Biology subjects

Hemberger, H.

Publications and source records attributed to Hemberger, H..

3 recordsLinked to original sources

bioRxiv2023

The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA

We recently identified glycoRNA--a previously undescribed glycoconjugate--which consists of RNAs modified with secretory N-glycans and presented on the cell surface. While previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging periodate oxidation and aldehyde ligation (rPAL) and Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. The sensitivity and robustness of rPAL provided the first evidence of a direct glycan-RNA linkage, and its flexibility will enable further characterization of glycoRNA biology.

molecular biology↗

bioRxiv2023

RNA binding proteins and glycoRNAs form domains on the cell surface for cell penetrating peptide entry

The composition and organization of the cell surface determine how cells interact with their environment. Traditionally, glycosylated transmembrane proteins were thought to be the major constituents of the external surface of the plasma membrane. Here, we provide evidence that a group of RNA binding proteins (RBPs) are present on the surface of living cells. These cell surface RBPs (csRBPs) precisely organize into well-defined nanoclusters that are enriched for multiple RBPs, glycoRNAs, and their clustering can be disrupted by extracellular RNase addition. These glycoRNA-csRBP clusters further serve as sites of cell surface interaction for the cell penetrating peptide TAT. Removal of RNA from the cell surface, or loss of RNA binding activity by TAT, causes defects in TAT cell internalization. Together, we provide evidence of an expanded view of the cell surface by positioning glycoRNA-csRBP clusters as a regulator of communication between cells and the extracellular environment.

cell biology↗

bioRxiv2023

Rapid and sensitive detection of native glycoRNAs

Chemical tools enable precise characterization of many biopolymers, including glycoconjugates. Metabolic chemical reporters enabled the discovery of glycoRNAs, however they have certain limitations due the requirement of having living cells to incorporate the modified sugar. Here we develop a periodate oxidation and aldehyde ligation method to detect and characterize native sialoglycoRNAs, termed rPAL. With optimized RNA biochemistry to enhance recovery and analysis of small RNAs, we show rPAL is at least an order of magnitude more sensitive than previous methods for detecting sialoglycoRNAs. These improvements allow rPAL to detect sialoglycoRNA from human clinical samples as demonstrated by defining the abundance and patterns of sialoglycoRNAs from sorted populations of peripheral blood mononuclear cells. The sensitivity, robustness, and flexibility of rPAL will allow greater access towards characterizing glycoRNA biology.

molecular biology↗