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N o v e l s i t e s o f A -t o -I R N A e d i t i ng i n t h e m a m ma l i an b ra i n Johan Ohlson Novel sites of A-to-I RNA editing in the mammalian brain Johan Ohlson Stockholm University The cover picture shows an immunolabeling experiment of the α3 subunit of the GABAA receptor, using an anti-α3 antibody in non-permeabilized HEK 293 cells. ©Johan Ohlson, Stockholm 2007 ISBN 978-91-7155-498-7 Printed in Sweden by Printers name, City 20XX Distributor: Stockholm University Library ABSTRACT The number of protein-coding genes are likely not sufficient to account for the complexity of higher organisms. It is plausible that the proteome is responsible for the complexity of an organism. An important mechanism that increases the protein variability is posttranscriptional modifications that alter the pre-mRNA sequence from that encoded in the genome. In this thesis work I have been focusing on a posttranscriptional process where adenosine (A) is deaminated to inosine (I), Ato-I RNA editing. Inosine is read as a guanosine (G) by the translation machinery, editing within coding regions can therefore give rise to more than one protein isoform from a single gene. A-to-I RNA editing is catalyzed by members of the ADAR enzyme family. ADARs have been found in all metazoans tested and two active ADAR proteins, ADAR1 and ADAR2, have been found in mammals. However, recoding by A-to-I editing is a rarely found event in mammals. To detect novel substrates for A-to-I editing we developed an experimental approach to pull down ADAR2 substrates using immunoprecipitations. The captured RNAs were identified by microarray analysis. In this thesis two novel substrates for A-to-I editing are presented that were found using our IP-array approach, in combination with bioinformatic techniques. The transcript coding for the GABAA receptor subunit α3 (Gabra-3) was found to be selectively edited by both ADAR1 and ADAR2. Editing of Gabra-3 recodes an isoleucine to a methionine and it was found to have a negative effect on the Gabra-3 assembly into the receptor. Moreover, the mouse specific CTN-RNA that codes for the CAT2 Transcribed NuclearRNA was shown to be hyper-edited by ADAR2. In conclusion, this thesis work has resulted in an experimental method that extracts ADAR substrates. Two novel editing substrates were discovered. Our data adds additional evidence to the fact that RNA editing is of principal significance for a functional brain. List of papers included in the thesis The thesis is based on the following articles, which will be referred to by their Roman numerals in the text. I. Johan Ohlson, Mats Ensterö, Britt-Marie Sjöberg and Marie Öhman (2005) A method to find tissue-specific novel sites of selective adenosine deamination. Nucleic Acids Res. 33:e167 II. Johan Ohlson, Jakob S. Pedersen, David Haussler and Marie Öhman (2007) Editing modifies the GABAA receptor subunit α3. RNA 13:698-703 III. Johan Ohlson, Chammiran Daniel, Petra Björk, Henrik Boije, Finn Hallböök and Marie Öhman. A-to-I editing regulates GABAA receptor assembly. Manuscript IV. Johan Ohlson, Kannanganattu V. Prasanth, Helene Wahlstedt, David L. Spector and Marie Öhman. ADAR2 edits the CTN-RNA that is retained in the nucleus. Manuscript Paper not included in the thesis Johan Ohlson and Marie Öhman (2007) A method to find sites of selective adenosine deamination. Methods Enzymol. 2007;424:289-300 Reprints were made with the permission from the copyright holders. Contents ABSTRACT ..........................................................................................................v Introduction ....................................................................................................... 13 A historical introduction............................................................................................... 13 ADAR1 ....................................................................................................................... 14 Discovering ADAR2 and ADAR3 ................................................................................ 15 ADAR2 ....................................................................................................................... 15 ADAR3 ....................................................................................................................... 16 ADAR in the evolution................................................................................................. 16 Substrate specificity for selective A-to-I editing........................................................... 17 Dimer formation or not ................................................................................................ 18 Crystal structure of ADAR2......................................................................................... 19 The flip-out model....................................................................................................... 19 Editing selectivity ........................................................................................................ 19 Roles for the different ADAR domains ........................................................................ 20 Editing site complementary sequence is required for editing ...................................... 