Modulating Alternative Splicing of the MAO-A Gene Transcript
Monoamine Oxidase A (MAO-A) or notoriously known as the “warrior gene”, has recently contributed to the notion that genes can influence an individual’s behavior. Recently, studies have shown that differential expression of MAO-A have been correlated with various psychiatric diseases and developmental disorders. The MAO-A disease related studies have focused predominantly in males, due to the fact this gene is located on the X chromosome. The under expression of MAO-A, or the presence of a variant allele which encodes for a less functional enzyme, is believed to be associated with autism, antisocial disorder, ADHD, and aggressive behavior. The over expression of MAO-A is associated with major depression disorder.
MAO-A is a flavoenzyme tethered to the outer membrane of the mitochondria and plays a vital role in the deamination of dietary monoamines and neurotransmitters. The proposed dimensions of MAO-A protein structure and mechanism involved in substrate deamination is not well understood, however it has been found that MAO-A contains an active site for FAD prosthetic group, which is known to carry out oxidative reactions. These amine groups are either safely removed and fed into the urea cycle as ammonia, or the backbones are subsequently utilized in protein synthesis. MAO-A has been identified in 160 different tissues, however scientific articles have largely investigated its role in placenta, neurons, placenta, and endothelial cells.
The goal of this experiment was to see if chemical compounds can be used to manipulate MAO-A expression, or induce alternative splicing events. Other compounds can be used to induce changes in gene expression and alternative splicing of MAO-A transcript as potential therapies in place of MAO inhibitors (MAOi) which are commonly used as antidepressants with dangerous side effects. The splice variants can also be used in determining essential protein domains or used as diagnostic tools for psychiatric diseases.
Materials and Methods
HepG2 cells, which are derived from liver carcinoma cells, were treated with various compounds for 24 hours.The control group comprised of untreated HepG2 cells that were handled in the same environmental conditions as the experimental cells.
RT-PCR was performed using primer pairs spanning exon 10 and exon 16. RT-PCR products were visualized on a 1% agarose electrophoresis gel. These bands were gel purified using Qiaquick Gel extraction kit and further sequenced by Genewiz.Identification of splice variants were determined by ApE alignment tool and NCBI BLAST.
RT-PCR products generated from HepG2 cells treated with compound 135 produced two alternatively spliced variants visualized on an agarose electrophoresis gel (Fig 2). Genewiz validated that the two bands were distinctly different RT-PCR products. NCBI Blast determined that the top band with the expected size of 449, included exons 11-16 (Fig 2). However, the lower band RT-PCR product was 445 bp and had low homology when analyzed with NCBI BLAST. ApE alignment tool was used to compare sequences between the suspected variant and the MAO-A gene sequence. These results indicated that the variant contained all the exons as the expected product, except for exon 14 (Fig 3).
ExPASY translate tool was used to determine the open reading frame of the spliced variant. Exon 14 exclusion generated a frameshift mutation that resulted in a premature stop codon in exon 15(Fig 4). NCBI was used to ascertain which protein domains were truncated. The variant encodes for a truncated protein in the transmembrane domain (Fig 4).
Treating HepG2 cells with compound #135 induced alternative splicing in the MAO-A gene transcript. The alternatively spliced variant excluded exon 14 which resulted in a frameshift mutation that coded for a truncated protein. The normal MAO-A transcript encodes a protein that is approximately 527 amino acids long. However the spliced variant encodes a truncated protein that consists of 460 amino acids. This splice variant has not been recorded on the NCBI database.
The truncated protein generated from the splice variant may be less functional. This suggests that the regulation of alternative splicing in the MAO-A gene transcript can be used to regulate MAO-A protein activity. Those with MAO-A associated disorders, such as major depression disorder, can use compounds that induce alternative splicing of MAO-A transcripts that encode less functional protein products.
Figure 1- A schematic of the human MAO-A gene drawn to scale, including 16 exons and 15 introns.
Figure 2- Agarose gel electrophoresis image of RT-PCR products from cells that were untreated (a) and treated with compound #135 (b). RT-PCR products were amplified using primers that spanned exon 10 and exon 16 (c).
Figure 3- Schematic of normal and alternative spliced variants isolated from cells treated with compound #135.
Figure 4- The protein domains for both the normal splice transcript that included all sixteen exons, and the variant transcript of MAO-A that excluded exon 14.
MAO-A plays a vital role in the deamination of primary amine substrates and neurotransmitters. Abnormal MAO-A activity has been linked to various psychiatric disorders. Chemical inhibitors have been used as treatments to modulate MAO-A activity in the past. However, there are many dangerous side effects associated with their use. Various compounds were used to induce alternative splicing in HepG2 liver carcinoma cells. By observing alternative splicing and differential expression of MAO-A we can better understand its role in psychiatric health and investigate potential treatments.
A special thank you to Dr. Rubin who gave me the opportunity and support for this project. I would also like to thank Anthony Evans and Catharina Grubaugh for their endless guidance and patience throughout this project. Their dedication and work ethic is inspiring.