No abstract

RNA editing by members of the ADAR (adenosine deaminases acting on RNA) family leads to site-specific conversion of adenosine to inosine (A-to-I) in precursor messenger RNAs. Editing by ADARs is believed to occur in all metazoa, and is essential for mammalian development. Currently, only a limited number of human ADAR substrates are known, whereas indirect evidence suggests a substantial fraction of all pre-mRNAs being affected. Here we describe a computational search for ADAR editing sites in the human transcriptome, using millions of available expressed sequences. We mapped 12,723 A-to-I editing sites in 1,637 different genes, with an estimated accuracy of 95%, raising the number of known editing sites by two orders of magnitude. We experimentally validated our method by verifying the occurrence of editing in 26 novel substrates. A-to-I editing in humans primarily occurs in noncoding regions of the RNA, typically in Alu repeats. Analysis of the large set of editing sites indicates the role of editing in controlling dsRNA stability.

We identify a set of 575 human genes that are expressed in all conditions tested in a publicly available database of microarray results. Based on this common occurrence, the set is expected to be rich in "housekeeping" genes, showing constitutive expression in all tissues. We compare selected aspects of their genomic structure with a set of background genes. We find that the introns, untranslated regions and coding sequences of the housekeeping genes are shorter, indicating a selection for compactness in these genes. 1The amazing diversity of the human body stems from the different expression patterns of genes in different tissues. Although most genes show constitutive expression in only a subset of tissues, some gene products are required for the maintenance of the basal cellular function and are constitutively found in all human cells. These genes are called housekeeping genes (HK genes) [1]. HK genes can be used to calibrate measurements of gene expression [2].They might also help to define the minimal gene complement needed for a human cell [1].Several attempts have been made recently to define the complete set of HK genes [3,4].Microarrays are often used to identify sets of genes that are expressed either ubiquitously or in specific tissues or conditions. However, the technique is technically demanding and prone to artifacts, so independent evidence is often required to confirm the results. In principle, identifying the set of HK genes using microarray data is straightforward; one need only look for genes that are expressed in all tissues and all experimental conditions.Employing such an approach has so far resulted in two lists of HK genes [3,4]. However, problems in probe design, measurement noise and other artifacts introduce inevitable errors in such lists. Because a northern blot experiment for each gene in each tissue is impractical, an independent test is needed to validate any list of HK genes. Here, we report a validation test that uses a recently discovered property of highly expressed genes.The transcription process is both slow and costly; it takes 50 milliseconds [5,6] and two ATP molecules [7] approximately to transcribe a nucleotide. This might be expected to provide selective pressure to make genes as short as functionally possible. The more copies of a gene required for the organism, the stronger this pressure should be. The first demonstration of this principle [8] showed that genes with a large number of expressed sequence tags (ESTs) in public libraries (and hence most mRNAs) have a significantly shorter average intron length than those with fewer ESTs.Here, an implication of this principle is used to validate a set of HK genes. The HK genes, which are transcribed in all somatic cells and under all circumstances, are by nature highly expressed, and therefore should be selected to have shorter introns. We used a recently published database of microarray experiments [9] to identify a set of HK genes. As a further validation step, we checked the Gene Ontology (GO) annotation of thes...

RNA molecules transmit the information encoded in the genome and generally reflect its content. Adenosine-to-inosine (A-to-I) RNA editing by ADAR proteins converts a genomically encoded adenosine into inosine. It is known that most RNA editing in human takes place in the primate-specific Alu sequences, but the extent of this phenomenon and its effect on transcriptome diversity are not yet clear. Here, we analyzed large-scale RNA-seq data and detected~1.6 million editing sites. As detection sensitivity increases with sequencing coverage, we performed ultradeep sequencing of selected Alu sequences and showed that the scope of editing is much larger than anticipated. We found that virtually all adenosines within Alu repeats that form double-stranded RNA undergo A-to-I editing, although most sites exhibit editing at only low levels (<1%). Moreover, using high coverage sequencing, we observed editing of transcripts resulting from residual antisense expression, doubling the number of edited sites in the human genome. Based on bioinformatic analyses and deep targeted sequencing, we estimate that there are over 100 million human Alu RNA editing sites, located in the majority of human genes. These findings set the stage for exploring how this primate-specific massive diversification of the transcriptome is utilized.

Modifications of RNA affect its function and stability. RNA editing is unique among these modifications because it not only alters the cellular fate of RNA molecules but also alters their sequence relative to the genome. The most common type of RNA editing is A-to-I editing by double-stranded RNA-specific adenosine deaminase (ADAR) enzymes. Recent transcriptomic studies have identified a number of 'recoding' sites at which A-to-I editing results in non-synonymous substitutions in protein-coding sequences. Many of these recoding sites are conserved within (but not usually across) lineages, are under positive selection and have functional and evolutionary importance. However, systematic mapping of the editome across the animal kingdom has revealed that most A-to-I editing sites are located within mobile elements in non-coding parts of the genome. Editing of these non-coding sites is thought to have a critical role in protecting against activation of innate immunity by self-transcripts. Both recoding and non-coding events have implications for genome evolution and, when deregulated, may lead to disease. Finally, ADARs are now being adapted for RNA engineering purposes.