A Study of RNA Editing in Human Peripheral Blood Mononuclears and Endotheliocytes by NextGen Sequencing

Abstract

RNA editing is a process of post-transcriptional modification of the nucleotide sequence in a transcript that leads to a change in the information content of the RNA. The alteration of nucleotides in the mRNA can have several consequences at the molecular level, including modified protein products as well as creation or deletion of splice sites. Two types of RNA editing have been known to take place in mRNA: Adenosine (A) to Inosine (I) and Cytidine (C) to Uridine (U), both of which involve deamination of the nucleotide with the help of deaminase enzymes. The “A to I” RNA editing is facilitated by deaminating enzymes known as ADARs (Adenosine Deaminase that Act on RNA). ADARs specifically recognize double stranded RNA structure or RNA duplex structure as their substrate. Though previous studies have shown some evidence of low level sporadic occurrence of RNA editing in other human tissues, brain remains to be the only tissue where RNA editing has been studied systematically. In this study, we investigated “A to I” RNA editing in human PBMCs (peripheral blood mononuclear cells). We were also interested in looking at RNA editing in the umbilical endotheliocytes that are capable of producing interferons. Specifically, the cDNA derived from PBMCs and endothelial cells, was sequenced by NextGen sequencing. The PBMCs’ genomic DNA was sequenced by Sanger sequencing and the sequences were analyzed. Since inosine is recognized as guanosine (G) by most enzymes, A-to-I substitution leads to A-to-G transition in the edited substrate and therefore, any kind of “A to I” RNA editing event will be detected as A to G changes in the transcripts. The multiple sequence alignment tool SeqmanPro was used to assemble all the transcripts together, which revealed that the A to G change frequency was significantly high over 14% in TLR2 at a specific site, in most of the samples of PBMCs and endotheliocytes. At the same consensus position 26, the PMBCs genomic DNA possessed an Adenosine, which was confirmed by the distinct peak in the chromatogram obtained from Sequencher software. The presence of these A to G mismatches within the transcripts, therefore, suggests that “A to I” RNA editing takes place in PBMCs and in endothelial cells.