Enzyme-mediated proximity labeling identifies small RNAs in the endoplasmic reticulum lumen

In eukaryotic cells, the subcellular targeting of RNA controls many fundamental aspects of cellular physiology. RNA molecules are broadly distributed throughout the nucleoplasm and cytoplasm, but are conventionally believed to be excluded from the secretory pathway compartments, including the endoplasmic reticulum (ER). Recent discovery of RNA N-glycan modification (glycoRNAs) has challenged this view, but direct evidence of RNA localization in the ER lumen has been lacking. In this study, we applied enzyme-mediated proximity labeling to profile the ER lumen-localized RNAs in human embryonic kidney 293T cells and rat cortical neurons. Our dataset revealed the presence of small non-coding RNAs in the ER lumen, including U RNAs, sco/sca RNAs and Y RNAs, which partially overlap with glycoRNAs. These findings shed light on novel RNA targeting mechanism and raise interesting questions regarding the biological functions of ER lumenal RNAs.