PROJECT SUMMARY RNA modifications are chemical changes made to transcripts that can regulate their processing, structure, and stability. Recent advances in modification detection techniques have revealed the presence of RNA modifications in mRNA and lncRNA, expanding the known regulatory potential of these modifications beyond their canonical roles in tRNA and rRNA. RNA modifications have been found to modulate the expression of oncogenes and tumor suppressors alike, demonstrating the need to better understand the basic mechanisms of this process so that specific and effective cancer therapeutics can be developed. A critical gap in the literature is the spatial context of modified transcripts; many studies use RNA from whole cells and may miss key mechanisms by averaging the effects of RNA modifications across the transcriptome. I will bring a new perspective to RNA modification biology by focusing my work on a single subcellular context: the paraspeckle. Paraspeckles are stress-inducible nuclear bodies that are assembled on the lncRNA NEAT1, and both this transcript and the paraspeckle itself have been implicated in human cancers. I have used mass spectrometry and sequencing to identify novel RNA modifications on NEAT1, and I hypothesize that these and other modifications on NEAT1 contribute to the assembly of functional paraspeckles. Critically, my preliminary results indicate that the modification profile of NEAT1 differs between cell lines, so I will perform experiments in lines from two cancers marked by overexpression of NEAT1 and one where NEAT1 is downregulated, so that I can look for common mechanisms as well as patterns in the differences between them. In Aim 1, I will focus on NEAT1 directly. I propose the expansion of my current mass spectrometry and sequencing methods so that I can assemble a more complete map of RNA modifications on NEAT1, including the validation and quantification of modifications at single-base resolution. I will mutate identified modification sites, then measure the effects on NEAT1 stability and isoform distribution by qPCR and effects on protein interactions through crosslinking and proteomic analysis. In Aim 2, I will investigate the paraspeckle. I will use both an unbiased genome-wide screen using a paraspeckle reporter system and a targeted microscopy screen of known RNA modification enzymes to identify novel regulators of the paraspeckle. I will make catalytically inactive mutants of the top hits from these screens and perform modification-sensitive RNA sequencing to determine whether these enzymes are modifying NEAT1, other components of the paraspeckle, or upstream regulators, and use fluorescence recovery after photobleaching to measure modification-specific changes in paraspeckle dynamics. Together, these aims will discover and characterize RNA modifications that have a role in paraspeckle formation, revealing insights in an unexplored area of RNA cell biology.

PROJECT NARRATIVE Chemical modification of RNA is often misregulated in cancer, and whether the modifications promote or suppress cancer progression can be specific to the cell-type and transcript. My work focuses on the long noncoding RNA, NEAT1, and the nuclear body that it scaffolds, the paraspeckle, which have been studied in a cancer context for over a decade but only recently examined for RNA modifications. My preliminary data reveals unexplored, cell-line specific RNA modifications on NEAT1, and my proposed research will map and determine the function of these and other RNA modifications on NEAT1 and the paraspeckle in cancer lines with characteristic over- or under-expression of NEAT1.