The overall goal of this protocol is to engineer artificial splicing factors that can specifically manipulate alternative splicing of a given target. This method can help to answer key questions in the field of RNA processing, such as the regulation and the misregulation of alternate splicing in cancer cells. The main advantage of this technique is its flexibility.
We can design artificial factors that either promote or inhibit different type of alternate splicing in any given genes. Demonstrating this procedure will be Huan-Huan, a research associate of my laboratory, and Qianyun, a postdoc in my laboratory. Wild-type PUF DNA is used as a template for generating the customized PUF domain scaffold.
Use PCR primers containing PUF sequences that specifically recognize different RNA nucleotides in each position. For each PUF repeat, use four different primers to recognize a different base on each position. In round one PCR, use the primer pairs shown to generate coding sequences for four PUF repeats, universal bridge fragments, and cap fragments.
In round two, use the PCR products generated in the last step as templates with the primer combinations shown to generate coding sequences for RNA recognition codes R-one to R-four, and R-five to R-eight. In round three, use the products from round two and bridge four to five as templates with the R-one forward primer and R-eight reverse primer to generate coding sequences for R-one to R-eight without caps. In round four PCR, use R-one to R-eight, five-prime end cap and three-prime end cap as templates, to generate coding sequences for complete PUF domains.
To begin transfection of HEK-293T cells, mix the liposomal transfection reagent by gently inverting the bottles. Then dilute two microliters of liposomal transfection reagent in 50 microliters of reduced serum medium per transfection. Mix gently, and then incubate for five minutes at room temperature.
Next, dilute 04 micrograms of glycine-rich domain expression vector, and 0.2 micrograms of the modular splicing reporter plasmid in 50 microliters of reduced serum medium in a sterile tube. Then dilute 0.4 micrograms of RS-effector domain expression vector, and 0.2 micrograms of PGZ-three reporter plasmid in 50 microliters of reduced serum medium. Lastly, dilute 0.4 micrograms of PGL-RS-PUF expression vector and 0.2 micrograms of PEZ-1B or PEZ-2F reporter plasmids in 50 microliters of reduced serum medium in a sterile tube.
After five minutes of incubation at room temperature, gently mix the diluted liposomal transfection reagent with the diluted plasmid mixtures. Incubate the final mixtures for 20 minutes at room temperature. Then add the transfection mixtures containing the expression vectors to each well of a 24-well plate seeded with HEK-293 cells, and incubate for at least 12 hours in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide.
After 12 hours of incubation, harvest the transfected cells by trypsinization and centrifugation. Following centrifugation, discard the medium and add 0.5 milliliters of RNA extraction buffer per tube. Add 0.1 milliliters of chloroform per 0.5 milliliters of RNA extraction buffer to each sample.
Following the incubation, centrifuge the tubes for 15 minutes at 12, 000 times G and four degrees Celsius. Then transfer the aqueous base to a fresh tube. Add 0.25 milliliters of isopropanol per 0.5 milliliters of RNA extraction buffer used in the initial homogenization.
Mix by vortexing, and incubate at room temperature for 10 minutes. After a centrifugation as before, the RNA precipitate is usually visible on the bottom of the tube. Discard the supernatant, and wash the RNA pellet with 0.5 milliliters of 75%ethanol per 0.5 milliliters of RNA extraction buffer.
Vortex vigorously, and then centrifuge at 7, 500 times G for five minutes at four degrees Celsius. After the spin, remove the supernatant, and air-dry the RNA pellets. Dissolve the RNAs in 50 microliters of RNase-free water.
Next, add two microliters of five units per microliter DNase-one, seven microliters of 10X buffer, and 11 microliters of water to each 50-microliter RNA solution. After the incubation, heat the solutions to 70 degrees Celsius for 15 minutes to inactivate the DNase. After performing a reverse transcriptase reaction to synthesize cDNA from each sample, add the components of the body labeled PCR reaction in the order shown on screen to PCR tubes.
Prepare one reaction per sample. Run the RT-PCR program on the thermal cycler. Finally, resolve the PCR products by electrophoresis through a 10%polyacrylamide gel with TBE buffer.
Use a fluorescence scanner to visualize the products, and measure the amount of each spliced isoform using densitometry software. For the immunofluorescence assay to measure apoptosis, prepare a transfection mix containing glycine-rich domain expression plasmids as previously shown, and transfect HeLa cells grown on polylysine-coated glass cover slips in a six-well plate. 24 hours after transfection, fix the cells on the cover slips with one milliliter of 4%paraformaldehyde in 1x PBS for 20 minutes at room temperature.
Then gently wash the cells on the cover slips with two milliliters of 1x PBS for five minutes. Next, permeabilize the cells with 0.2%Triton X-100 in 1x PBS for 10 minutes. After washing three times with 1x PBS as before, block non-specific binding with 3%bovine serum albumin in 1x PBS for 10 minutes.
Dilute the flag antibody one to 1, 000 in 3%BSA in PBS, and pipette 30 microliters of the diluted flag antibody onto parafilm sheet. Use forceps to remove a cover slip. Carefully dry off excess buffer with lab wipes, and then place the cover slip cell-side down on the anti-flag solution.
Incubate for one hour at room temperature. Following the incubation, add approximately 500 microliters of 1x PBS to the cell-side of the cover slip until it floats on top of the solution. Return it to the six-well plate with 1x PBS in the wells.
Incubate with secondary antibody on parafilm as before for 15 minutes. Wash as before, and then mount the cover slips on microscope slides using mounting medium containing DAPI. Visualize the cells using a fluorescence microscope, and photograph them using a digital camera.
Using immunofluorescence microscopy, many cells expressing Gly-PUF-531 had fragmented nuclear DNA, indicating that they were undergoing apoptosis. As a control, cells expressing Gly-PUF wild-type had less fragmented nuclear DNA when compared to Gly-PUF-531 transfected cells. Here, immunofluorescence microscopy using the anti-flag antibody merged with DAPI-stained cells shows that the Gly-PUF-531 localized predominantly in the nuclei of transfected cells.
Gly-PUF wild-type also localized in the nuclei of transfected cells, suggesting that these ESFs dissociate from their targets when the fully-spliced mRNAs are transported into the cytoplasm. The percentage of apoptotic cells with fragmented nuclear DNA was measures from randomly chosen fields of fluorescence microscopy images. Once mastered, the construction of PUF scaffolds with RNA binding specificity can be done in two days if it is performed properly.
After watching this video, you should have a good understanding of how to specifically manipulate alternative splicing in human cells by engineering artificial factors.