21 ADAR and gene regulation ......................................................................................... 22 Site selective editing substrates.................................................................................. 23 The glutamate receptors ....................................................................................... 23 The serotonin receptor .......................................................................................... 26 The potassium channel ......................................................................................... 26 ADAR2 .................................................................................................................. 26 The endothelin B receptor ..................................................................................... 27 The tyrosine phosphatase, non-receptor type 6 gene............................................ 27 The hepatitis delta virus and the polyomavirus...................................................... 27 Other candidates for A-to-I editing......................................................................... 28 miRNAs ................................................................................................................. 28 Hyper-editing .............................................................................................................. 29 Why receptors? .......................................................................................................... 30 ADAR is required for normal life ................................................................................. 31 RNA editing and human disease phenotypes ............................................................. 32 Methods to detect novel editing sites .......................................................................... 32 Present Investigation ........................................................................................ 34 Aim ............................................................................................................................. 34 Paper I - the method ................................................................................................... 34 Paper II - finding novel substrates for A-to-I editing .................................................... 36 Paper III - the function of GABA receptor editing ........................................................ 38 Paper IV – CTN-RNA, a novel substrate for ADAR editing ......................................... 39 Future studies ................................................................................................... 41 Detecting novel sites of selective editing .................................................................... 41 Knockin mice .............................................................................................................. 41 Other objectives using the IP-array method ................................................................ 42 Concluding remarks.......................................................................................... 43 Acknowledgement ............................................................................................ 44 References ........................................................................................................ 46 Abbreviations A ADAR CTN-RNA dsRBM dsRNA ECS G GABAA Gabra-3 GluR I mRNA NES NLS Pre-mRNA RT-PCR Adenosine Adenosine deaminase that acts on RNA CAT2 Transcribed Nuclear-RNA Double stranded RNA binding motif Double stranded RNA Editing complementary sequence Guanosine gamma-aminobutyric acid type A GABAA receptor subunit α3 Glutamate receptor Inosine Messenger RNA Nuclear export signal Nuclear localization signal Precursor messenger RNA Reverse transcription followed by polymerase chain reaction amplification 12 Introduction A historical introduction When the mitochondrial cytochrome oxidase (cox) subunit II gene from trypanosome was investigated something extraordinary was found. Four extra nucleotides were found in the mRNA of the cox II gene that were not present in the genomic sequence. Four uridines were inserted into the mRNA, creating a functional protein. This finding suggested a novel posttranscriptional modification named RNA editing, referring to the insertion of uridine in the mRNA (Benne et al., 1986). Initially, modification of mRNA by RNA editing was thought to only occur in protozoa. Two forms of apolipoprotein (apo-) B, a shorter apo-B48 and a longer apo-B100, were examined in mammals (Powell et al., 1987). By comparing human cDNA clones from the small intestine and liver cells, with the genomic DNA sequences, a difference between the sequences at nucleotide position 6666 was found. The sequence TAA was found in the intestinal cDNA while the genomic sequence and the liver cDNA showed a CAA at the same position. Interestingly, as TAA is read as a stop codon by the translational machinery this transcript coded for the shorter apo-B48 while the CAA transcript coded for the longer apo-B100 product. This mRNA nucleotide modification was found to be tissue specific, which correlates with the presence of apo-B48 in the small intestine and the presence of apo-B100 in the liver. It