Presentations

Abstract

Introduction

Glycosylated RNA (glycoRNA) is an emerging field in molecular biology. Carbohydrate modifications are vital for protein and lipid stability, transport, and localization. Glycosylation influences cell functions like growth, differentiation, apoptosis, and immune response, potentially impacting human disease. Carolyn Bertozzi's lab was the first to show that nucleic acids can also be glycosylated and isolating glycoRNAs from human and mouse cell lines using unbiased bioorthogonal chemistry methods. Yet, the biological significance of glycoRNAs remains unknown. We will investigate glycoRNA in the context of human prostate cancer (PCa). PCa is the 2nd leading cancer-related cause of male deaths. More effective diagnostic and therapeutic options are required to increase patient survivorship. Our goal is to develop glycoRNA as novel PCa clinical tools. We were the first to show that human prostate cells express glycoRNA. We found that glycoRNA of the small (<200 nt) noncoding RNA class correlated with cancer progression. Human prostate cell lines with no (RWPE-1) or low malignancy (LNCaP) expressed higher levels of glycoRNA compared to aggressive/metastatic PCa cell lines. Our glycosidase studies and lectin northern blot analysis indicated that glycoRNA carries both N-linked and O-linked modifications. This project will further characterize this heterogeneous glycoRNA population in the prostate and explore if glycoRNA is evolutionarily conserved using the C. elegans genetic model. The hypothesis to be tested is that glycosylated RNA function to maintain prostate homeostasis and has cancer protective roles. We predict that glycoRNA dysregulation will correlate with malignancy/androgen sensitivity in prostate cells. We aimed to 1) Test a wider range of metabolic labeling regents to better characterize N-linked and O-linked modifications carried by prostate glycoRNA; 2) Perform subcellular fractionation studies to determine glycoRNA localization; and 3) Develop C. elegans as a new genetic model to study glycoRNA biogenesis.

Methods

LNCaP cells were grown in media with 100 μM azidosugar for 48 hours to label carbohydrate moieties with a reactive azide-group used for downstream ligation click chemistry, and compared to untreated controls. Cells were harvested for RNA isolation using rigorous methods (acid-phenol extraction, silica column purification, DNase I, Proteinase K, Mucinase, LiCl ethanol precipitation) and RNA was separated into large (>200 nt) and small (<200 nt) fractions (mirVana miRNA Isolation Kit). RNA was reacted with DBCO-biotin at 55˚C for 10 min to covalently ligate a biotin tag onto azide-labeled glycans. Biotin-labeled RNA was separated on a denaturing agarose gel, transferred onto a nitrocellulose membrane, and crosslinked. The northern blot was hybridized with a fluorescent-tagged streptavidin for visualization (Odyssey scanner). For subcellular fractionation studies, LNCaP cells were metabolically labeled and fractionated into cytoplasmic, membrane, soluble nuclear, chromatin bound nuclear, and cytoskeletal fractions (Thermo Subcellular Fractionation Kit). RNA was isolated for click chemistry and glycoRNA imaging. Western blot verified subcellular marker expression of the fractions. For C. elegans experiments, mixed-stage wild type N2 nematodes were grown on NGM plates containing 1mM azidosugars for 1 week, lysed in liquid Nitrogen with mortar/pestle, and prepared for glycoRNA visualization.

Results

Northern blot indicated small (but not large) glycoRNA expression when LNCaP cells were metabolically labeled with Ac4GalNAz (predominantly O-linked) and Ac4ManNAz (predominantly N-linked), but no/low glycoRNA expression when treated with Ac4GlcNAz (n=4). Our stringent RNA isolation procedures were confirmed to be free of mucin-contamination. GlycoRNA was predominantly noted in the membrane fraction of LNCaP cells labeled with Ac4GalNAz or Ac4ManNAz. GlycoRNA expression correlated with the plasma membrane marker PSMA (Prostate Specific Membrane Antigen) by western blot. C. elegans were verified to express small glycoRNA when grown on Ac4GalNAz (but not Ac4GlcNAz) plates. As expected, no glycoRNA was detected using Ac4ManNAz (sialic acid precursor) plates, since C. elegans do not make sialic acid.

Conclusion

We verified that glycoRNA is heterogeneous and carries both N-linked and O-linked modifications. Moving forward, we will determine how these sugar motifs change in non-malignant vs metastatic PCa cells and will identity glycoRNA by LC-MS/MS and RNA seq. Fractionation studies showed that prostate glycoRNA localized to the cell membrane, and suggests external signaling that could influence disease progression. More refined membrane fractionation, lectin array, and J2 antibodies studies are needed. This is the first report showing that C. elegans express glycoRNAs, implying conserved biological significance. This project will provide novel insights into cancer-associated glycoRNA and lead to new PCa clinical targets.