GSE116008
Atlas of Subcellular RNA Localization Revealed by APEX-seq
We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome. Overall design: APEX-seq libraries for both labeled targets and unlabeled controls were generated for the following constructs: (1) NLS (nucleus), (2) NIK (nucleolus), (3) LMA (nuclear lamina), (4) NucPore (nuclear pore), (5) NES (cytosol), (6) ERM (ER membrane cytosol facing), (7) KDEL (ER lumen), (8) OMM (Outer mitochondrial membrane), and (9) MITO (mitochondrial matrix). 4 libraries were prepared for each construct (2 biological replicates for labeled targets, 2 biological replicates for unlabeled controls). Furthermore, we generated drug-perturbation libraries for OMM and NES using the following treatments: (1) cycloheximide, (2) puromycin and (3) m-chlorophenylhydrazone (CCCP). In addition, nocodazole-perturbation libraries for OMM and NES were generated following drug treatment for: (1) 3 minutes, (2) 6 minutes, (3) 9 minutes, (4) 30 minutes and (5) 2 hours. We also generated nuclear and cytosol fractionation RNA-seq libraries (2 replicates each) following a protocol by Gagnon et al. (Nature Protocols, 2014).