Isolamento de Subpopulações Subcelulares de Ribossomo Baseado em Biotinilação Iluminada Aprimorada por Localização Específica de Etiquetas Peptídicas Recombinantes

Our research investigates how ribosome composition and mRNA translation vary across subcellular domains to regulate local protein synthesis. Current methods lack spatial and temporal precision. ALIBi addresses this through optogenetically controlled proximity labeling of ribosomes.

To begin, obtain L31FTA mouse embryonic stem cells and transfect them with ALIBi, GFP control, or full length BirA plasmids. After 24 to 48 hours of transfection, pre-chill the centrifuge to four degrees Celsius. With the lights off, move the transfected plates to a temporary 37-degree Celsius incubator.

To prevent temperature fluctuations, once the transilluminator is placed in the main incubator, using 70%ethanol, wipe down the blue light transilluminator. After connecting the transilluminator to power, place it in the main incubator. Allow the incubator conditions to reequilibrate, while continuing the setup.

Warm an aliquot of media to 37 degrees Celsius for dosing with biotin and cyclohexamide, or CHX. If preparing control samples without supplemental biotin, warm a separate aliquot for CHX-only dosing. Chill an aliquot of DPBS intended for CHX dosing.

Prepare the RNase removal buffer, salt wash, lysis buffer, and wash buffers. Using a pipette, mix buffers containing dense or viscous components to ensure even mixing. Chill the prepared buffers on ice before use.

Re-suspend the stock bottle of streptavidin beads by gentle pipetting or rocking. Transfer 450 microliters of beads into a protein low binding tube for each 10 centimeter plate of cells. Place the re-suspended beads on the magnetic rack for one minute, or until the beads have fully migrated.

Aspirate and discard the bead storage buffer without touching the bead pellet. Wash the beads two times with 900 microliters of RNAs removal buffer. Wash the beads once with 900 microliters of salt wash.

Wash the beads two times with 900 microliters of wash I buffer. Store the beads in the wash I buffer at four degrees Celsius while performing activation and cell lysis. Add biotin and CHX to the warmed aliquot of media and keep at 37 degrees Celsius until use.

Add CHX to the cold DPBS aliquot and keep it chilled until use. Aspirate the media from the transfected plates. Immediately add 10 milliliters of warm media containing CHX with or without Biotin to each plate.

Place negative control plates in a dark incubator. Place the remaining plates directly on top of the transilluminator. Turn on the transilluminator and incubate the plates for 15 minutes.

Wash each plate two times with the 10 milliliters of cold DPBS containing CHX. Place the plates on ice between and after washes. After aspirating the second wash, tilt the plate and aspirate any residual DPBS.

Keeping the plate flat on ice, add two milliliters of ice-cold lysis buffer and incubate the plate on ice for five minutes. Tap the plate two to three times during incubation to dislodge cells. Using a micropipette, pipette the lysate up and down across the plate surface to dislodge any remaining cells.

Transfer the lysate to a chilled labeled protein low binding tube. In difficult to lyse organelles, sonicate all samples for 45 seconds at four degrees Celsius. And if required, transfer the lysate into a larger tube before sonication.

Transfer the sonicated lysate into a two-milliliter tube. Incubate the lysate on ice for 15 minutes. Centrifuge the lysate at 17, 000 G for three minutes at four degrees Celsius.

Transfer the supernatant into a chilled protein low binding tube without disturbing the pellet. Using a magnetic rack, pellet the washed strepped avident beads. Aspirate the wash one buffer.

Transfer 1, 200 microliters of clarified lysate to the tube containing the washed beads. Pipette up and down gently to re-suspend the beads. Store the remaining clarified lysate at minus 80 degrees Celsius.

Incubate the lysate with the beads at four degrees Celsius in the dark on a tube rotator for one hour. During the incubation, prepare the Tobacco Etch Virus, or TEV elution buffer. Pellet the beads on a magnetic rack.

Transfer the supernatant to a chilled protein low binding tube. Store the supernatant at minus 80 degrees Celsius. Wash the beads two times with 900 microliters of wash I buffer and once with 900 microliters of wash II buffer.

Add 450 microliters of TEV elution buffer to the beads. Incubate the beads in TEV elution buffer for one hour at room temperature on a tube rotator. Pellet the beads on the magnetic rack.

Transfer the elute to a protein low binding tube. Store the eluate at minus 80 degrees Celsius if proteomics analysis or Western blot is intended. Cells containing all system components showed a L31-FTA band, which was stronger with full length burray and faint or absent when components were omitted.

Endogenous biotinilated carboxylase proteins were detected in all lysates at 70 to 80 kilodaltons and 120 to 130 kilodaltons. ANTV5 probing confirmed expression of the split bore fragments. Biotinillated L31FTA decreased in the flow through relative to the lysate.

No biotinillated L31-FTA was present in the eluate following TEV-mediated elution. Anti-flag probing showed a cleaved L31-FTA band at a lower molecular weight than intact L31-FTA ribosomal protein. S12 probing demonstrated the presence of the small ribosomal subunit in the eluate.

Anti-V5 probing showed that proteins that do not bind the ribosome were absent from the eluate. When 2.5 fold more lysate was used with the same 450 microliters of streptavidin coated beads, a substantial amount of biotinilated L31-FTA remained in the flow through. Endogenous biotinilated proteins were partially depleted in the flow through.

Endogenous biotinillated proteins were absent from the eluet, but were recovered after heating post-dilution beads in lambly buffer. Anti-flag probing detected cleaved L31-FTA at a lower molecular weight than intact L31-FTA. Anti-V5 probing showed that the split burray fragments were absent from the eluate.

ALIBi enables the identification of ribosome-bound proteins and mRNAs in specific subcellular regions, revealing differences in translation regulation. The key challenge is maintaining specific proximity labeling through careful control of targeting construct expression and illumination conditions. Following this protocol, affinity purified ribosomes and associated mRNAs can be analyzed using quantitative proteomic and transcriptomic approaches.