was concluded that the stop codon (TAA) arises as a result of coor posttranscriptional RNA editing involving a single C-to-U substitution. Within the same year, two laboratories that were working on a method to block mRNA translation by antisense RNA found that antisense inhibition did not work in developing embryos (Bass & Weintraub, 1987; Rebagliati & Melton, 1987). The double stranded RNA (dsRNA) in embryos was sensitive to a single-strand specific ribonuclease. These results suggested an activity that unwinds RNA-RNA hybrids. The RNA duplex was found to be not completely unwound and not able to rehybridize. Moreover, the unwinding activity was shown to permanently alter the base pairing properties of the dsRNA by converting up to 40% of the adenosine residues to inosines (Bass & Weintraub, 1988; Wagner et al., 1989). A new form of posttranscriptional modification was found, the A-to-I editing (figure 1). This was later shown to be the most widespread type of RNA editing in multi-cellular organisms. 13 Figure 1. A-to-I RNA editing. Adenosine (A) is converted to inosine (I) by hydrolytic deamination. Inosine is read as a guanosine (G) by the translation machinery. Using directly combined high-performance liquid chromatography mass spectrometry (LC/MS) to examine the A-to-I editing reaction revealed that water serves as the oxygen donor in the conversion from adenosine to inosine, in other words a hydrolytic deamination reaction (Polson et al., 1991). The enzyme catalyzing the deamination of adenosine to inosine was found to be a double-strand RNA specific adenosine deaminase, later named ADAR1. The ADAR1 protein was purified and characterized from Xenopus laevis (Hough & Bass, 1994), calf thymus (O'Connell & Keller, 1994) and bovine nuclear extract (Kim et al., 1994a) and the editing activity was verified on synthetic dsRNAs. To clone the ADAR1 gene parts of the amino acid sequence were obtained and corresponding degenerate probes were used to screen cDNA libraries. The ADAR1 gene was obtained from human, rat and bovine (Kim et al., 1994b; O'Connell et al., 1995). ADAR1 ADAR1 is a 1226 amino acid protein consisting of a catalytic deamination domain located at the C-terminal end, three double-stranded RNA binding motifs (dsRBMs), two Z-DNA binding domains and a nuclear localization signal (NLS) (Kim et al., 1994b; O'Connell et al., 1995; Herbert et al., 1997) 14 (figure 2). The mammalian ADAR1 is expressed from at least three different promoters, one interferon inducible promoter that is giving rise to a 150 kDa protein (ADAR1p150) (George & Samuel, 1999a) and two other promoters giving rise to shorter 110 kDa proteins (ADAR1p110) (George & Samuel, 1999b; Kawakubo & Samuel, 2000). The 110 kDa proteins are exclusively nuclear whereas the 150 kDa protein, that carries a nuclear export signal (NES) (Poulsen et al., 2001), is present both in the cytoplasm and the nucleus of human cells (Patterson & Samuel, 1995). To increase the variation, the full-length ADAR1 is subjected to alternative splicing creating two additional isoforms (Liu et al., 1997). ADAR1 has been found in various amounts in all tissues examined (O'Connell et al., 1995). Discovering ADAR2 and ADAR3 The most prominent sites of selective A-to-I editing have been found in the mRNA coding for glutamate receptor subunit B (GluR-B). There are at least two sites that are edited selectively in the coding region of the GluR-B transcript, the Q/R site that causes a glutamine (Q) to arginine (R) codon change and the R/G site that causes an arginine (R) to glycine (G) codon change in the amino acid sequence (review by (Seeburg et al., 1998)). Cotransfectional analyzes using a reporter GluR-B gene and an ADAR1 expression vector revealed that the R/G site but not the Q/R site was efficiently edited by the ADAR1 protein (Melcher et al., 1996b). This suggested that additional dsRNA deaminases might exist with Q/R site specificity. To investigate if there was another dsRNA adenosine deamination enzyme capable of catalyzing editing at the Q/R site, a DNA probe for the catalytic domain of ADAR1 was synthesized. Using this DNA probe a rat-brain cDNA library was screened by low-stringency hybridization. Cloned cDNAs were inserted into an eukaryotic expression vector and co-transfected into HEK 293 cells along with the reporter GluR-B gene. RT-PCR products were analyzed for editing at the Q/R site and 90% editing was successfully created. An ADAR1-related cDNA was cloned and later named ADAR2 (Melcher et al., 1996b). A cDNA clone with sequence similarity to both ADAR1 and ADAR2 was also discovered during this period (Melcher et al., 1996a). However, this protein later named ADAR3 has not been shown to have RNA editing activity. Human homologs for ADAR2 (O'Connell et al., 1997) and ADAR3 (Chen et al., 2000) were later identified. ADAR2 ADAR2 can efficiently edit the Q/R-site of GluR-B. It is a protein of about 90 kDa sharing 31% overall sequence identity with ADAR1 in rat. The protein carries two dsRBMs, aligning best with the first and the third dsRBM of 15 ADAR1, one NLS and a catalytic deaminase domain (Melcher et al., 1996b) (figure 2). ADAR2 lacks the Z-DNA binding motifs found in the N terminal region of ADAR1. Just like ADAR1, ADAR2 is subjected to alternative