A computational screen for site selective A-to-I editing detects novel sites in neuron specific Hu proteins

Mats Ensterö,Daniel Lundin,Bei Wang,Örjan Åkerborg,Marie Öhman,Terrence S Furey,Jens Lagergren

doi:10.1186/1471-2105-11-6

Abstract

BackgroundSeveral bioinformatic approaches have previously been used to find novel sites of ADAR mediated A-to-I RNA editing in human. These studies have discovered thousands of genes that are hyper-edited in their non-coding intronic regions, especially in alu retrotransposable elements, but very few substrates that are site-selectively edited in coding regions. Known RNA edited substrates suggest, however, that site selective A-to-I editing is particularly important for normal brain development in mammals.ResultsWe have compiled a screen that enables the identification of new sites of site-selective editing, primarily in coding sequences. To avoid hyper-edited repeat regions, we applied our screen to the alu-free mouse genome. Focusing on the mouse also facilitated better experimental verification. To identify candidate sites of RNA editing, we first performed an explorative screen based on RNA structure and genomic sequence conservation. We further evaluated the results of the explorative screen by determining which transcripts were enriched for A-G mismatches between the genomic template and the expressed sequence since the editing product, inosine (I), is read as guanosine (G) by the translational machinery. For expressed sequences, we only considered coding regions to focus entirely on re-coding events. Lastly, we refined the results from the explorative screen using a novel scoring scheme based on characteristics for known A-to-I edited sites. The extent of editing in the final candidate genes was verified using total RNA from mouse brain and 454 sequencing.ConclusionsUsing this method, we identified and confirmed efficient editing at one site in the Gabra3 gene. Editing was also verified at several other novel sites within candidates predicted to be edited. Five of these sites are situated in genes coding for the neuron-specific RNA binding proteins HuB and HuD.

Highlights

Several bioinformatic approaches have previously been used to find novel sites of ADAR mediated adenosines to inosines (A-to-I) RNA editing in human
Prediction of RNA stem structures within mouse genes Previous work in our laboratory have included the use of Mouse Genome 430A 2.0 Array (Affymetrix) to experimentally detect novel sites of A-to-I editing [18]
We first extracted the genome sequences corresponding to the genes on the above mentioned microarray from the mouse genome assembly release 8 (Mm8) [21]

Summary

Introduction

Several bioinformatic approaches have previously been used to find novel sites of ADAR mediated A-to-I RNA editing in human. On the pre-mRNA level, alternative splicing is a well-known mechanism altering transcripts This type of alternative processing is important in the nervous system, where it helps determine the properties of many types of neurons [1]. The known substrates for site selective editing typically have a functional significance due to non-synonymous alteration of a codon that alters the amino acid sequence. Both RNA strands of a substrate stem often show high conservation of sequence as well as structure in species from human to chicken [5,6,7]. Even though only a handful of substrates have been identified, editing has proven to be important for the function of the developing brain in both invertebrates [9] and vertebrates [10,11,12]

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