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

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• mRNA translation is compartmentalized, with subcellular mRNA localization crucial for protein function.
• Current techniques for isolating and characterizing localized ribosomes are limited, hindering understanding of translational regulation.
• The composition of the translational machinery is key to regulating localized mRNA translation.

Purpose of the Study:

• To develop a novel method for specifically labeling and isolating ribosomes from any subcellular compartment.
• To enable detailed characterization of the molecular components involved in localized mRNA translation.
• To advance the study of how ribosome composition regulates protein synthesis in specific cellular locations.

Main Methods:

• AviTag-specific Location-restricted Illumination-enhanced Biotinylation (ALIBi) combines epitope tagging and optogenetically activated split-biotin ligase.
• CRISPR/Cas9 editing fuses AviTag and FLAG tags to ribosomal proteins.
• Light-activated biotin ligase specifically labels nearby AviTagged ribosomes for affinity purification.
• Non-denaturing elution using TEV protease allows downstream analysis of proteins and mRNAs.

Main Results:

• ALIBi successfully labels and isolates ribosomes from targeted subcellular compartments.
• Purified ribosomes are suitable for downstream proteomic and RNA sequencing analyses.
• The method allows for characterization of ribosome-associated proteins and mRNAs.

Conclusions:

• ALIBi provides a powerful tool for studying the regulation of localized mRNA translation.
• This method overcomes previous technical limitations in ribosome isolation and characterization.
• ALIBi facilitates a deeper understanding of how cellular compartments control protein synthesis.