Glycans modify lipids and proteins to mediate inter-and intramolecular interactions across all domains of life. RNA, another multifaceted biopolymer, is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammalian cells use RNA as a third scaffold for glycosylation in the secretory pathway. Using a battery of chemical and biochemical approaches, we find that a select group of small noncoding RNAs including Y RNAs are modified with complex, sialylated N-glycans (glycoRNAs). These glycoRNA are present in multiple cell types and mammalian species, both in cultured cells and in vivo. Finally, we find that RNA glycosylation depends on the canonical N-glycan biosynthetic machinery within the ER/Golgi luminal spaces. Collectively, these findings suggest the existence of a ubiquitous interface of RNA biology and glycobiology suggesting an expanded role for glycosylation beyond canonical lipid and protein scaffolds. MAINGlycans have been shown to regulate a wide array of critical biological processes, ranging from cell-cell contacts to host-pathogen interactions, and even the organization of multicellular organisms(1). In a traditionally adjacent field of study, RNA represents another biopolymer that is central to all known life. While the building blocks of RNA are canonically limited to four bases, post-transcriptional modifications (PTMs) can dramatically elaborate the chemical diversity of RNA, with >100 identified PTMs(2-4). The cellular role for RNA is more complex than that of a simple messenger. For instance, RNAs function as scaffolds, molecular decoys, enzymes, and network regulators across the nucleus and cytosol(5-7). With the exception of a few monosaccharide-based tRNA modifications (8,9), there has been no evidence of a direct interface between these two fields of biology.

Antibody-drug conjugates (ADCs) selectively deliver highly toxic chemotherapeutic agents to target antigen-expressing cells and have become an important cancer treatment in recent years.However, the molecular mechanisms by which ADCs are internalized and activated within cells remain unclear. Here we use CRISPR-Cas9 screens to identify genes that control the toxicity of ADCs. Our results demonstrate critical roles for a range of known and novel endolysosomal trafficking regulators in ADC toxicity. We identify and characterize C18orf8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of CRISPR screens with ADCs bearing a noncleavable linker versus a cleavable valine-citrulline (VC) linker, we show that a subset of late endosomal and lysosomal regulators are selectively essential for toxicity of noncleavable linker ADCs. We further show that cleavable VC linkers are rapidly processed upon internalization and therefore surprisingly appear to bypass the requirement of lysosomal delivery. Lastly, we show that inhibition of sialic acid biosynthesis sensitizes cells to ADC treatment by increasing the rate of ADC internalization. This sensitization was observed using several ADCs targeting different antigens in diverse cancer cell types, including the FDA-approved ADC trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal novel regulators of endolysosomal trafficking, provide important insights to guide future ADC design, and identify candidate combination therapy targets as well as potential mechanisms of ADC resistance.