Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

Stress granules are dynamic non-membrane bound organelles made up of untranslating messenger ribonucleoproteins (mRNPs) that form when cells integrate stressful environmental cues resulting in stalled translation initiation complexes. Although stress granules dramatically alter mRNA and protein localization, understanding these complexes has proven to be challenging through conventional imaging, purification, and crosslinking approaches. We therefore developed an RNA proximity labeling technique, APEX-Seq, which uses the ascorbate peroxidase APEX2 to probe the spatial organization of the transcriptome. We show that APEX-Seq can resolve the localization of RNAs within the cell and determine their enrichment or depletion near key RNA-binding proteins. Matching both the spatial transcriptome using APEX-seq, and the spatial proteome using APEX-mass spectrometry (APEX-MS) provide new insights into the organization of translation initiation complexes on active mRNAs, as well as revealing unanticipated complexity in stress granule contents, and provides a powerful approach to explore the spatial environment of macromolecules. APEX-eIF4E, APEX-eIF4A, APEX-GFP, C1-APEX, and APEX-CBX1 under Naive conditions were used to assess within and between compartment differnces were performed in duplicate with their corresponding input samples using a Hiseq4000. RNA from APEX-eIF4A under heatshock or hippuristanol samples were performed in duplicate using a Hiseq4000