splicing resulting in different isoforms (Gerber et al., 1997; Lai et al., 1997; Rueter et al., 1999). ADAR2 is mainly expressed in the brain, but has also been detected in other tissues at lower extent (Melcher et al., 1996b). ADAR3 ADAR3 is a 82 kDa protein, closely sequence-related to ADAR2 in rat. Both proteins contain two dsRBMs followed by the deaminase domain. ADAR3 differs from ADAR2 in the N terminal region where it has a longer argininerich sequence (Melcher et al., 1996a) (figure 2). ADAR3 also has an ssRNA binding motif. The binding affinity for dsRNA was shown to be lower for ADAR3 than for ADAR1 and ADAR2. No editing activity was observed after co-transfecting of the ADAR3 protein with the known ADAR1- and ADAR2 substrates (Melcher et al., 1996a; Chen et al., 2000) and there are currently no known editing substrates for ADAR3. It is possible that ADAR3 targets a very selected set of RNA substrates that differ from the already known ADAR1 and ADAR2 substrates, since it may utilize its combined ssRNA- and dsRNA-binding activities. The ADAR3 protein was found to be exclusively expressed in the brain. The expression pattern and the lack of editing substrates make this protein distinct from the other two ADAR gene family members (Melcher et al., 1996a; Chen et al., 2000). Alternative splice forms of ADAR3 have not been described. ADAR in the evolution A-to-I editing activity on dsRNA has been found in every metazoan tested. It is believed that ADAR evolved from ADAT (adenosine deaminase that acts on tRNA), an adenosine deaminase highly specific for tRNAs (Review by (Gerber & Keller, 2001)) (figure 2). Bioinformatic analysis has identified ADAR homologs in fish, revealing the conservation of the ADAR gene family members in vertebrates through evolution (Slavov et al., 2000a; Slavov et al., 2000b). Moreover, ADAR has also been found in invertebrates. The most well studied are the Drosophila ADAR (dADAR), which is homologous to the mammalian ADAR2 protein (Palladino et al., 2000a), and two C. elegans and briggsae, ADR-1 and ADR-2 (Tonkin et al., 2002). However, ADAR has not been identified in plants, fungi or yeast. 16 Figure 2. A schematic figure indicating the major functional motifs in the deaminases ADAR1, ADAR2, ADAR3, dADAR, Adr-1, Adr-2 and ADAT. The positions of the nuclear localization export signal (NES), the Z-DNA binding domains, the arginine (R) rich domain, the dsRNA binding motifs, the ssRNA binding motif, the nuclear localization signal (NLS) and the deaminase domain are indicated. Substrate specificity for selective A-to-I editing The ADAR proteins have, as mentioned before, multiple copies of doublestranded RNA-binding motifs (dsRBMs) in their N-terminal region. ADAR1 carries three copies of dsRBMs whereas ADAR2 and ADAR3 have two. The dsRBM consists of approximately 65 amino acid residues found in many other dsRNA-binding proteins, like the dsRNA dependent protein kinase (PKR) and Dicer (reviewed in (Doyle & Jantsch, 2002)). Several highresolution structures of other dsRBMs bound to RNA targets have revealed a highly conserved dsRBM-RNA structure (Ryter & Schultz, 1998; Ramos et al., 2000; Blaszczyk et al., 2004; Wu et al., 2004). A characteristic α-β-β-βα fold has been shown for the dsRBM. In contrast to the DNA double helix the double stranded RNA consists of a wide and shallow minor grove and a major grove that is narrow and deep. The dsRBM interacts with the sugarphosphate backbone in a sequence independent manner spanning two minor grooves and the intervening major groove on the dsRNA. The binding site spans 16 base pairs of the RNA, making contact at three distinct locations 17 (Ryter & Schultz, 1998). In accordance, the ADAR enzymes bind duplex RNA of a defined length in a largely sequence independent fashion. A recent study of the structure of the two dsRBMs from ADAR2 verified the predicted α-β-β-β-α fold (Stefl et al., 2006). Furthermore, binding studies between the dsRBMs of ADAR2 and a selectively edited RNA substrate revealed that the binding is selective (Stephens et al., 2004). In agreement with this, footprinting studies show that ADAR2 binds the stem surrounding the edited GluR-B R/G site at a discrete region whereas binding to a mutant substrate lacking two of the internal loops is non-specific (Öhman et al., 2000). The deaminase domain has also been shown to be important for determine the substrate specificity for site selective A-to-I editing. By swapping deaminase domains between ADAR1 and ADAR2, it was shown that some substrate specificity also is determined by the catalytic domain (Wong et al., 2001). Dimer formation or not The deaminase domain of ADAR is similar to the deaminase domain of E. coli cytidine deaminase and Apo B cytidine deaminase. The knowledge that both E. coli and Apo B cytidine deaminase form homodimers (Navaratnam et al., 1998), led to the suggestion that the ADAR proteins might form homodimer complexes as well. However, this hypothesis has been under debate. Investigating the formation of complexes between ADAR monomers revealed that both ADAR1 and ADAR2 form a stable, enzymatically active homodimer complex whereas ADAR3 remains in a monomeric, enzymatically inactive form (Cho et al., 2003). Another report supporting ADAR dimerization