An extra double-stranded RNA binding domain confers high activity to a squid RNA editing enzyme

RNA editing by adenosine deamination is particularly prevalent in the squid nervous system. We hypothesized that the squid editing enzyme might contain structural differences that help explain this phenomenon. As a first step, a squid adenosine deaminase that acts on RNA (sqADAR2a) cDNA and the gene that encodes it were cloned from the giant axon system. PCR and RNase protection assays showed that a splice variant of this clone (sqADAR2b) was also expressed in this tissue. Both versions are homologous to the vertebrate ADAR2 family. sqADAR2b encodes a conventional ADAR2 family member with an evolutionarily conserved deaminase domain and two double-stranded RNA binding domains (dsRBD). sqADAR2a differs from sqADAR2b by containing an optional exon that encodes an “extra” dsRBD. Both splice variants are expressed at comparable levels and are extensively edited, each in a unique pattern. Recombinant sqADAR2a and sqADAR2b, produced in Pichia pastoris, are both active on duplex RNA. Using a standard 48-h protein induction, both sqADAR2a and sqADAR2b exhibit promiscuous self-editing; however, this activity is particularly robust for sqADAR2a. By decreasing the induction time to 16 h, self-editing was mostly eliminated. We next tested the ability of sqADAR2a and sqADAR2b to edit two K(+) channel mRNAs in vitro. Both substrates are known to be edited in squid. For each mRNA, sqADAR2a edited many more sites than sqADAR2b. These data suggest that the “extra” dsRBD confers high activity on sqADAR2a

Identification of Widespread Ultra-Edited Human RNAs

Adenosine-to-inosine modification of RNA molecules (A-to-I RNA editing) is an important mechanism that increases transciptome diversity. It occurs when a genomically encoded adenosine (A) is converted to an inosine (I) by ADAR proteins. Sequencing reactions read inosine as guanosine (G); therefore, current methods to detect A-to-I editing sites align RNA sequences to their corresponding DNA regions and identify A-to-G mismatches. However, such methods perform poorly on RNAs that underwent extensive editing (“ultra”-editing), as the large number of mismatches obscures the genomic origin of these RNAs. Therefore, only a few anecdotal ultra-edited RNAs have been discovered so far. Here we introduce and apply a novel computational method to identify ultra-edited RNAs. We detected 760 ESTs containing 15,646 editing sites (more than 20 sites per EST, on average), of which 13,668 are novel. Ultra-edited RNAs exhibit the known sequence motif of ADARs and tend to localize in sense strand Alu elements. Compared to sites of mild editing, ultra-editing occurs primarily in Alu-rich regions, where potential base pairing with neighboring, inverted Alus creates particularly long double-stranded RNA structures. Ultra-editing sites are underrepresented in old Alu subfamilies, tend to be non-conserved, and avoid exons, suggesting that ultra-editing is usually deleterious. A possible biological function of ultra-editing could be mediated by non-canonical splicing and cleavage of the RNA near the editing sites

Transcript- and tissue-specific imprinting of a tumour suppressor gene

The Bladder Cancer-Associated Protein gene (BLCAP; previously BC10) is a tumour suppressor that limits cell proliferation and stimulates apoptosis. BLCAP protein or message are downregulated or absent in a variety of human cancers. In mouse and human, the first intron of Blcap/BLCAP contains the distinct Neuronatin (Nnat/NNAT) gene. Nnat is an imprinted gene that is exclusively expressed from the paternally inherited allele. Previous studies found no evidence for imprinting of Blcap in mouse or human. Here we show that Blcap is imprinted in mouse and human brain, but not in other mouse tissues. Moreover, Blcap produces multiple distinct transcripts that exhibit reciprocal allele-specific expression in both mouse and human. We propose that the tissue-specific imprinting of Blcap is due to the particularly high transcriptional activity of Nnat in brain, as has been suggested previously for the similarly organized and imprinted murine Commd1/U2af1-rs1 locus. For Commd1/U2af1-rs1, we show that it too produces distinct transcript variants with reciprocal allele-specific expression. The imprinted expression of BLCAP and its interplay with NNAT at the transcriptional level may be relevant to human carcinogenesis

Evidence for large diversity in the human transcriptome created by Alu RNA editing

Adenosine-to-inosine (A-to-I) RNA editing alters the original genomic content of the human transcriptome and is essential for maintenance of normal life in mammals. A-to-I editing in Alu repeats is abundant in the human genome, with many thousands of expressed Alu sequences undergoing editing. Little is known so far about the contribution of Alu editing to transcriptome complexity. Transcripts derived from a single edited Alu sequence can be edited in multiple sites, and thus could theoretically generate a large number of different transcripts. Here we explored whether the combinatorial potential nature of edited Alu sequences is actually fulfilled in the human transcriptome. We analyzed datasets of editing sites and performed an analysis of a detailed transcript set of one edited Alu sequence. We found that editing appears at many more sites than detected by earlier genomic screens. To a large extent, editing of different sites within the same transcript is only weakly correlated. Thus, rather than finding a few versions of each transcript, a large number of edited variants arise, resulting in immense transcript diversity that eclipses alternative splicing as mechanism of transcriptome diversity, although with less impact on the proteome

