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

Hu Zhu,George J Jurjus,George J Jurjus,Wesley K Kroeze,Daniel J Urban,James C Overholser,Matthew T Mcpheeters,Piotr Mieczkowski,Jared Blashka,Grazyna Rajkowska,Gouri J Mahajan,Lesa Dieter,Bryan L Roth,Zefeng Wang,Patrick F Sullivan,Craig A Stockmeier,Craig A Stockmeier,Alfred Lewin

doi:10.1371/journal.pone.0043227

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.

Highlights

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional modification of RNA transcripts catalyzed by ADARs (Adenosine Deaminases Acting on RNA)
Three independent sets of normal human brain samples were used to quantify RNA editing: the first represents sample derived from the cerebral cortex and cerebellum pooled from 10 normal humans (Clontech); the second comprises one subject with 5 different brain regions sampled (Stanley Medical Research Institute; SMRI); and the third is of 5 normal humans with two brain regions sampled (Human Brain Collection Core, Center for Psychiatric Neuroscience (CPN), University of Mississippi Medical Center)
DNA fragments containing the possible editing sites were amplified by reverse transcription–polymerase chain reaction (RT-PCR), and each amplicon was subjected to ultra-high-throughput sequencing (uHTS) using an Illumina Genome Analyzer II platform

Summary

Introduction

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional modification of RNA transcripts catalyzed by ADARs (Adenosine Deaminases Acting on RNA). In the first and second sets of normal human brain samples, we reanalyzed the frequency of RNA editing of 36 sites from category I in the cerebral cortex and cerebellum.

Results

Conclusion