Histone demethylases and regulation of chromatin and transcription in eukaryotes

摘要：细胞生存需要维持基因组的完整性，而基因组的完整性不断受到威胁 细胞内和外部的DNA损伤。DNA损伤的一个普遍来源是紫外线 光，产生环丁烷嘧啶二聚体（CPD）和6-4嘧啶-嘧啶酮（6-4 PP） 加合物核苷酸切除修复（NER）途径主要负责去除和修复 修复这些损伤。NER途径的两个不同分支有助于修复：转录 偶联修复（TCR）识别转录活性位点的CPD，而全局基因组修复（GRP） (GGR)标记基因组其他部分的病变。跨病变合成（TLS）与 绕过S期未修复的病变。许多这些成分的突变会导致 光敏性疾病，特别是着色性干皮病和Cockayne综合征。 最近，我们发现腺苷6位（6 mA）的RNA甲基化是一个令人兴奋的 DNA修复的新贡献者6 mA RNA在以下损伤部位被快速和瞬时诱导： 紫外线照射，我们已经确定了RNA甲基转移酶胃L3负责 这个修改。我们的初步结果表明，胃L3可能参与TCR分支， NER途径，作为转录区域的修复，在胃L3敲除中特异性受损 (KO)细胞因此，在UV暴露后，通过胃L3 KO损失6 mA RNA降低了细胞存活， 其被野生型但不是无催化活性的胃L3拯救。这项工作的目标是 确定6 mA在DNA修复中的作用和作用机制。我们将确定6 mA是否 RNA的功能是在全基因组范围内修复转录和/或非转录区域， 它参与了NER的哪些途径/步骤。我们还将探讨其在TLS和修复其他 DNA损伤类型，以确定6 mA RNA在DNA损伤反应中的作用范围 （DDR）。其次，我们将研究6 mA标记转录本的假设， DNA模板降解，和/或6 mA影响选择性剪接或翻译 修饰的转录物，特别是那些编码对DNA修复或细胞存活重要的蛋白质的转录物。 最后，我们将识别和表征识别6 mA RNA的“阅读器”蛋白质，以便 研究这些阅读器在将6 mA RNA连接到DDR中起机械作用的假设。 我们发现RNA修饰介导DNA修复，揭示了一个新的令人兴奋的方面 以及我们对细胞如何保持基因组完整性的认识。该研究在 这一应用将为6 mA RNA如何调节DDR提供重要的新见解， 这些发现对于认识和治疗光敏性疾病具有重要意义。

ABSTRACT: Cell viability requires maintenance of genome integrity, which is continuously threatened by intracellular and external sources of DNA damage. One prevalent source of DNA damage is UV light, which generates cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone (6-4 PP) adducts. The nucleotide excision repair (NER) pathway is mainly responsible for the removal and repair of these lesions. Two different branches of the NER pathway contribute to repair: transcription coupled repair (TCR) recognizes CPDs at transcriptionally active loci, while global genomic repair (GGR) marks lesions in other parts of the genome. Trans-lesion synthesis (TLS) operates in parallel to bypass any unrepaired lesions in S phase. Mutations in many of these components cause photosensitivity diseases, notably Xeroderma Pigmentosa and Cockayne Syndrome. Recently, we have identified methylation of RNA at the 6 position of adenosines (6mA) as an exciting new contributor to DNA repair. 6mA RNA is rapidly and transiently induced at damage sites following UV exposure, and we have identified the RNA methyltransferase METTL3 as being responsible for this modification. Our preliminary results suggest that METTL3 may participate in the TCR branch of the NER pathway, as repair of transcribed regions is specifically compromised in METTL3 knock-out (KO) cells. Accordingly, loss of 6mA RNA by METTL3 KO decreased cell survival after UV exposure, which was rescued by wild-type, but not catalytically-inactive, METTL3. The goals of this work are to determine the role and mechanism of action of 6mA in DNA repair. We will determine whether 6mA RNA functions to repair transcribed and/ or non-transcribed regions genome-wide, and identify in which pathways/ steps of NER it participates. We will also explore its role in TLS and repair of other types of DNA damage to determine how broadly 6mA RNA operates in the DNA damage response (DDR). Secondly, we will investigate the hypotheses that 6mA marks transcripts arising from damaged DNA templates for degradation, and/ or that 6mA influences alternative splicing or translation of modified transcripts, particularly of those encoding proteins important for DNA repair or cell survival. Finally, we will identify and characterize "reader" proteins that recognize 6mA RNA, in order to investigate the hypothesis that these readers play a mechanistic role in linking 6mA RNA to the DDR. Our discovery of an RNA modification mediating DNA repair has uncovered a new and exciting facet to the DDR and to our knowledge of how cells maintain genome integrity. The research proposed in this application will provide important new insight into how 6mA RNA regulates the DDR, and our findings will be important for understanding and treating photosensitivity diseases.