Noah Ziemba
RNA Sequencing Analysis
This was a research project conducted through the University of Pittsburgh Vascular Medicine Institute to investigate the functions of the proteins mARC1 and mARC2. In order to adapt to the virtual environment, I obtained data from online databases about the mARC protein's expression in different human tissues. This data was then sorted and statistical analysis done to determined where the mARC proteins were most highly expressed in the body.
Problem
The study of mARC enzymes plays an important role in understand human physiology; however, much is still unknown about these enzymes. The natural function and physiological substrate of mARC have yet to be uncovered. There are two mARC paralogs, mARC1 and mARC2, located on human chromosome 1. These proteins are molybdenum enzymes, a group of enzymes characterized by a molybdenum cofactor near the active site. mARC is an oxidoreductase that acts on a range of substrates, primarily N-hydroxylated compounds (Tejada-Jimenez et al., 2018). The mARC enzymes have also been shown to reduce nitrite to nitric oxide (NO) (Sparacino-Watkins et al., 2014).
(Sparacino-Watkins)
(Schneider et al., 2018)
The current literature on mARC suggests roles in detoxification, lipid metabolism, and NO signaling. More evidence is needed to determine if these hypotheses are true. To establish the physiological function of mARC, the location of where the mARC proteins are expressed needs to be determined. The Human Protein Atlas is an open access data bank established to expand scientist’s knowledge on human protein expression (Uhlén et al., 2015). The database utilizes a variety of technologies and projects to provide data on RNA and protein levels in cells and tissues throughout the body. The Genotype-Tissue Expression project (GTEx) is an NIH funded project which collects RNA sequence data from human tissues. The Human Protein Atlas reports data from the GTEx project that includes information on the molybdenum enzymes, mARC1 and mARC2.
Methods
RNA sequencing data was collected from GTEx portal through the Human Protein Atlas (HPA)’ Tissue Atlas database. The mARC1 and mARC2 transcriptional data was obtained for the following tissues: liver, lung, heart - LV, heart – atrial appendage, kidney, visceral fat, subcutaneous fat. Samples were collected according to the standard operating procedures for biobanks laid out by the National Institute of Health. Donors included males and females ranging from ages 20-79. The GTEx project sequences samples using the Illumina TrueSeq library construction protocol with an Illumina TrueSeq RNA sequencer. The data was collected in transcripts per kilobase million (TPM), which normalizes by gene length followed by sequencing depth. The TPM values were then scaled to a sum of one million for further normalization to yield units of pTPM.
(Uhlen et al., 2015)
The information was organized according to donor age and gender in Excel. Graphpad Prim was used to analyze the data, with a significant probability of .05. A two-way ANOVA test was used to compare mARC expression across gender and age groups for each tissue. Multiple t-tests were done to compare transcription levels between male and females for each age group. For the kidney, sample size was limited, and ANOVA and multiple t-tests could not be run due to limitations on power. Instead, a paired t-test was performed to compare the differences in expression between males and females.
Results
mARC2 levels were found to be highest in the liver, lung, and kidney. mARC1 transcription was found to be highest in fat, liver, and lung. Comparing the two proteins, mARC2 levels were higher than mARC1 in all tissues except fat.
In a tissue such as the liver, where mARC is highly expressed, no differences in age or gender were found. The increased levels of mARC2 over mARC1 can be noticed between the two graphs.
In a tissue such as the subcutaneous fat, where mARC is also highly expressed, females expressed significantly more mARC1 than mARC2. Multiple significant differences across age groups were also discovered.
Discussion and Conclusion
The results of this experiment aligned with previous studies which found high levels of mARC in the kidney, liver, and fat. The high levels of mARC in the fat and liver support hypotheses that the proteins could be involved in process such as detoxification or lipid metabolism. In the future, I plan to examine mARC expression across cell types using the Tabula Muris database, and to carry out gene regulation studies using Illumina's Correlation Engine. With these studies I hope to gain a better understanding of how the mARC proteins function in the human body.