。到目前为止，还没有对这些酶对trna的特异性进行测试。因此，产生tRNA一半的植物tRNA内切酶(s)仍有待鉴定和表征。鉴定和分析tRNA半生成酶将提供增加或减少活植物中tRNA半生成的手段，并揭示其在植物生长中的潜在作用。提出的目标是：i)研究通过韧皮部分配的tRNA一半的功能，ii)鉴定和功能表征tRNA产生酶的一半，以及iii)建立一个不同于siRNA和miRNA的韧皮部小ncrna的综合数据库。

The general aim of the project is to study the biogenesis and function of tRNA halves allocated via the vasculature of flowering plants. In plants it is well established that the phloem serves as system-wide delivery pathway for nutrients and hormones. However, in the last decade this view was challenged by the discovery that the phloem also allocates specific RNA molecules. Such phloem delivered RNAs include messenger RNA (mRNA), silencing-induced RNA (siRNA) and micro RNA (miRNA). Furthermore, these phloem allocated RNA molecules were shown to act as important long distance signals effecting growth. In the course of our studies we identified a considerable number of full length and fragments of small non-coding RNAs (ncRNA) such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snoRNA), and other unknown small ncRNA in the phloem. Although our study on the PS ncRNAs was not comprehensive with respect to the number of analyzed and identified transcripts, we uncovered an interesting bias in the tRNA pool found in the phloem exudate. A number of distinct and essential tRNAs are missing, which suggests a selective delivery into the phloem stream. In addition, we detected high amounts of specific tRNA fragments (tRNA halves) exclusively in the phloem. Emphasizing the significance of our finding we demonstrated that, in contrast to the native leaf RNA pool, the native phloem RNA pool inhibits in a non-specific fashion translation. In line with a later tRNA halves study performed on human cells, we could show that tRNA halves contribute to this inhibition. Such tRNA halves are also observed in other organisms such as yeast and human cells. A number of studies suggest that tRNA halves appear during developmental switches and under stress conditions. Apart from this a role for the tRNA halves remains to be shown in all organisms. In yeast a specialized RNase T2 and in mammalians an RNase A related enzyme, named angiogenin, cut specific tRNAs at their anticodon loop and produce tRNA halves. In plants angiogenin related RNase enzymes do not exist, however, genes encoding RNases T2 related enzymes are abundant (e.g. five in A. thaliana). Up to now these enzymes are not tested regarding their specificity towards tRNAs. Hence, the plant tRNA endonuclease(s) producing tRNA halves remains to be identified and characterized. The identification and analysis of the tRNA halves producing enzyme will provide the means to increase or decrease the production of tRNA halves in living plants and to unravel their potential role in plant growth.The proposed aims are: i) to study the function of tRNA halves allocated via the phloem, ii) to identify and functionally characterize the tRNA halves producing enzyme, and iii) to build a comprehensive database of the phloem small ncRNAs distinct from siRNA and miRNA.