A UV-Based ChIRP Method For The Verifiable Identification of Proteins That Directly Interact With LncRNA: From Cultured Cells To Mouse Testis

Our research developed cPDiRT, a UV cross-linking method to map directly in RNA and proteins interactions in both the cultured cells and the native tissues such as testes and carving TUG1's molecular mechanisms in spermatogenesis. This protocol enables direct RNA protein into action mapping a wider range of systems from cultured cells to intact native tissues. To begin, wash the GC2 cells three times with PBS to remove residual culture medium.

Then, add three milliliters of PBS to the dish and distribute it evenly before cross-linking. Irradiate the cells in a 254-nanometer ultraviolet cross-linker at a dose of 4, 000 millijoules per square centimeter. Collect the cells in PBS and centrifuge the cell suspension at 3000G for five minutes at four degrees Celsius.

After discarding the supernatant, weigh the pellet and confirm a minimum pellet mass of 500 milligrams per sample. Collect testes from four-week-old male C57BL6 mice. Wash the decapsulated testicular tissue twice with 5 to 10 milliliters of DMEM.

Then, incubate 100 milligrams of tissue with 10 milliliters of DMEM containing one milligram per milliliter collagenase IV at 37 degrees Celsius. Continue the digestion for 5 to 15 minutes until the seminiferous tubules are released. Then, centrifuge the suspension at 1000G for one minute at four degrees Celsius and remove the supernatant.

Wash the pellet with 5 to 10 milliliters of DMEM. Centrifuge the suspension again at 1000G for one minute at four degrees Celsius. And re-suspend the pellet in 0.25%trypsin containing one milligram per milliliter deoxyribonuclease I.Incubate the suspension at 37 degrees Celsius for 5 to 15 minutes.

Then, add an equal volume of DMEM supplemented with 10%FBS to stop the enzymatic digestion. Filter the suspension through a 70-micrometer cell strainer. Centrifuge the cells at 2000G for five minutes at four degrees Celsius.

Discard the supernatant and wash the pellet with 5 to 10 milliliters of PBS. Centrifuge the suspension again at 2000G for five minutes at four degrees Celsius. Re-suspend the single cell suspension in PBS and distribute it evenly onto a 15-centimeter culture dish.

Cross-link the testicular cells as demonstrated earlier for GC2 cells. Omit irradiation for the control cells. Collect the cross-linked cells.

Centrifuge the suspension at 3000G for five minutes at four degrees Celsius and discard the supernatant. Ensure a minimum pellet mass of 500 milligrams by pooling testicular cells isolated from at least 10 to 12 mice. After weighing the pellet, re-suspend it in cell lysis buffer at a ratio of 100 milligrams of pellet per one milliliter of buffer.

After vortexing, sonicate the sample using a focused ultrasonicator or a water bath sonicator until the lysate becomes visually clear. Then, aliquot approximately 10 microliters of the lysate into microcentrifuge tubes for RNA and protein analysis. Add 30 microliters of pre-washed magnetic beads to the lysate.

Incubate the samples at 37 degrees Celsius for 30 minutes with gentle mixing in a hybridization oven. Place the samples on a magnetic stand for approximately one minute or until the solution becomes clear. Transfer the cleared lysate to a fresh tube.

Place the new tube on the magnetic stand. Then, transfer the lysate to another tube to remove any residual beads. Add two volumes of hybridization buffer tempered at 37 degrees Celsius to each sample.

Add biotinulated DNA probes at a ratio of one microliter of 100 micromolar probe stock per one milliliter of lysate. Incubate the samples at 37 degrees Celsius with end-over-end rotation for 12 to 16 hours in a hybridization oven. Immediately before use, wash 100 microliters of streptavidin magnetic beads three times with cell lysis buffer.

And remove the residual buffer after the final wash. Briefly centrifuge the hybridization reaction tubes. Then, add the hybridized sample to the washed beads and mix gently.

Return the bead suspension to the original tube and incubate at 37 degrees Celsius with mixing for 30 to 40 minutes. After incubation, briefly centrifuge the samples. Place the tubes on a magnetic stand for one to two minutes, or until the solution becomes clear.

Then, discard the supernatant. To wash the beads, add one milliliter of the wash buffer and incubate the tubes in the hybridization oven at 37 degrees Celsius for five minutes. After five washes, transfer 1-10%of the beads to a fresh tube for RNA analysis.

Finally, add 40 microliters of 1X SDS sample buffer to the remaining beads. Boil the samples at 95 degrees Celsius for 30 minutes to elude the proteins and reverse the cross-links. Recover the proteins for downstream mass spectrometry analysis.

ChIRP mass spectrometry analysis identified more than 190 proteins enriched relative to the negative control in GC2 cells. At a UV dose of 4, 000 millijoules per square centimeter, cPDiRT-MS identified a more selective protein set. More than two-thirds of these proteins overlapped with those identified by conventional ChIRP-MS.

Most proteins identified with at least two unique peptides were reproducibly detected across biological replicates in both GC2 cells and mouse testes. Gene ontology analysis of the reproducibly identified proteins revealed biological processes broadly consistent with the known functions of TUG1. Comparison of the GC2 cell and mouse testes data sets identified 15 proteins that were commonly enriched.

Enhanced cross-linking immunoprecipitation sequencing revealed clear and specific binding peaks on the TUG1 transcript for PABPC1 and CCT3. cPDiRT allows researchers to identify and measure proteins that directly interact with specific linked RNA in both the culture cells and the native tissues. The most important challenge is achieving efficient UV cross-linking while ensuring high specificity for direct RNA and the protein interactions.

Our protocol will support future studies to apply this approach to other RNAs and the tissues in restating direct RNA protein into action in development and the reproduction.