Enzyme-Mediated Proximity Labeling Identifies Small RNAs in the Endoplasmic Reticulum Lumen

In eukaryotic cells, the subcellular targeting of RNAcontrolsmany fundamental aspects of cellular physiology. Despite broad distributionsthroughout the cytoplasm, RNA molecules are conventionally believedto be excluded from the secretory pathway compartments including theendoplasmic reticulum (ER). Recent discovery of RNA N-glycan modification (glycoRNAs) has challenged this view, but directevidence of RNA localization in the ER lumen has been lacking. Inthis study, we applied enzyme-mediated proximity labeling to profilethe ER lumen-localized RNAs in human embryonic kidney 293T cells andrat cortical neurons. Our data set reveals the presence of small non-codingRNAs in the ER lumen, including U RNAs and Y RNAs, which raises interestingquestions regarding their transport mechanism and biological functionsin the ER.