suggests that a Drosophila ADAR monomer first binds to the dsRNA, followed by a second monomer binding and an enzymatically active dimer is formed (Gallo et al., 2003). However, when the deaminase domain of ADAR2 was crystallized it was proposed that each ADAR domain is monomeric with no indication for dimer formation (Macbeth et al., 2005). Fluorescently tagged ADAR1 and ADAR2 expression vectors were cotransfected into HeLa cells and analyzed by fluorescence resonance energy transfer (FRET) with and without RNase. This study revealed that ADAR1 and ADAR2 form both homo- and heterodimers, independent of RNA (Chilibeck et al., 2006). Moreover, recently it was shown that ADAR1 and ADAR2 form homodimers mediated by protein-protein interactions, independent of dsRNA (Valente & Nishikura, 2007). Functional dsRBMs on both proteins within the homodimer was found to be required for editing activity, indicating that a dimer formation is essential for A-to-I editing. In conclusion, these studies point to the fact that dimer formation of ADAR is required for editing, even though some studies are contradictory. Additional, kinetic studies are required to confirm whether a monomer or a dimer is formed on the substrate. 18 Crystal structure of ADAR2 In 2005, Macbeth et al. crystallized and determined the structure of the catalytic domain of human ADAR2. Four amino acids, H394, C451, C516 and E396, of ADAR2 were shown to be involved in the coordination of a zinc atom and the formation of the catalytic centre. For the catalytic mechanism, the zinc ion activates the water molecule that is proposed to carry out the hydrolytic deamination. The occurrence of the zinc ion correlates with the suggestion that ADAR is a metalloenzyme. The most surprising discovery in the determined structure was that inositol hexakisphosphate (IP6), a highly negatively charged metabolite, was found to be embedded very close to the catalytic centre. Sequence alignments suggested that not only ADAR2, but also the closely related enzyme adenosine deaminase that acts on tRNA-1 (ADAT1), binds IP6. Experiments in a Saccharomyces cerevisiae strain deficient of IP6, revealed that IP6 has a crucial role during the deaminase reaction for both ADAR2 and ADAT1 (Macbeth et al., 2005). IP6 has previously been described to be involved in a number of cellular processes, including mRNA export (York et al., 1999), chromatin remodeling (Shen et al., 2003) and DNA repair (Hanakahi & West, 2002). Currently, it is not understood why this cofactor is required for ADAR and ADAT activity. The flip-out model The structure of the catalytic domain of hADAR2 reveled that the reactive nucleotide within the editing substrate must adopt a conformation that removes the reactive base from the helical stack before it can access the zinccontaining active site (Macbeth et al., 2005). The conformational change of the editing substrate, referred to as the flip-out model, was suggested several years ago by the Bass lab (Hough & Bass, 1997). Experimental support for this model was revealed by using RNAs fluorescently base-labeled with a 2aminopurine (2-AP) at different positions, including a known editing site. A nucleotide position-specific conformational change in the RNA editing substrate was observed during an editing reaction catalyzed by ADAR2 (Stephens et al., 2000). Moreover, the flip-out model has been further studied for adenosines in different duplex RNA sequence environments. By using molecular dynamics simulations, adenosine at the R/G-site of GluR-B was found to be more prone to move out of the helical stack than other adenosines present in the modelled duplex (Hart et al., 2005). Editing selectivity The ADAR enzymes can create two types of editing, site selective editing and hyper-editing (promiscuous editing). The extent of editing in a particular 19 RNA, largely depends on the length and structure of the double stranded region (Lehmann & Bass, 1999). RNAs with short stem-loop structures interrupted by mismatches, bulges or internal loops are often edited at selective single sites (Polson & Bass, 1994; Lehmann & Bass, 1999). Longer extensively double stranded structures are often edited at multiple sites, referred to as hyper-editing. ADAR1 and ADAR2 can promiscuously edit up to 60% of the adenosines in a perfect duplex RNA in vitro (Bass & Weintraub, 1988; Wagner et al., 1989; Nishikura et al., 1991; Polson & Bass, 1994). This type of editing has been found within introns and untranslated regions (UTRs) of mRNAs, preferentially in repetitive Alu sequences (Morse et al., 2002; Athanasiadis et al., 2004; Blow et al., 2004; Levanon et al., 2004). It is not known how ADARs discriminate between certain adenosines for selective editing, but studies using scanning force microscopy have shown that the ADAR2 protein prefers to bind to a selectively edited substrate over a random complete double stranded sequence of several hundred base pairs (Klaue et al., 2003). No common sequence preference for the edited substrate has been found. However, in vitro studies reveal that both the sequence and the structure determines a preferred editing site (Dawson et al., 2004). Other studies have shown that there is a certain neighbor and mismatch preference for the adenosine to be edited. A similar 