Characterization of prostate-derived glycoRNA in the context of cancer progression

Noncoding RNA harbors a range of post-transcriptional modifications (m6A, pseudouridine, uridine tailing) important for RNA folding, stability, transport, and localization. Dysregulation of these RNA modifications is associated with human diseases, ex., cancer, inflammation, fibrosis, and viral infection. Noncoding RNA carrying a new class of glycosylation modifications was recently noted in cultured human cells metabolically labeled using bioorthogonal chemistry methods normally employed for glycosylated protein enrichment. Nothing is known about the clinical significance of glycoRNA in prostate cancer (PCa). As high-grade forms of PCa often lead to poor prognoses, it is imperative to improve patient stratification screening tools and treatment options to increase patient survivorship. We hypothesize that RNA requires carbohydrate modifications to maintain organ homeostasis and proper immune surveillance, and we predicted that the glycosylated state of RNA could serve as a biomarker correlating with prostate disease progression and patient prognosis. A clearer understanding of how PCa-associated glycoRNA is regulated during disease progression will provide valuable insight into its function in the prostate. We confirmed that prostate glycoRNA exists using a panel of human prostate cell lines that differed in androgen response and metastatic status. We employed a stringent RNA isolation workflow and validated the existence of carbohydrate-modified RNA using two independent approaches: metabolic labeling using azide click chemistry for biotin-tag detection & lectin northern blot analysis. Small (<200 nucleotide) noncoding RNA were found to carry both N-linked and O-linked sugars, with sensitivity to RNase, endoglycosidases (PNGase F, O-glycosidase) and glycosylation inhibitors. As predicted, glycoRNA expression correlated with PCa progression. GlycoRNA was preferentially enriched in human prostate cell lines and syngeneic panels with no/low malignancy compared to metastatic PCa lines. Subcellular fractionation studies showed glycoRNA localization at the plasma membrane of prostate cells, indicating a signaling role. LC-MS/MS analysis showed glycans containing core-fucosylation and alpha2-3/alpha2-6 sialylation. Several glycans displayed LacNAc and GalNAc extensions, suggesting lectin binding and biological function. We have profiled a large panel of glycosylation-associated enzymes in prostate cell lines to gain insight into glycoRNA regulation. This work will provide novel insight into how RNA modifications impact PCa progression and identify first-in-class clinical tools to improve patient outcomes.