Quantitative Analysis of Focused A-To-I RNA Editing Sites by Ultra-High-Throughput Sequencing in Psychiatric Disorders

Abstract

A-to-I RNA editing is a post-transcriptional modification of single nucleotides in RNA by adenosine deamination, which thereby diversifies the gene products encoded in the genome. Thousands of potential RNA editing sites have been identified by recent studies (e.g. see Li et al, Science 2009); however, only a handful of these sites have been independently confirmed. Here, we systematically and quantitatively examined 109 putative coding region A-to-I RNA editing sites in three sets of normal human brain samples by ultra-high-throughput sequencing (uHTS). Forty of 109 putative sites, including 25 previously confirmed sites, were validated as truly edited in our brain samples, suggesting an overestimation of A-to-I RNA editing in these putative sites by Li et al (2009). To evaluate RNA editing in human disease, we analyzed 29 of the confirmed sites in subjects with major depressive disorder and schizophrenia using uHTS. In striking contrast to many prior studies, we did not find significant alterations in the frequency of RNA editing at any of the editing sites in samples from these patients, including within the 5HT2C serotonin receptor (HTR2C). Our results indicate that uHTS is a fast, quantitative and high-throughput method to assess RNA editing in human physiology and disease and that many prior studies of RNA editing may overestimate both the extent and disease-related variability of RNA editing at the sites we examined in the human brain.

Whole transcriptome RNA-Seq allelic expression in human brain

“…In addition, the variability of RNA editing is not significantly different within the 10 regions as compared to the 13 DLPFC, suggesting RNA editing is tightly regulated at these 12 sites. These findings could be driven by our stringent criteria for designating RNA editing sites, but other studies have found similarly consistent levels of intra- versus inter-individual adenosine-to-inosine editing [40] and at greater depth in the brain [41]. …”

Functional Status of the Serotonin 5-HT2C Receptor (5-HT2CR) Drives Interlocked Phenotypes that Precipitate Relapse-Like Behaviors in Cocaine Dependence

“…Partially edited isoforms exhibit profiles between these two extremes. Although these multiple isoforms are predicted as key in 5-HT 2C R function, there are no published methods to separately analyze expression of isoform-specific proteins; however, high-throughput sequencing provides the opportunity to evaluate expression of mRNA for all 5-HT 2C R isoforms across phenotypes (Morabito et al, 2010;Zhu et al, 2012).…”

“…RNA extracted from the mPFC collected from HI and LI rats was analyzed for efficiency of editing at each of the five editing sites (A, B, E, C, D) as well as the frequency of mRNA variants using high-throughput multiplexed transcript analysis (Morabito et al, 2010;Zhu et al, 2012); all 32 possible mRNA variants were detected in the rat mPFC (Supplementary Table 2). A main effect of phenotype (F (1,56) ¼ 9.86, po0.01), site (F (4,56) ¼ 22744.6, po0.001), and a phenotype  site interaction (F (4,56) ¼ 3.22, po0.05) on editing efficiency at the five editing sites was observed; HI rats expressed higher levels of editing at the E and D sites vs LI rats (Figure 3d; po0.05).…”

A-to-I RNA editing does not change with age in the healthy male rat brain

“…In this study, only male rats were analyzed, so sex specific effects cannot be ruled out. However, no differences in RNA editing have been reported to date between sexes (Zhu et al 2012), and no differences in gender were observed during aging in humans (Nicholas et al 2010). …”