5’ neighbor preference of the deaminated adenosine (A=U>C=G) has been determined for both ADAR1 and ADAR2. ADAR2 also has a 3’ neighbor preference which is U=G>C=A (Polson & Bass, 1994; Lehmann & Bass, 2000). An A:C mismatch or an A-U basepair is preferred between the reactive adenosine and the opposite nucleotide, before A:A or A:G (Wong et al., 2001; Källman et al., 2003). Even though ADAR1 and ADAR2 have similar neighbor and mismatch preferences their substrate selectivities differ. For instance, both enzymes can edit the R/G site of the glutamate receptor subunit B but the Q/R site is edited only by ADAR2, indicating that ADAR1 and ADAR2 display distinct but overlapping selectivities. Also, the different isoforms of ADAR1 (Liu & Samuel, 1999) and ADAR2 (Gerber et al., 1997; Lai et al., 1997) were shown to vary regarding their editing activity. Furthermore, two alternative splice forms of the hADAR2, containing or lacking an Alu repetitive element cassette in the deaminase domain, showed the same substrate specificity but had a two-fold difference in their specific editing activity (Gerber et al., 1997). Roles for the different ADAR domains The dsRBMs in ADAR2 have not only been shown to contribute to the editing site selectivity (Stephens et al., 2004), the dsRBMs have also been shown to increase the flexibility of the RNA duplex and in that way assist the base flipping mechanism (Yi-Brunozzi et al., 2001). Moreover, ADAR2 has been shown to exist in an auto-inhibited conformation until both of its 20 dsRBMs bind to the editing substrate. After the interaction the autoinhibition is relieved and the deaminase domain is placed over the editing site prepared to catalyze the deamination reaction (Macbeth et al., 2004). However, the largest variability among ADARs is found at their amino terminal regions. The Z-DNA binding domains are only found in ADAR1 and have been proposed to regulate editing activity. Mutations in the Z-DNA binding domains decreased the editing efficiency even though the editing substrate lacks Z-forming sequences (Herbert & Rich, 2001). However, as previously mentioned the deaminase domain of ADAR1 and ADAR2 have been shown to play a dominant role in defining substrate specificity (Wong et al., 2001). The different domains of the ADAR enzyme are not only important for the activity and selectivity, they also determine the localization of ADAR in the cell. The full-length ADAR1, containing the nuclear export signal (NES), is localized predominantly in the cytoplasm whereas the Nterminally truncated form of ADAR1 localizes exclusively to the nucleus (Patterson & Samuel, 1995; Poulsen et al., 2001; Desterro et al., 2003). ADAR2 lacking the NES is exclusively nuclear (Desterro et al., 2003; Sansam et al., 2003). Within the nucleus both ADAR1 and ADAR2 accumulates in the nucleolus. The localization of the ADAR proteins to the nucleolus is dependent on RNA, probably by interactions with ribosomal RNA in a non-specific manner. Once a specific editing substrate appears, the ADAR proteins can relocate from the nucleolus to the editing substrate elsewhere in the cell (Desterro et al., 2003). This suggests that the nucleolus is a place for storage to possibly prevent non-specific editing. Editing site complementary sequence is required for editing It is well established that A-to-I RNA editing requires a double stranded RNA structure. Studies of the glutamate receptor subunit B revealed that a proximal part of the intron downstream of the Q/R site is required for the editing. This part of the intron contains an imperfect inverted repeat to the exon sequence centered around the unedited CAG codon at the Q/R site (Higuchi et al., 1993). This suggested that only the pre-mRNA and not the mRNA could be a substrate for editing. In vitro studies revealed that the editing site complementary sequence (ECS) can be as short as 15 bp (Nishikura et al., 1991; Herbert & Rich, 2001). In figure 5 some examples of site selectively edited substrates are shown. As illustrated in the figure, the distance separating the edited adenosine from the ECS can vary in different substrates, from several thousand nucleotides to a loop with a handful bases. Most of the known selectively edited sites are located within an exon with the ECS in the downstream intron. Nevertheless, there are a few exceptions. One example is the pre-mRNA of ADAR2, where the double-stranded struc- 21 ture is formed within an intron (fig. 5f). Another example is the transcript of the intronless human potassium channel gene hKv1.1, where the doublestranded structure is formed by the intronless transcript (Bhalla et al., 2004) as shown in figure 5e. ADAR and gene regulation ADAR has been shown to target double-stranded RNA structures within coding sequences, introns and 5’ and 3’ untranslated regions (UTRs) as summarized in figure 3. Since inosine is read as a guanosine by the translation machinery (Basilio et al., 1962), editing within coding regions can alter the meaning of the codon creating more than one protein isoforms from one gene. As already mentioned, site selective editing can change the codon during the translation, like the Q/R editing site in the GluR-B transcript, where a CAG codon for glutamine (Q) is almost completely edited to CIG encoding