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.