ABSTRACT Glioblastoma (GBM) is the most common and deadliest primary malignant brain tumor. Most GBM research has focused on protein-coding genes and less on non-coding transcripts that make up 98% of cellular RNA. Transcribed Ultra-Conserved Regions (TUCRs) are a group of 481 transcripts that are 100% conserved across multiple species. They are highly resistant to variation and are commonly deregulated in cancer, suggesting regulatory and functional importance. Some evidence suggests that most TUCRs are long non-coding RNAs (lncRNAs) that are highly conserved, unlike most other lncRNAs that are usually poorly conserved. TUCRs are largely understudied in cancer and not at all in GBM. As of the date of submission of this R21 application, there were no published studies on TUCRs in GBM. In preliminary work, we performed the first analysis of TUCR expression in GBM using The Cancer Genome Atlas (TCGA) RNA-Seq data and identified 194 TUCRs that are differentially expressed relative to normal brain. Many of these TUCRs correlated with patient survival. This project aims to identify and characterize TUCRs that are differentially expressed in GBM and to uncover their functions and mechanisms of action. We propose three specific aims. In Aim 1, we will identify and characterize TUCRs that are differentially expressed in GBM. Candidate TUCRs will be identified from TCGA RNA-Seq data analyses and prioritized based on their differential expression and correlation with survival. Their full transcript sequences will be uncovered and their lncRNA status verified. In Aim 2, we will uncover the functions of select TUCR lncRNAs in GBM. The top 20 ranked TUCR lncRNAs from aim 1 will be used for this aim. TUCR lncRNAs will be overexpressed and knocked down/out in GBM cell lines and stem cells. The effects of the TUCRs on cell proliferation, survival, invasion, migration and stem cell differentiation as well as on in vivo tumor growth in an RCAS/Tva immune competent mouse model of GBM will be analyzed. In Aim 3, we will identify the mechanism of action for select TUCR lncRNAs in GBM. TUCRs that exert regulatory effects on GBM malignancy as determined in aim 2 will be prioritized. We will identify the subcellular localization of each TUCR lncRNA and use a multipronged approach consisting of bioinformatics and experimental determination of protein and nucleic acid binding partners followed by functional rescue experiments to uncover the mechanisms of action of TUCR lncRNAs. Successful completion of this project would represent the first comprehensive analysis of TUCRs in GBM and generate new knowledge on the mechanisms of GBM malignancy.