an arginine (R) (Higuchi et al., 1993). Editing within non-coding regions has the potential to affect different RNA processing events. A-to-I editing has the potential to create or delete splice donor (AU) and acceptor (AG) sites, editing can therefore result in alternative splicing or intron retention. One example is the auto-editing of the intronic dsRNA sequence of the Adar2 pre-mRNA, resulting in the formation of an alternative 3’-splice acceptor site. An AA dinucleotide is altered to an AI dinucleotide, which is recognized as an AG dinucleotide, typically found at the 3’ splice site. The alternative splicing, as a result of A-to-I editing, creates a frameshift that has the potential to regulate ADAR2 expression (Rueter et al., 1999). Moreover, the adenosine-rich poly(A)tail is a potential site for editing. One example is a mouse polyomavirus transcripts, which is edited at the poly(A)tails. Editing at these sites regulates its life cycle (Gu et al., 2006). The interferon-inducible ADAR1 protein (ADAR1p150) has been suggested to play a role in viral defense by promiscuous deamination of viral double stranded RNAs in the cytoplasm. The hepatitis C virus was shown to be specifically eliminated by ADAR1 editing upon interferon induction (Taylor et al., 2005). In mammals, a highly conserved and abundant nuclear protein, p54nrb, binds to inosine-containing RNAs. The p54nrb-protein forms a nuclear retained complex with the splicing factor PSF and inner nuclear matrix structural protein matrin-3 (Zhang & Carmichael, 2001). Nuclear retention was described for the cellular CAT2 Transcribed Nuclear-RNA (CTNRNA). The highly edited CTN-RNA was found to interact with the nuclear protein p54nrb and thereby be retained in the nucleus (Prasanth et al., 2005). Hyper-edited RNAs can also be impeded from translation by cytoplasmic endonuclease degradation (Scadden & Smith, 2001). The endonuclease cleavage was shown to be promoted by Tudor staphylococcal nuclease (Tudor-SN), a known subunit of the RNA interference silencing complex (Scadden, 2005). 22 ADAR has the potential to regulate gene expression not only by editing, but also by specific interactions. The long form of ADAR1 (ADAR1p150) was shown to associate with the nuclear factor 90 protein family (NF90) of dsRNA-binding proteins. The NF90 proteins have been demonstrated to regulate gene expression at multiple levels including transcription (Reichman & Mathews, 2003; Reichman et al., 2003) and translation (Xu & Grabowski, 1999; Xu et al., 2000). ADAR1p150 was shown to bind the NF90 protein complex, and regulate NF90-mediated gene expression independently of RNA editing (Nie et al., 2005). Figure 3. Possible effects of A-to-I editing of pre-mRNAs in the cell nucleus. The location of sites for editing are indicated on the pre-mRNA and their potential consequences are in boxes. Intron sequences are in light grey while exon and untranslated regions are in dark grey. Site selective editing substrates The glutamate receptors The rapid excitatory neurotransmission in the vertebrate central nervous system is mediated by the ionotropic glutamate receptor channels. The glutamate-gated ion channels are assembled from four glutamate receptor 23 (GluR) subunits. The glutamate receptors are classified into three subtypes; α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), Nmethyl-D-aspartate (NMDA) and kainite receptors (KA). Mammals express four AMPA receptor subunits (GluR-A, -B, -C and –D), five NMDA receptor subunits (NR1, NR2A, NR2B, NR2C and NR2D) and five KA receptor subunits (KA-1, KA-2, GluR-5, GluR-6 and GluR-7). Each subunit is a glycosylated polypeptide of approximately 900 amino acid residues, encoded by a separate gene (reviewed by (Seeburg et al., 1998)). The glutamate receptor subunits contain four hydrophobic regions (M1 – M4) determining the transmembrane topology and the channel architecture. The M2 region occupies the inner channel pore and an arginine (R) residue within the M2 region controls the Ca2+ permeability (Hollmann et al., 1991; Burnashev et al., 1992). Studies of the AMPA receptor revealed that the arginine in the M2 region is only found in GluR-B, whereas there is a glutamine (Q) in the homologous position in GluR-A, -C and –D (Sommer et al., 1991). An arginine in the M2 region makes the AMPA channels impermeable to Ca2+ whereas glutamine in that position renders high Ca2+ permeability. Sequence analyzes showed that also the KA receptor subunits GluR5 and GluR-6 exist with either glutamine or arginine in the M2 region (Bettler et al., 1990; Egebjerg et al., 1991). Analyzing the genomic M2 sequence of the GluR-5 and GluR–6 revealed that each gene contains a glutamine at the position where both glutamine and arginine have been found in the transcript (Sommer et al., 1991). The same pattern was shown for GluRB, although all characterized GluR-B cDNAs had an arginine codon in their M2 sequence (Boulter et al., 1990; Sakimura et al., 1990). The conversion from CAG coding for glutamine to CGG coding for arginine of GluR-B, GluR-5 and GluR-6 are generated posttranscriptionally by RNA editing (figure 4). RT-PCR on rat brain revealed almost complete editing (99%) at the Q/R site of the GluR-B (Sommer et al., 1991; Higuchi et al., 1993). The Q/R-site of