Cerebral ischemia significantly alters the expression and/or function of transcriptional and translational mechanisms including various classes of noncoding RNAs, epigenetics and epitranscriptomics. My research in the past 20 years evaluated these mechanisms that are central in promoting either secondary brain damage or recovery after stroke with a goal to design novel therapies. In the 7 years, I will focus on studying the role of various RNAs in promoting post-stroke brain damage. The goal is to understand the mechanisms as well as identifying new therapeutic targets to minimize the secondary brain damage and to promote functional recovery after stroke. In this R35, I propose 4 projects. Project 1: In a currently funded RO1 grant we are evaluating the functional significance of an epigenetic modification called DNA hydroxymethylation (5hmC). Our studies so far showed that stroke leads to induction of 5hmC in many prosurvival genes that induces their expression. We intriguingly observed that many lncRNA induced after stroke also show increased 5hmC levels. In this project, we will continue the studies to understand the significance of 5hmC induction in lncRNAs to post-stroke functional outcomes. Project 2: RNAs can be tagged by >150 distinct chemical modifications, which are collectively defined as epitranscriptomic modifications that form an additional layer of post- transcriptional gene regulation. Among them, methylation of the adenosine at N6-position (N6- methyladenosine; m6A) is the most abundant modification in the brain. Our studies show that focal ischemia downregulates m6A demethylase FTO leading to increased abundance of m6A-tagged mRNAs. Many of these are inflammatory and apoptotic. Furthermore, activation of FTO promotes RNA demethylation and neuroprotection. We will use molecular tools to evaluate the functional significance and mechanisms of increased m6A methylation of RNAs in brain damage after stroke. Project 3: RNAs can also undergo the epitranscriptomics modification of glycation and the neural glycoRNAs interact with microglial Siglec receptors to dampen inflammation following CNS insults. We will study the significance of glycoRNAs and the mechanism of action in brain after stroke. Project 4: Various classes of RNAs collaborate in their actions. We particularly observed that circular RNAs interact with lncRNAs to modulate mRNAs and microRNAs. We will analyze the role of RNA networks in relation to ischemic brain damage using 2 examples. Project 4A: The first one is the interaction of a circRNA (circPUM1) with a lncRNA (NORAD) to control a mRNA that codes an RNA binding protein (Pum1) and a mRNA that codes BNIP2. Project 4B: An ongoing RO1 is studying the role of miR-7 in controlling -Synuclein in post-stroke brain. Although mature miR-7 levels decrease after stroke, levels of premiR-7a and 7b are not altered. As circRNA CDR1as binds and stabilizes miR-7, we are planning to study the interactive role of this CDR1as-miR-7--...