Coilin is a Mediator of m6A RNA Methylation

项目摘要 存活和增殖是在肿瘤发生期间利用的基本细胞活动。的关键细胞 支持存活和增殖的通路是核因子-κ B（NF-κB）信号通路，其 在癌细胞中也非常活跃，并且是导致治疗耐药性的一个著名因素， 在各种癌症中观察到的现象，包括急性淋巴细胞白血病（ALL）。发生率为 大约每百万人中有30例，ALL是儿童中最常见的癌症，也是儿童死亡的最常见原因。 20岁以前死于癌症。在最近的一项研究中，显示卷曲蛋白的表达升高， Cajal小体（CB）标记蛋白是ALL患者的一个负面预后因素。我们实验室最近的工作 发现coilin是microRNAs（miRNAs）生物发生的正调控因子，miRNAs是一类非编码的 负调节基因表达的RNA。然而，这种功能的机制尚不清楚。另一 miRNA生物合成的正调节因子是N6-甲基腺苷（m6 A）RNA甲基化，一种可逆的修饰 由写入器安装，由擦除器删除，并由读取器执行。我们的实验室假设线圈是 通过调节m6 A RNA甲基化来影响miRNA的生物合成，我们已经产生了令人兴奋的结果。 初步数据显示卷曲蛋白抑制导致m6 A writer胃L3表达的降低， 阅读器hnRNPA 2B 1和擦除器ALKBH 5蛋白，以及它们在细胞内的错误定位和它们在细胞内的错误定位。 全球m6 A RNA水平降低。此外，我们实验室发表的工作发现，卷曲蛋白正调节 NF-κB激活剂miR-517的表达。由于NF-κB通路既受细胞因子的调节，又是细胞因子的调节因子， 几种miRNAs的生物起源，并考虑到先前发表的关于卷曲蛋白的工作，我们 提出了一个中心假设，即卷曲蛋白支持m6 A RNA甲基化，并通过甲基化调节miRNA 生物合成并促进细胞的存活和增殖。这一假设将通过使用 各种原代和转化细胞系，以及我们新产生的CRISPR/Cas9工程改造的线圈蛋白斑马鱼 模型为了检验我们的中心假设，我们已经产生了两个具体的目标，我们将对此进行检验。 假设：1）卷曲蛋白指导m6 A的最佳合成、翻译后修饰和定位 RNA写入器、读取器和擦除器;以及2）卷曲蛋白介导m6 A RNA甲基化，通过该甲基化，miRNA生物合成 并且存活和增殖途径受到调节。这项工作将描述三种新的CRISPR工程 同时也研究了coilin介导m6 A RNA甲基化的新途径 并调节miRNA的生物合成。这项提案中的发现将突出卷曲蛋白在促进 存活和增殖，以及卷曲蛋白在癌症如ALL中的潜在预后或治疗作用。

PROJECT SUMMARY Survival and proliferation are fundamental cell activities that are exploited during oncogenesis. A key cellular pathway that supports survival and proliferation is the nuclear factor-Kappa B (NF-κB) signaling pathway, which is also hyperactive in cancer cells and is a noted element that contributes to therapeutic resistance, a phenomenon observed in various cancers, including acute lymphoblastic leukemia (ALL). With an incidence of about 30 cases per million, ALL is the most common cancer among children and the most frequent cause of death from cancer before 20 years of age. In a recent study, it was shown that the elevated expression of coilin, the Cajal body (CB) marker protein, is a negative prognostic factor in ALL patients. Recent work by our lab has discovered that coilin is a positive regulator in the biogenesis of microRNAs (miRNAs), a class of noncoding RNAs that negatively regulate gene expression. However, the mechanism for this function is unclear. Another positive regulator of miRNA biogenesis is N6-methyladenosine (m6A) RNA methylation, a reversible modification that is installed by writers, removed by erasers, and acted on by readers. Our lab hypothesized that coilin was influencing miRNA biogenesis through the regulation of m6A RNA methylation and we have generated exciting preliminary data showing that coilin suppression results in a decrease in the expression of m6A writer METTL3, reader hnRNPA2B1, and eraser ALKBH5 proteins, as well as a their mislocalization within the cell and a reduction in global m6A RNA levels. Additionally, work published by our lab found that coilin positively regulates the expression of miR-517, a NF-κB activator. Since the NF-κB pathway is both regulated by and a regulator of the biogenesis of several miRNAs, and in consideration of previously published work on coilin, we have generated the central hypothesis that coilin supports m6A RNA methylation through which it regulates miRNA biogenesis and facilitates the survival and proliferation of the cell. This hypothesis will be tested with the use of various primary and transformed cell lines, and our newly generated, CRISPR/Cas9-engineered coilin zebrafish models. To test our central hypothesis, two specific aims have been generated for which we will test the hypotheses that: 1) Coilin directs the optimal synthesis, posttranslational modification, and localization of m6A RNA Writers, Readers, and Erasers; and 2) Coilin mediates m6A RNA methylation by which miRNA biogenesis and survival & proliferative pathways are regulated. This work will characterize three novel CRISPR-engineered zebrafish coilin lines while also investigating a novel pathway by which coilin mediates m6A RNA methylation and regulates miRNA biogenesis. Findings in this proposal will highlight a novel role for coilin in facilitating survival and proliferation, as well as a potential prognostic or therapeutic role for coilin in cancers, such as ALL.