Genetic algorithm learning as a robust approach to RNA editing site prediction

BACKGROUND: RNA editing is one of several post-transcriptional modifications that may contribute to organismal complexity in the face of limited gene complement in a genome. One form, known as C → U editing, appears to exist in a wide range of organisms, but most instances of this form of RNA editing have been discovered serendipitously. With the large amount of genomic and transcriptomic data now available, a computational analysis could provide a more rapid means of identifying novel sites of C → U RNA editing. Previous efforts have had some success but also some limitations. We present a computational method for identifying C → U RNA editing sites in genomic sequences that is both robust and generalizable. We evaluate its potential use on the best data set available for these purposes: C → U editing sites in plant mitochondrial genomes. RESULTS: Our method is derived from a machine learning approach known as a genetic algorithm. REGAL (RNA Editing site prediction by Genetic Algorithm Learning) is 87% accurate when tested on three mitochondrial genomes, with an overall sensitivity of 82% and an overall specificity of 91%. REGAL's performance significantly improves on other ab initio approaches to predicting RNA editing sites in this data set. REGAL has a comparable sensitivity and higher specificity than approaches which rely on sequence homology, and it has the advantage that strong sequence conservation is not required for reliable prediction of edit sites. CONCLUSION: Our results suggest that ab initio methods can generate robust classifiers of putative edit sites, and we highlight the value of combinatorial approaches as embodied by genetic algorithms. We present REGAL as one approach with the potential to be generalized to other organisms exhibiting C → U RNA editing

Identification of enhancers of A to I editing in functional in vivo yeast screen

Adenosin Deaminierung von Ribonukleinsäure (RNA) ist ein weitverbreiteter Mechanismus auf posttranskriptioneller Ebene, der zu einer massiven Zunahme der Variabilität des Metazoentranskriptoms führt. Die A zu I Editierung wird von sogenannte Adenosin Deaminasen (ADARs = adenosine deaminases that act on RNA) durchgeführt. Diese Deaminierungen führen zu Veränderungen von Kodons und Spleißmustern und bewirken dadurch eine Steigerung der Zahl von Protein-Isoformen. Zusätzlich spielen ADARs eine wichtige Rolle in der Biogenese, Stabilität und der Zielmolekülauswahl von mikroRNA (miRNA) Molekülen. Die meisten bekannten kodierenden Substrate für ADARs sind Transkripte von Proteingenen, die im Zentralnervensystem exprimiert werden. Darunter fallen z.B.: Glutamatrezeptoren, der Serotonin 2C Rezeptor oder Gamma-Aminobutiryl-Säure Typ A Rezeptor. Nicht überraschend sind Veränderungen im Editierungsmuster einiger dieser Zielsequenzen mit neurodegenerativen Erkrankungen verbunden, wie z.B. Depression, Schizophrenie oder ALS (Amyotrophe laterale Sklerose) sowie Hirntumoren. Daher ist es entscheidend mehr über die Aktivitätskontrolle von ADARs zu erfahren, um etwaige Therapieansätze zu entwickeln. Die Funktionen und Aktivitäten von Deaminasen sind sehr gut bekannt, Mechanismen der Regulation von A zu I Editierung sind bisher jedoch noch sehr wenig untersucht. Die Regulation einiger ADAR Isoformen passiert auf transkriptioneller Ebene, es gibt aber zunehmend Hinweise darauf, dass es noch weitere Regulationsmechanismen gibt. Die Aufklärung einiger solcher Regulationsmechanismen war das Hautpziel dieser Dissertationsarbeit. Zu diesem Zweck wurde ein in vivo Editing System in Hefe entwickelt, mit dessen Hilfe es möglich war, zelluläre Verstärkermoleküle im RNA-Editierungsprozess zu identifizieren. Das Protein DSS1 wurde dabei als besonders aktiver Verstärker isoliert. Die Eigenschaften von DSS1 wurden anschließend in einem Expressionssystem im Säugerhintergrund an unterschiedlichen Editierungssubstraten bestätigt, wodurch auch die Funktionalität des neu entwickelten Screening Systems bewiesen werden konnte.Adenosine to inosine editing of RNA is a widespread posttranscriptional mechanism increasing the variety of the transcriptome in metazoa. Adenosine deamination is performed by adenosine deaminases that act on RNA (ADARs). A to I editing can change codons or splice patterns and lead to a higher diversity in protein isoforms. Additionally ADARs play an important role in miRNA biogenesis, stability and choice of their silencing target. Most known coding editing substrates are the transcripts of proteins expressed in the central nervous system like: glutamate receptors, serotonin 2C receptor or gamma-aminobutyric acid type A receptor. Not surprisingly, changes in editing patterns at some of these sites are linked to neurodegenerative diseases like: schizophrenia, depression or ALS (amyotrophic lateral sclerosis) and also to brain tumours. It is important to learn how the activity of ADARs is controlled in attempt to cure some of these diseases. Although much is known about function and activity of the enzymes, regulation of A to I editing is not well understood so far. Some ADAR isoforms are regulated at the transcriptional level but an increasing amount of evidence suggests other mechanisms of regulation in addition. The aim of this thesis was answering some of the open questions concerning the regulation of ADAR activity. A functional yeast in vivo editing system was developed and successfully employed in identification of cellular enhancers of RNA editing. In this screen the protein DSS1 could be identified as strong enhancer of editing. The properities of this protein were confirmed in the mammalian context on various editing substrates proving the functionality of the developed screening system

