RNA Modifications and Turnover during Viral-induced Decay

项目总结 在应激条件下，细胞需要对环境线索做出快速反应以适应基因 表情景观。基因表达级联的一个特殊方面正在成为最重要的 这种快速变化的来源是对RNA周转的操纵。然而，是什么决定了RNA的衰退或稳定性 对时间敏感和快速的方式还没有完全理解。近年来，转录后修饰 已经成为包括RNA稳定性在内的一系列RNA功能的有力和动态调节器。然而， 关于转录后修饰在应激条件下的调节或对 基因表达的快速变化。本提案重点关注转录后RNA修饰N6- 甲基腺苷(M6A)，旨在确定m6A状态如何在面对 广泛的核糖核酸腐烂。我们的中心假设是，这种修饰有助于区分 目标是从那些幸免于难的人那里快速降级。为了检验这一假设，我们使用了一个强大的工具 因为我们正在利用一种非常有效的病毒核酸酶。这种内切酶来自KSHV病毒 (卡波西肉瘤相关疱疹病毒)，其本身能够触发高达80%的总mRNA 哺乳动物细胞的降解。然而，到目前为止，还不清楚是什么使mrna变得敏感或耐药。 这种普遍存在的核酸酶。在这个提案中，我们将使用这个病毒系统来查询宿主转录组 对这一大规模的RNA衰变事件的反应。我们将结合RNA-SEQ和M6A-RIP SEQ战略来评估 M6A景观在退化或幸存的mRNA中的多样性程度。然后我们将监控M6A如何 机械响应和/或受到细胞中RNA稳定状态的这种重构的影响。尤其是，我们的 重点将放在m6A读取器上，这些读取器可能直接参与解码马厩上的m6A标记 MRNAs。最后，由于RNA在应激条件下和/或对外界刺激的反应中衰退是一种 异质性过程中，我们将把我们对m6A调节RNA稳定性的探索扩展到其他来源 一种新的定位定向CRISPR CAS系统用于病毒核酸酶以外的RNA降解。总而言之，我们 预计这些研究将揭示一种适应宿主细胞的新型传感机制 环境对RNA稳定性影响较大。由于RNA周转的调节是许多人的核心 在细胞中的过程，了解转录后修饰可能如何有助于这种复杂 平衡应该揭示致病细胞和正常细胞的新途径。

PROJECT SUMMARY Under stress conditions, the cells needs to rapidly respond to environmental clues to adapt the gene expression landscape. One particular aspect of the gene expression cascade that is emerging as a prime source for this rapid change is the manipulation of RNA turnover. Yet, what dictates RNA decay or stability in a time-sensitive and fast manner is not fully understood. In the recent years, post-transcriptional modifications have emerged as potent and dynamic regulator of a range of RNA functions including RNA stability. However, little is known about the regulation of post-transcriptional modifications in stress conditions or in response to rapid changes in gene expression. This proposal focuses on the post-transcriptional RNA modification N6- methyladenosine (m6A) and aims to determine how m6A status may control RNA fate in the face of widespread RNA decay. Our central hypothesis is that this modification helps discriminate mRNAs that are targeted for fast degradation from those that are spared. To test this hypothesis, we are using a powerful tool as we are taking advantage of a very potent viral nuclease. This endonuclease comes from the KSHV virus (Kaposi's Sarcoma Associated Herpesvirus) and has the ability, by itself, to trigger up to 80% of total mRNA degradation in mammalian cells. However, to date, it is unclear what renders an mRNA susceptible or resistant to this pervasive nuclease. In this proposal, we will use this viral system to query the host transcriptome response to this massive RNA decay event. We will combine RNA-seq and m6A-RIP seq strategies to assess how diverse the m6A landscape is among degraded or spared mRNAs. We will then monitor how the m6A machinery responds and/or is affected this re-structuring of the RNA steady state in the cell. In particular, our emphasis will be on the m6A readers that may directly be involved in decoding the m6A marks on the stable mRNAs. Finally, because RNA decay under stress conditions and/or in response to external stimuli is an heterogenous process, we will expand our exploration of m6A regulation of RNA stability to other sources of RNA degradation beyond viral nucleases using a novel site directed CRISPR Cas system. Taken together, we anticipate that these studies will shed light on a novel type of sensing mechanism that adapts the host cell environment to large changes in RNA stability. Since the regulation of RNA turnover is at the core many processes in the cell, understanding how post-transcriptional modifications may contribute to this complex balance should reveal novel pathways both in pathogenic and normal cells.