Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.
Kinetic CRAC can be used to study temporal associations between RNA and RNA binding proteins on an unprecedented short timescale. When combined with our crosslinker, kinetic CRAC is able to crosslink live cells within seconds. This reduces UV damage responses and facilitates the study of stress responses at minute scale resolution.
Kinetic CRAC is a very long protocol that can be challenging for new users. Additionally, working with RNA poses its own challenges. To prevent RNA degradation, one should filter sterilize all solutions and make sure that the pipettes and lab bench are kept clean.
Visual demonstration of this method is critical because some of the steps are not very intuitive. This includes UV crosslinking and harvesting of cells, as well as extraction of crosslinked RNAs from the gel. To perform UV crosslinking on microorganisms in solution, inoculate 3.5 liters of the desired medium with yeast to a starting OD 600 of 0.05, and grow the culture overnight at 30 degrees Celsius with continuous shaking at 180 rpm.
Once the cells reach the desired OD, pour 500 milliliters of the culture straight into the Vari-X-linker crosslinker and irradiate it with 250 millijoules of 254 nanometer UV.After crosslinking, use a vacuum filtration device to filter the cells. Roll up the membrane with the filtered cells, and place it in a 50 milliliter conical tube labeled T0.Filter the remaining cells over six different filters, and drop the membranes in three liters of prewarmed stress-inducing medium, mixing them vigorously with a pipette for 50 seconds. Continue crosslinking the cells in 500 milliliter volumes at the desired time points, and store the samples at minus 80 degrees Celsius.
To crosslink adherent cells, transfer the quartz culture dish with the cells to the specialized tray, and irradiate it with 300 millijoules of 254 nanometer UV.Immediately place the dish on ice. Remove the growth medium from the dish, and add 10 milliliters of ice cold PBS. Collect the cells by scraping, and transfer them into a 15 milliliter conical tube.
Then pellet the cells by centrifugation at 300 G for five minutes at four degrees Celsius. After washing the cells with PBS, remove the PBS and snap freeze the cell pellet on dry ice. Store the sample at minus 80 degrees Celsius as needed.
Set the centrifuge to four degrees Celsius, and prepare two rows of 1.5 milliliter tubes per sample for elution. Quickly spin the columns with the nickel beads to get rid of the void volume. Then place the columns in the first row of elution tubes, and fill them with 200 microliters of elution buffer.
After two minutes, quickly spin the columns and transfer them to the second row of tubes. Then repeat the elution as previously demonstrated. When finished, combine all eluates in a five milliliter tube, and add two microliters of 20 milligrams per milliliter glycogen.
Then add 100 microliters of trichloroacetic acid to the sample, and vortex it for 30 seconds. After washing the sample with acetone, resuspend it in 30 microliters of protein loading buffer. Then, check the radioactivity using the Geiger counter.
Heat the sample for 10 minutes at 65 degrees Celsius, and load it on a one millimeter four to 12%precast Bis-Tris gel. Run the gel for 90 minutes at 125 volts in MOPS buffer. When finished, wrap the gel in cling film, and secure it to the inside of a light tight cassette with tape.
After exposing the gel to the auto radiographic film, develop the film by cutting away the cling wrap covering the gel, making sure to not move the gel and offset the image. Place the film over the gel, and excise the band of interest. Put the gel slice in a two milliliter tube, and crush it with a P1000 pipette tip.
Then add proteinase K buffer and proteinase K.Incubate the sample at 55 degrees Celsius with vigorous shaking. Run the cDNA on a precast 6%TBE gel at 100 volts for one hour, using an appropriate ladder for the quantification of short DNA fragments. When finished, place the gel in a liquid tight container with enough TBE to cover it, and add an appropriate amount of SYBR Safe dye.
Stain the gel for 15 minutes at room temperature. Then replace the SYBR containing buffer with fresh TBE. Wash the gel for 10 minutes with gentle shaking.
Drain the TBE, and place the gel in a transparent folder. Cut the folder to an appropriate size, and image the gel with an image quant. Excise the DNA fragments between 175 and 400 base pairs, and put the gel piece in a 1.5 milliliter tube.
Smash the gel with a P1000 pipette tip, and add 400 microliters of water. Then incubate it for one hour at 37 degrees Celsius while shaking. Freeze the sample on dry ice for 10 minutes.
Then repeat the incubation at 37 degrees Celsius. Insert two glass microfiber filters inside a filter column placed in a tube. Then, use a cut P1000 pipette tip to transfer the TBE gel suspension into the filter unit and spin it down at 17, 000 G for 30 seconds.
This protocol was used to monitor Nrd1-RNA interactions in yeast cells subjected to a glucose starved environment. Samples were crosslinked before the shift to medium with no glucose, as well as one, two, four, eight, 14, and 20 minutes after. The autoradiographs from the experiment showed an intense signal at the expected molecular weight of Nrd1, representing the protein bound to short radio labeled RNAs not amenable for sequencing.
The signal above this band, which is the protein crosslinked to longer RNA fragments, was isolated. To amend the kinetic CRAC protocol for mammalian cells, a special stage was developed to allow UV irradiation of dishes with adherent cells. The efficiency of this setup was measured through crosslinking and capture of stably tagged GFP RBM7.
The crosslinker was able to recover protein RNA complexes from mammalian cells using 254 nanometer UV irradiation at efficiencies comparable to a widely used UV irradiation device. Development of a UV-permeable quartz Petri dish during crosslinking further enhanced the recovery of protein RNA complexes. Nrd1 data shows the protein binds to many non-coding RNA transcripts, indicating that it is involved in the degradation of these transcripts.
Binding to transcripts encoding HXT6 and HXT7, both of which are upregulated during glucose starvation, was also demonstrated. Kinetic CRAC allows one to measure the dynamic interactions between protein RNAs during cell development, stress responses, and assembly of macromolecular complexes, such as the ribosome or spliceosome.