Establishing stable cell lines for the generation of interaction profiles of proteins involved in RNA editing

Adenosin zu Inosin Editierung von Ribonukleinsäuren (RNA) ist eine konservierte post-transkriptionelle Modifikation in höheren Metazoen. Die hydrolytische Desaminierung von Adenosin zu Inosin wird von einer Enzymfamilie durchgeführt, welche als Adenosin-Deaminasen bekannt sind (ADARs) und doppelsträngige RNA desaminieren. Durch RNA-Editierung wird die Sequenz eines primären Transkriptes verändert, was dramatische Auswirkungen haben kann: Editierung der mRNA eines kodierenden Transkriptes kann zu alternativem Splicing und Aminosäurensubstitutionen im translatierten Protein führen. Abgesehen davon gehören nicht-codierende RNAs und repetitive Sequenzen, wie Alu-Elemente und micro RNAs, zu den Hauptsubstraten von ADARs. Editierung dieser Substrate kann Einfluss auf die Expression von Genen haben, weil die Sequenz regulatorischer Elemente und kleiner RNAs verändert wird. Durch aktuelle Studien wurden bereits viele Substrate für ADAR-vermittelte Editierung identifiziert. Jedoch sind Auswirkungen der Editierung auf die Funktion vieler prozessierter Substrate noch nicht bekannt. Zusätzlich zu den unbekannten Konsequenzen der RNA-Editierung gibt auch die Regulation dieses Mechanismus Rätsel auf. In den vergangenen Jahren wurden in unserer Arbeitsgruppe einige Kandidaten identifiziert, welche als mögliche Regulatoren für RNA-Editierung in Frage kommen. In diesem Projekt haben wir versucht, diese Kandidaten stabil in Säugerzellen zu exprimieren, um durch Aufreinigungsmethoden und anschließende massenspektrometrische Analyse Interaktionsnetzwerke dieser Kandidaten aufzuklären. Der zweite Teil dieser Arbeit behandelt zwei Substrate für RNA-Editierung: das zytoskelettale Protein Filamin A und BLCAP, ein Protein, assoziiert mit der Entstehung von Blasenkrebs. Editierte und nicht editierte Versionen dieser Proteine wurden stabil in Säugerzellen exprimiert. Durch Aufreinigungsexperimente unter nativen Bedingungen und anschließender massenspektrometrischer Analyse konnten einige Proteine identifiziert werden, welche mit diesen ADAR-Substraten interagieren.Adenosine to inosine RNA editing is a posttranscriptional modification highly conserved in higher metazoa. The hydrolytic deamination of adenosine to inosine is catalysed by a family of enzymes, known as adenosine deaminases that act on double-stranded RNA (ADARs). Changing the sequence of a primary transcript by RNA editing can have dramatic consequences: Editing of the pre-mRNA of a coding transcript can lead to alternative splicing events and may cause amino acid substitutions in the translated protein, as the triplet codon becomes changed. Apart from that, most of the known substrates of RNA editing are non-coding RNAs and repetitive elements, as Alu elements in untranslated regions of the transcript, and micro RNAs. These editing events can influence gene expression, as the sequence of regulatory elements or the target specificity of small RNAs is altered. To date, on-going studies have identified many targets of ADAR editing. However, very little is known about the consequences of the editing events. In addition to the consequences of editing on its targets, mechanisms of regulation of A to I editing are still unclear. In the last few years several candidates for regulators of ADAR activity have been identified in our lab. In this thesis we stably expressed these candidates in mammalian cell lines for purification assays. Subsequent mass spectrometric analysis of purified complexes led to the identification of proteins interacting with editing regulator candidates, what may help to clarify regulatory networks involved in A to I RNA editing. The second part of this project deals with two protein-coding targets of RNA editing: The effect of RNA editing on the interaction profiles of the cytoskeletal cross-linker Filamin A, and the bladder cancer associated protein BLCAP is investigated. Edited and unedited versions of both proteins were stably expressed in mammalian cell lines. Purification of the two targets of RNA editing under native conditions led to the identification of interacting proteins after mass spectrometric analysis