Stefan Maas

Widespread A-to-I RNA Editing of Alu-Containing mRNAs in the Human Transcriptome

RNA editing by adenosine deamination generates RNA and protein diversity through the posttranscriptional modification of single nucleotides in RNA sequences. Few mammalian A-to-I edited genes have been identified despite evidence that many more should exist. Here we identify intramolecular pairs of Alu elements as a major target for editing in the human transcriptome. An experimental demonstration in 43 genes was extended by a broader computational analysis of more than 100,000 human mRNAs. We find that 1,445 human mRNAs (1.4%) are subject to RNA editing at more than 14,500 sites, and our data further suggest that the vast majority of pre-mRNAs (greater than 85%) are targeted in introns by the editing machinery. The editing levels of Alu-containing mRNAs correlate with distance and homology between inverted repeats and vary in different tissues. Alu-mediated RNA duplexes targeted by RNA editing are formed intramolecularly, whereas editing due to intermolecular base-pairing appears to be negligible. We present evidence that these editing events can lead to the posttranscriptional creation or elimination of splice signals affecting alternatively spliced Alu-derived exons. The analysis suggests that modification of repetitive elements is a predominant activity for RNA editing with significant implications for cellular gene expression.

Molecular diversity through RNA editing: a balancing act

RNA editing by adenosine deamination fuels the generation of RNA and protein diversity in eukaryotes, particularly in higher organisms. This includes the recoding of translated exons, widespread editing of retrotransposon-derived repeat elements and sequence modification of microRNA (miRNA) transcripts. Such changes can bring about specific amino acid substitutions, alternative splicing and changes in gene expression levels. Although the overall prevalence of adenosine-to-inosine (A-to-I) editing and its specific functional impact on many of the affected genes is not yet known, the importance of balancing RNA modification levels across time and space is becoming increasingly evident. In particular, transcriptome instabilities in the form of too much or too little RNA editing activity, or misguided editing, manifest in several human disease phenotypes and can disrupt that balance.

RNA editing: a driving force for adaptive evolution?

Genetic variability is considered a key to the evolvability of species. The conversion of an adenosine (A) to inosine (I) in primary RNA transcripts can result in an amino acid change in the encoded protein, a change in secondary structure of the RNA, creation or destruction of a splice consensus site, or otherwise alter RNA fate. Substantial transcriptome and proteome variability is generated by A‐to‐I RNA editing through site‐selective post‐transcriptional recoding of single nucleotides. We posit that this epigenetic source of phenotypic variation is an unrecognized mechanism of adaptive evolution. The genetic variation introduced through editing occurs at low evolutionary cost since predominant production of the wild‐type protein is retained. This property even allows exploration of sequence space that is inaccessible through mutation, leading to increased phenotypic plasticity and provides an evolutionary advantage for acclimatization as well as long‐term adaptation. Furthermore, continuous probing for novel RNA editing sites throughout the transcriptome is an intrinsic property of the editing machinery and represents the molecular basis for increased adaptability. We propose that higher organisms have therefore evolved to systems with increasing RNA editing activity and, as a result, to more complex systems.

Novel Exon of Mammalian ADAR2 Extends Open Reading Frame

Background The post-transcriptional processing of pre-mRNAs by RNA editing contributes significantly to the complexity of the mammalian transcriptome. RNA editing by site-selective A-to-I modification also regulates protein function through recoding of genomically specified sequences. The adenosine deaminase ADAR2 is the main enzyme responsible for recoding editing and loss of ADAR2 function in mice leads to a phenotype of epilepsy and premature death. Although A-to-I RNA editing is known to be subject to developmental and cell-type specific regulation, there is little knowledge regarding the mechanisms that regulate RNA editing in vivo. Therefore, the characterization of ADAR expression and identification of alternative ADAR variants is an important prerequisite for understanding the mechanisms for regulation of RNA editing and the causes for deregulation in disease. Methodology/Principal Findings Here we present evidence for a new ADAR2 splice variant that extends the open reading frame of ADAR2 by 49 amino acids through the utilization of an exon located 18 kilobases upstream of the previously annotated first coding exon and driven by a candidate alternative promoter. Interestingly, the 49 amino acid extension harbors a sequence motif that is closely related to the R-domain of ADAR3 where it has been shown to function as a basic, single-stranded RNA binding domain. Quantitative expression analysis shows that expression of the novel ADAR2 splice variant is tissue specific being highest in the cerebellum. Conclusions/Significance The strong sequence conservation of the ADAR2 R-domain between human, mouse and rat ADAR2 genes suggests a conserved function for this isoform of the RNA editing enzyme.

Identification of a selective nuclear import signal in adenosine deaminases acting on RNA

The adenosine deaminases acting on RNA (ADARs) comprise a family of RNA editing enzymes that selectively modify single codons within RNA primary transcripts with often profound impact on protein function. Little is known about the mechanisms that regulate nuclear RNA editing activity. Editing levels show cell-type specific and developmental modulation that does not strictly coincide with observed expression levels of ADARs. Here, we provide evidence for a molecular mechanism that might control nuclear import of specific ADARs and, in turn, nuclear RNA editing. We identify an in vivo ADAR3 interaction partner, importin alpha 1 (KPNA2) that specifically recognizes an arginine-rich ADAR3 sequence motif and show that it acts as a functional nuclear localization sequence. Furthermore, whereas KPNA2, but not KPNA1 or KNPA3, recognizes the ADAR3 NLS, we observe the converse binding specificity with ADAR2. Interestingly, alternative splicing of ADAR2 pre-mRNA introduces an ADAR3-like NLS that alters the interaction profile with the importins. Thus, in vivo RNA editing might be regulated, in part, through controlled subcellular localization of ADARs, which in turn is governed by the coordinated local expression of importin α proteins and ADAR protein variants.

