Characterization of N-glycosylation and its functional role in SIDT1-Mediated RNA uptake

The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multipass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site- specific N-glycosylation information and its functional contribution remain unclear. In this study, we use high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the Nglycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system with potential implications for therapeutic applications.