Rationale:
Some people have severe epilepsy which can be the result of MELAS (Mitochondrial Myopathy, Encephalopathy, Lactic acidosis, and Stroke-like episodes) or MERRF (Myoclonus Epilepsy with Ragged Red Fibers). MELAS and MERRF are both caused by mutations in mitochondrial DNA, which results in defective oxidative phosphorylation.
The mitochondria is a crucial organelle of the cell that produces most of the ATP via oxidative phosphorylation (OXPHOS). OXPHOS complexes are put together from proteins made in the nucleus along with proteins made from mitochondrial DNA. It also encodes for 22 tRNA’s.
Mitochondrial RNA polymerase transcribes these genes into a single polycistronic transcript. It is then cut to liberate individual RNAs.
Human Mitochondrial RNA Polymerase (POLRMT) undergoes a phenomenon called transcriptional pausing. Transcriptional pausing occurs when RNA polymerase is transcribing and it stops in the middle of the process for some biological mechanism to occur, such as RNA folding.
Over this summer, basic science research was performed in an attempt to understand transcriptional pausing on human mitochondrial tRNA phenylalanine, and disease-causing mutation sequences.
Methods:
SHAPE (Selective 2’-Hydroxyl Acylation followed by Primer Extension) is a technique which allows for the structural analysis of RNA. RNA undergoes a chemical modification with a 1M7, a SHAPE probe which bonds with the flexible nucleotides of RNA, and DMS.
RT-PCR was then used to turn probed RNA back into cDNA. The cDNA will tell where modifications have taken place because reverse transcriptase will have misincorporated a nucleotide when it is transcribed.
Gel electrophoresis was utilized to make sure that desired products were obtained after undergoing chemical modifications.
After desired products were confirmed by gel electrophoresis and gel imaging, the samples were gel purified so that the products could be worked with for future steps.
Step 1 PCR was then performed, which refers to cDNA that was amplified so that we can increase its quantity for sequencing.
Lastly, Step 2 PCR adds unique barcodes to DNA products so that multiple samples can be pooled together for sequencing.
Results:
RNA was transcribed successfully from the wild type and mutant DNA, by both T7 and POLRMT. These RNA products were probed successfully, and were successfully Step 1 and Step 2 PCR’d. These SHAPE products were then sent out for sequencing.Conclusions:
In conclusion, tRNA sequences were successfully probed, from both the wild type and mutant. These samples were then prepared for sequencing so that the structural differences between the wild type and mutant can be seen.Funding: American Epilepsy Society Predoctoral Research Fellowship