Posttranscriptional recoding by RNA editing.

The posttranscriptional recoding of nuclear RNA transcripts has emerged as an important regulatory mechanism during eukaryotic gene expression. In particular the deamination of adenosine to inosine (interpreted by the translational machinery as a guanosine) is a frequent event that can recode the meaning of amino acid codons in translated exons, lead to structural changes in the RNA fold, or may affect splice consensus or regulatory sequence sites in noncoding exons or introns and modulate the biogenesis of small RNAs. The molecular mechanism of how the RNA editing machinery and its substrates recognize and interact with each other is not understood well enough to allow for the ab initio delineation of bona fide RNA editing sites. However, progress in the identification of various physiological modification sites and their characterization has given important insights regarding molecular features and events critical for productive RNA editing reactions. In addition, structural studies using components of the RNA editing machinery and together with editing competent substrate molecules have provided information on the chemical mechanism of adenosine deamination within the context of RNA molecules. Here, I give an overview of the process of adenosine deamination RNA editing and describe its relationship to other RNA processing events and its currently established roles in gene regulation.

Altered editing in RNA editing adenosine deaminase ADAR2 gene transcripts of systemic lupus erythematosus T lymphocytes

Adenosine Deaminases that act on RNA (ADARs) edit gene transcripts through site‐specific conversion of adenosine to inosine by hydrolytic deamination at C6 of the adenosine. ADAR2 gene transcripts are substrates for the ADAR1 and ADAR2 enzymes and their expression is regulated by editing at the −u200a1 and −u200a2 sites. Our previous experiments demonstrated up‐regulation of type I interferon (IFN) inducible 150u2003kDa ADAR1 in systemic lupus erythematosus (SLE) T cells. In this study we investigate the role of ADAR1 and ADAR2 in editing of ADAR2 gene transcripts of healthy controls and SLE patients. The ADAR2 gene transcripts were cloned into pCR2.1‐TOPO vectors. A total of 150 clones from SLE and 150 clones from controls were sequenced. Sequence analysis demonstrated A to I editing at −u200a1, +u200a10, +u200a23 and +u200a24 in normal T cells. In SLE clones site‐selective editing of the −u200a2 site was observed as a result of type I IFN‐inducible 150u2003kDa ADAR1 expression. These results are confirmed by analysing ADAR2 transcripts of normal T cells activated with type I IFN‐α. Editing of the +u200a23 and +u200a24 sites was decreased in SLE T cells compared to normal controls. In addition to A to G changes, U to C discrepancies were observed in normal and SLE T cells. In SLE cells, positions −u200a6 and +u200a30 were frequently edited from U to C compared to normal controls. Taken together, these results demonstrate altered and site‐selective editing in ADAR2 transcripts of SLE patients. Based on these results, it is proposed that altered transcript editing contributes to the modulation of gene expression and immune functions in SLE patients.

Genome-wide evaluation and discovery of vertebrate A-to-I RNA editing sites.

RNA editing by adenosine deamination, catalyzed by adenosine deaminases acting on RNA (ADAR), is a post-transcriptional modification that contributes to transcriptome and proteome diversity and is widespread in mammals. Here we administer a bioinformatics search strategy to the human and mouse genomes to explore the landscape of A-to-I RNA editing. In both organisms we find evidence for high excess of A/G-type discrepancies (inosine appears as a guanosine in cloned cDNA) at non-polymorphic, non-synonymous codon sites over other types of discrepancies, suggesting the existence of several thousand recoding editing sites in the human and mouse genomes. We experimentally validate recoding-type A-to-I RNA editing in a number of human genes with high scoring positions including the coatomer protein complex subunit alpha (COPA) as well as cyclin dependent kinase CDK13.

Altered editing in cyclic nucleotide phosphodiesterase 8A1 gene transcripts of systemic lupus erythematosus T lymphocytes

The aetiopathogenesis of the abnormal immune response in systemic lupus erythematosus (SLE) remains incompletely understood. We and other investigators demonstrated altered expression of adenosine deaminase that act on RNA (ADAR) genes in SLE patients. Based on this information, we hypothesize that the altered expression and function of ADAR enzymes is a mechanism for the immunopathogenesis of SLE. ADARs edit gene transcripts through site‐specific conversion of adenosine to inosine by hydrolytic deamination at C6 of the adenosine. Thirteen SLE subjects and eight healthy controls were studied. We assessed the role of ADAR enzymes in editing of PDE8A1 gene transcripts of normal and SLE T cells. These studies demonstrated the occurrence of ADAR‐catalysed altered and site‐selective editing profile of specific sites in the PDE8A1 gene transcripts of normal and SLE T cells. Two hot spots for A to I editing were observed in the PDE8A1 transcripts of normal and SLE T cells. A fundamental finding of this study is A to I hypo‐editing followed by up‐regulation of PDE8A1 transcripts in SLE T cells. These results are confirmed by analysing PDE8A1 transcripts of normal T cells activated with type I interferon‐α. It is proposed that, the altered expression of ADAR enzymes tilt the balance of editing machinery and alter editing in SLE transcriptome. Such altered editing may contribute to the modulation of gene regulation and ultimately, immune functions in SLE and play an important role in the initiation and propagation of SLE pathogenesis.

IGFBP7’s susceptibility to proteolysis is altered by A-to-I RNA editing of its transcript

MT‐SP1 cleaves IGFBP7 by protease assay (View interaction).