Mapping Spatial Transcriptome With Light-activated Proximity-dependent RNA Labeling

The distribution of RNA molecules in eukaryotic cells is highly heterogeneous.The subcellular localization of specific RNA molecules is closely related to their biological functions,which can not only promote local protein synthesis,but also regulate transcription and support cellular structural.It is also important for many physiological processes such as cell proliferation,embryonic development,and neuroplasticity.With the development of new techniques such as fluorescence imaging,subcellular fractionation,sequencing and analysis,the study of RNA subcellular localization has been pushed to transcriptome-level.The combination of cell fractionation with microarray or high throughput sequencing can be used to study the local transcriptome of cell membrane,ER,mitochondria or axons.Meanwhile,modern imaging techniques combined with RNA reporter,FISH,in situ sequencing or other methods can be used to visualize RNA localization in fixed cells.Despite the progress made by these methods,a new technique is in urgent need,which can study the subcellular transcriptome in live cells or animals with high spatial-temproal resolution.In this study,we report a newly developed chemical biology technique,CAP-seq,which could label subcellular localized RNAs in living cells with high spatial resolution.CAP-seq relies on a photosensitizing protein,mini SOG,that can be activated by blue light illumination to produce photooxidative damage and label nucleobases in proximal RNAs.By affinity purification and high throughput sequencing of the labeled RNAs,the transcriptome near mini SOG is obtained.Using this technique,we systematically studied the transcriptome of several subcellular regions,including the mitochondrial matrix,the ER surface,and the outer mitochondrial membrane.30 m RNAs encoding oxidative phosphorylation proteins and 55 m RNAs encoding ribosomal proteins were detected near the outer mitochondrial membrane.These results not only support the model that mitochondrial proteins are locally translated,but also suggest that association of mitochondrial function with protein translation.Due to its specificity and simple operation,CAP-seq is a new technique suitable for studying subcellular transcriptome in many biological systems.