RNA Modifications and Turnover during Viral-induced Decay

项目摘要 在压力条件下，细胞需要对环境线索做出快速反应，以适应基因 表达景观。基因表达级联的一个特殊方面， 这种快速变化的来源是RNA周转的操纵。然而，是什么决定了RNA的衰变或稳定性， 时间敏感和快速的方式没有得到充分的理解。近年来，转录后修饰 已成为一系列RNA功能（包括RNA稳定性）的有效且动态的调节剂。然而，在这方面， 关于在胁迫条件下或响应于胁迫的转录后修饰的调节知之甚少。 基因表达的快速变化。该提案的重点是转录后RNA修饰N6- 甲基腺苷（m6 A），旨在确定m6 A状态如何控制RNA的命运 广泛的RNA衰变。我们的中心假设是，这种修饰有助于区分mRNA， 从那些幸免于难的人中迅速降解。为了验证这一假设，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。这种核酸内切酶来自KSHV病毒 （卡波西肉瘤相关疱疹病毒），并有能力，本身，触发高达80%的总mRNA 在哺乳动物细胞中降解。然而，到目前为止，还不清楚是什么使mRNA敏感或耐药 这种无处不在的核酸酶。在这个提议中，我们将使用这个病毒系统来查询宿主的转录组 对这种大规模RNA衰变事件的反应。我们将联合收割机RNA-seq和m6 A-RIP seq策略相结合， m6 A在降解或保留的mRNA中的多样性。然后我们将监视m6 a如何 机器响应和/或受到细胞中RNA稳态的这种重构的影响。特别是我们 重点将放在m6 A阅读器，可能直接参与解码的m6 A标记的稳定 mRNA。最后，由于RNA在应激条件下和/或响应于外部刺激而衰变， 我们将把我们对m6 A调节RNA稳定性的探索扩展到其他来源， 使用新的定点CRISPR Cas系统的病毒核酸酶以外的RNA降解。总之，我们 预期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稳定性的巨大变化。由于RNA周转的调节是许多基因的核心， 过程中的细胞，了解如何转录后修饰可能有助于这一复杂的 平衡应该揭示致病细胞和正常细胞中的新途径。

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.