Tiny RNAs as new potential biomarkers for gammaherpesvirus-driven neurological and central nervous system diseases

项目摘要 已知EB病毒（EBV）通过以下途径直接和间接损害大脑和中枢神经系统： 感染的淋巴细胞这些病毒在健康条件下是潜伏的，但在免疫功能低下的情况下会重新激活。 造成严重后果的个人。与此同时，越来越多的文献已经确定， miRNAs广泛参与典型的大脑发育、功能和功能障碍。在人类中，20~23- 核苷酸（nt）miRNAs被装载到四种Argonaute（AGO）蛋白中，形成RNA诱导的沉默复合物 （RISC）抑制与其miRNA互补的mRNA的翻译。因此，适当的基因调控， miRNAs对于神经系统的产生是不可或缺的，它们的非典型表达模式反映了 神经元疾病的发病机制。由于已知miRNA在血清、血浆和其他体液中循环， 它们被用作诊断许多疾病的生物标志物。在这方面，我们最近发现，前- 相关的miRNA被三种3′→5′核酸外切酶修剪成14-nt或更短的微小RNA（tyRNA）：干扰素- 刺激基因20 kDa（ISG 20）、三个引物修复核酸外切酶1和增强的RNAi 1。ISG 20高表达 在EBV复制过程中。值得注意的是，我们的双荧光素酶报告基因测定表明，14-nt tyRNA不再保留 基因沉默活性。这些结果表明，病毒感染在全球范围内将miRNAs转化为tyRNAs，从而使 基因表达有很大的不同。根据PubMed，miRNA的研究稳步增加， 已发表论文20多万篇。相比之下，只有大约10篇论文 讨论tyRNA，包括我们最近的工作，这表明对tyRNA知之甚少。与 在两个具体目标之后，我们将确定EBV感染的细胞的四个不同阶段中的tyRNA：1）无 感染，2）新生感染，3）潜伏期，和4）裂解再激活（目的1）。此外，我们将验证假设 疱疹病毒感染增强了神经发育障碍患者中tyRNA的产生， 特异性单点突变（Aim 2）。拟议研究的成功结果将提供 tyRNA作为新的潜在生物标志物的全面数据集。这项研究的长期目标是提供一个 建立一个新的前沿，了解疱疹病毒感染如何产生tyRNA， 基因表达失调。在拟议的项目中获得tyRNA的全面概况是至关重要的 第一步是进行深入研究。预期测定的tyRNA用作生物标志物， 使先前鉴定的miRNA生物标志物更有信息性。

PROJECT SUMMARY Epstein-Barr virus (EBV) is known to damage the brain and central nervous system directly and indirectly through infected lymphocytes. These viruses are latent in healthy conditions but reactivate in immunocompromised individuals with severe consequences. Meanwhile, increasingly diverse literature has established that microRNAs (miRNAs) are extensively engaged in typical brain development, function, and dysfunction. In humans, 20~23- nucleotide (nt) miRNAs are loaded into four Argonaute (AGO) proteins, forming RNA-induced silencing complexes (RISCs) to repress the translation of mRNAs complementary to their miRNAs. Thus, proper gene regulation by miRNAs is indispensable for generating the neurological system, and their atypical expression patterns reflect the pathogenesis of neuronal diseases. Since miRNAs are known to circulate in serum, plasma, and other bodily fluids, they are used as biomarkers for diagnosing many diseases. In this context, we recently discovered that AGO- associated miRNAs are trimmed to 14-nt or shorter tiny RNAs (tyRNAs) by three 3′→5′ exonucleases: interferon- stimulated gene 20 kDa (ISG20), three prime repair exonuclease 1, and enhanced RNAi 1. ISG20 is highly expressed during EBV replication. Notably, our dual-luciferase reporter assay demonstrated that 14-nt tyRNAs no longer retain gene-silencing activity. These results suggest that viral infection globally converts miRNAs to tyRNAs, thereby making the gene expression drastically different. According to PubMed, the study of miRNAs has steadily increased, and more than 200,000 papers have already been published. In contrast, there have only been about 10 papers discussing tyRNAs, including our recent work, which demonstrates that little is known about tyRNAs. With the following two specific aims, we will determine the tyRNAs in the four different stages of EBV-infected cells: 1) no infection, 2) de novo infection, 3) latency, and 4) lytic reactivation (Aim 1). In addition, we will validate the hypothesis that the herpesvirus infection enhances tyRNA generation in neurodevelopmental disorder patients whose AGO has specific single-point mutations (Aim 2). The successful outcome of the proposed study will provide the first comprehensive data sets of tyRNAs as new potential biomarkers. The long-term goal of this study is to provide a foundation to establish a new frontier in understanding how herpesvirus infection generates tyRNAs and thus dysregulates gene expression. Obtaining the comprehensive profile of tyRNAs in the proposed project is an essential first step to proceeding towards a deeper study. The determined tyRNAs are expected to serve as biomarkers and make previously identified miRNA biomarkers more informative.