GlycoRNAs in plants

Ribonucleic acid (RNA) is a polymeric molecule with a remarkable range of functions in biology. In addition to its central role in transferring the DNA encoded information in the form of messenger RNA to proteins, RNA is essential for many biological processes including protein biosynthesis, DNA replication, epigenetic control of gene expression and catalysis. Like other macromolecules, RNA is subjected to chemical modifications that regulate its function. Glycosylation is an abundant modification of macromolecules in all cells, but RNA modified with a covalent linkage of carbohydrates has been unknown until recently. In human and other mammalian cells, glycosylated RNAs have been discovered recently and we hypothesize that glycosylated RNA molecules (GlycoRNAs) exist also in plants. In this project, GlycoRNAs will be isolated from various plant species using affinity purification with antibodies that bind to carbohydrate structures. The composition of the carbohydrate structures and the nucleotide sequence of the modified RNAs will determined to obtain first insights on their biosynthesis and biological function. The anticipated discovery of GlycoRNAs in plants will open up new research avenues for RNA function and glycobiology. 1

Ribonucleic acid (RNA) is an ancient macromolecule with a remarkable range of biological functions. In addition to its central role in translating the information encoded by DNA into proteins in the form of messenger RNA (mRNA), RNA is essential for other aspects of protein biosynthesis, DNA replication, epigenetic control of gene expression, catalysis and many other functions in biology. Like other macromolecules, RNA is subject to post-transcriptional modifications that regulate its function. Glycosylation is a common modification of proteins, lipids and metabolites that can diversify their function through conjugation with different types of monosaccharides or more complex oligosaccharides. For decades, glycobiology and nucleic acid research were completely separate fields. Recent studies in mammalian cells have revealed the presence of several types of glycosylated RNAs (glycoRNAs) on the cell surface of mammalian cells. In particular, small non-coding RNAs can undergo N-glycosylation, and the glycans detected carry Golgi-processed complex-type N-glycans with fucose and sialic acid. These glycan modifications are typically found on secreted proteins in mammalian cells. Here we wanted to investigate whether glycosylated RNA molecules also exist in plants, which is quite challenging because the tools and protocols used for mammalian cells cannot be directly applied to whole plant tissues. To identify glycosylated RNA in plants, we purified RNA from the model plant Arabidopsis thaliana and the tobacco-relative Nicotiana benthamiana. We developed and performed several methods to enrich and detect potentially glycosylated RNAs in leaves and seedlings. We used previously generated glycoengineered plants that produce glycoconjugates carrying the type of sialic acid that is abundant on mammalian glycoRNAs, but not naturally present in plants, to overcome some of the caveats of plant glycoRNA enrichment. Using dot blots and Northwestern blot detection with lectins and carbohydrate-specific antibodies, we were finally able to detect glycosylated RNA variants in plants. The discovery of these glycosylated RNAs in plants will open new avenues of research aimed at characterising in detail the structural variation of the detected glycosylated RNAs, their abundance and distribution, as well as their biosynthetic pathway, with the ultimate goal of identifying novel biological functions for these biomolecules in plants.