Isolation of Subcellular Ribosome Subpopulations Based on Recombinant Peptide Tag-Specific Location-Restricted...

Growing evidence suggests that mRNA translation is a highly compartmentalized process within a cell, and that subcellular trafficking and localization of specific mRNAs is key to ensuring that proteins with compartment-specific functions are produced in the ideal milieu and in appropriate quantities. However, techniques for subcellular isolation and characterization of the ribosomes that translate these mRNAs have been limited to date, such that much remains unknown about how the composition of translational machinery contributes to regulation of localized mRNA translation. Here, we demonstrate AviTag-specific Location-restricted Illumination-enhanced Biotinylation (ALIBi), a method that combines epitope tagging, a newly developed optogenetically activated split-biotin ligase, and affinity purification to rapidly and specifically label and isolate ribosomes localized to any subcellular compartment of interest. First, CRISPR/Cas9 editing is used to fuse an AviTag peptide, a tobacco etch virus (TEV) protease cleavage site, and a FLAG epitope tag to a ribosomal protein. The split biotin ligase, fused to an organelle-targeting domain, is expressed in this cell line and is inactive under normal biotin concentrations. Upon activation by supplemental biotin and blue light illumination, the ligase biotinylates AviTagged ribosomes in the immediate vicinity, allowing for affinity purification of biotinylated ribosomes and associated proteins and mRNAs on streptavidin-coated beads. Non-denaturing elution via TEV protease cleavage yields samples suitable for downstream characterization of core ribosomal proteins, ribosome-associated proteins, and ribosome-bound mRNAs via RNA sequencing or mass spectrometry proteomics. In this protocol, we review design principles for fusing AviTag to a ribosomal protein and targeting the split biotin ligase enzyme to the organelle of interest. We demonstrate activation of the ALIBi system, cell lysis, affinity purification, and sample elution. Finally, we discuss typical results and troubleshooting.