Our studies call for development of labeling and analytical methods that are really SPECIFIC for glycoRNA.

Team effort with Sungchul Kim lab, @chirlmin.bsky.social Joo lab, and @pmiesen.bsky.social Miesen lab

These glycoproteins behave like glycoRNA biochemically and electrophoretically, so it may confound assays on glycoRNA. E.g. when doing #glycomics analysis, how much glycans are from the contaminant glycoproteins vs. from glycoRNA? This will critically affect your conclusions

We show that the total #RNA isolated from cell cultures can contain substantial non-RNA glycoconjugates, which was found out to be #glycoproteins (characterized in Kegel et al paper), and those glycoconjugates persist even after protease treatment and rounds of purification.

It’ll be interesting to explore whether there are interacting proteins specifically recognizing sialate OAc moieties, or how specific gangliosides cluster with different membrane proteins on cell surface or organelles.

Regarding where they may play a role, in addition to cell surface, previous literatures suggest gangliosides can function intracellularly in mitochondria and very likely Golgi. Langereis et al 2016 paper showed OAcSias largely colocalizes with Golgi markers.

Thanks for the question, Chris! How the OAc gangliosides function at molecular level is still largely unknown. We generally believe the sialate OAc group blocks lectin or enzyme recognition. But there has not been any endogenous protein that specifically binds OAcSia or these gangliosides.

There is an interesting read here (posted shortly after the very first preprint): doi.org/10.1242/prel...

Hi Dr. Erasmus, here is an updated version: www.biorxiv.org/content/10.1...

An independent study from @nathanaelbkegel.bsky.social et al. has led to alike conclusions. The common RNA prep may not give you the RNA as pure as you think. The results also call for the development of glycoRNA-specific labeling methods… A dual labeling?

www.biorxiv.org/content/10.1...

Suppose you are ready to load the glycan-labeled glycoRNA samples into gels. Get half of it, mix with RNases/nucleases, incubate, add sample loading buffer, denature, DIRECTLY load the mixture into gel. Do the rest the same as reported. GlycoRNA gets degraded, the N-glycoconjugate remains intact.

We hope this serves as a heads-up for the community, and propose a simple checkpoint experiment for whichever protocol you are employing👇

The non-RNA N-glycoconjugate, is an independent, separate, but potentially co-existing molecular entity in glycoRNA preparations. It even exhibits an apparent RNase sensitivity related to purification methods, but not digestion.