RNA turnover in plant stress responses

调节RNA的稳定性是基因表达调控的一个重要特征。看家基因通常产生高度稳定的RNA，而压力反应基因通常编码稳定性较低的RNA。这使得RNA快速周转；这一特征对植物特别重要，它们必须迅速适应不断变化的环境条件和压力。在植物中执行RNA降解的途径是众所周知的。然而，逆境如何破坏特定RNA的稳定性，以及如何控制RNA降解酶的靶标特异性在很大程度上是未知的。为了解决这些问题，我们将应用我们最近开发的ERIC测序技术来研究逆境和非逆境下的拟南芥植物。传统上，RNA随着时间的推移而衰退，RNA的半衰期是在用剧毒的转录抑制剂如放线菌素D和虫草素进行严酷处理后确定的。我们发展了一种新的方法(ERIC-SEQ)，它使用无毒的5-乙炔基尿苷(5-Eu)结合RNA测序来标记代谢RNA。这使得我们第一次能够在非侵入性的情况下确定植物中的RNA稳定性。我们将应用这项技术来研究核和叶绿体编码的RNA在不同的非生物胁迫条件下的稳定性。此外，我们还将研究特定的胞质和胞质RNA降解和稳定途径在选定的胁迫条件下的作用。这种方法使我们能够识别逆境特异的RNA序列特征，即(1)确保RNA在非生物逆境条件下的稳定和失稳，以及(2)确定RNA亚群的RNA降解途径的特异性。这些序列元件将被功能研究，连接到特定的内切酶，并用于识别新的反式调节RNA稳定因子。作为一项主要的交付成果，我们的方法将在转录组范围内确定应激期间RNA翻转对基因调控的影响。

Modulating the stability of RNAs is a key feature of gene expression regulation. While housekeeping genes usually produce highly stable RNAs, stress-responsive genes often encode RNAs with low stability. This allows rapid RNA turnover; a feature particularly important for plants, which have to adapt quickly to changing environmental conditions and stresses. The pathways that execute RNA degradation in plants are well understood. However, it is largely unknown how stresses destabilize specific RNAs, and how target specificity of RNA degrading enzymes is controlled.To address these questions, we will apply our recently developed ERIC-sequencing technology to stressed and unstressed Arabidopsis thaliana plants. Traditionally, RNA decay over time and RNA half-lives have been determined after harsh treatments with highly toxic transcriptional inhibitors such as actinomycin D and cordycepin. We developed a novel method (ERIC-seq), which employs metabolic RNA labeling using the non-toxic 5-ethynyl uridine (5-EU) combined with RNA sequencing. This allowed us for the first time to determine RNA stability non-invasively in plants. We will apply this technique to study the stability of nuclear and chloroplast-encoded RNAs under a diverse set of abiotic stress conditions. In addition, we will study the role of specific cytosolic and plastidic RNA degradation and stabilization pathways under selected stress conditions. This approach enables us to identify stress-specific RNA sequence features, that (1) ensure RNA stabilization and destabilization under abiotic stress conditions, and that (2) determine the specificity of RNA degradation pathways for subpopulations of RNAs. Such sequence elements will be functionally studied, linked to specific endonucleases, and used to identify novel trans-regulatory RNA stability factors. As a major deliverable, our approach will identify the impact of RNA turn-over on gene regulation during stress on a transcriptome-wide scale.