GluR-B was the first selective A-to-I editing site reported and certainly one of the most studied. The pre-mRNA of the GluR-B in the vicinity of the Q/R-site forms a long double stranded structure, interrupted by mismatches, a bulge and loops, through complementary base pairing between exon 11 and the downstream intron. The reactive adenosine at the Q/R-site forms a basepair with the opposite nucleotide (A-U) (Figure 5a). Interestingly, the secondary structure in the vicinity of the Q/R-site of GluR6 is different from that in GluR-B as seen in figure 5b. The suggested structure for GluR-6 carries a mismatch nucleotide opposite the reactive adenosine and the distance between the adenosine and the ECS is much longer for GluR-6 than in GluR-B. RNA editing was also found in the coding region between the M3 and M4 regions of the GluR-B, -C and –D (Lomeli et al., 1994). Here an arginine (R) encoded by AGA is converted to a glycine (G) encoded by GGA, referred to as the R/G site (figure 4). Alternation of this amino acid leads to faster recovery rates from desensitization. The R/G site is largely unedited in the 24 embryonic brain but editing increases after birth. The pre-mRNA in the vicinity of the R/G-site forms a shorter double stranded structure, interrupted by three mismatches and a short loop of five nucleotides between exon 13 and the downstream intron. The reactive adenosine is situated only one nucleotide from the 5’ splice site creating an A-C mismatch with the opposite nucleotide (figure 5c). Moreover, GluR-6 has been shown to be edited at two additional sites in the M1 region. This results in two amino acid changes isoleucine (I) to valine (V) and tyrosine (Y) to cysteine (C). Not only the Q/R-site, but also the I/V- and Y/C-site of GluR-6 impact the Ca2+ permeability (Kohler et al., 1993). Thus, eight positions in five subunits of the GluR family have been found to undergo A-to-I RNA editing (figure 4). The Ca2+ permeability of the glutamate receptor has been connected to presence or absence of the glutamate receptor subunit B (Kumar et al., 2002). Moreover, editing at the Q/R-site as well as at the R/G-site has been shown to diminish the assembly property of the glutamate receptor subunit B, and hence decrease the expression of the subunit at the synapse (Greger et al., 2006). The AMPA channels’ impermeabity to Ca2+, as a result of editing, is therefore more likely due to assembly difficulties than amino acid changes within the ion pore. In conclusion, A-to-I editing have probably several roles within the glutamate receptor, regulating the responsiveness of the receptor and controlling the receptor assembly. Figure 4. The editing sites of the Glutamate receptor subunits. GluR-B carries the Q/R- and the R/G-site. GluR-C and GluR-D carry the R/G-site. GluR-5 carries the Q/R-site and GluR-6 carries the I/V-, the Y/C- and the Q/R-site. 25 The serotonin receptor The mRNA encoding the 2C subtype of serotonin receptor (5-HT2C) is also subjected to editing (Burns et al., 1997). Five editing sites in close proximity to each other have been found within the 5HT2C transcript (Burns et al., 1997; Niswender et al., 1998), resulting in five distinct amino acid changes. These sites are targeted specifically and differently by ADAR1 and ADAR2. Theoretically, 24 different receptor variants can be produced from various combinations of the edited sites. Editing of the 5HT2C mRNA has been shown to dramatically reduce the responsiveness of the receptor (Burns et al., 1997). The pre-mRNA forms a double stranded structure interrupted by mismatches, bulges and loops. The intronic editing complementary sequence has been proposed to form A-U base pairs with four of the sites and an A-C mismatch at one site (figure 5d). The potassium channel In humans, the potassium channel subfamily Kv1.1 pre-mRNA is subjected to ADAR editing (Hoopengardner et al., 2003). The editing cause an amino acids change from isoleucine (I) to valine (V). The editing site was found to be within the pore of the K+ channel. Measuring the extent of recovery from inactivation revealed that the isoleucine to valine change results in a 20-fold increase in the recovery rate (Bhalla et al., 2004). The Kv1.1 gene is intronless, and hence forms the double stranded structure within the coding sequence. The double stranded structure is interrupted by several mismatches, bulges and loops forming the RNA hairpin at the editing site with only a six base pair duplex (Bhalla et al., 2004) (figure 5e). ADAR2 ADAR2 regulates its own expression by alternative splicing created by editing of its own pre-mRNA (figure 5f). Studies in rat brain showed that editing converts an intronic AA to an AI dinucleotide which mimics the highly conserved AG sequence normally found at 3’ splice junctions. The conversion creates a new splice site, adding 47 nucleotides to the ADAR2 coding region resulting in a frame shift upon translation. The frame shift leads to premature stop codons resulting in a truncated protein lacking both of the double stranded RNA binding motifs and the catalytic domain. It was shown that less than 5% of the ADAR2 proteins correspond to the truncated protein although 80% of the ADAR2 transcripts were subjected to editing and subsequent alternative splicing (Rueter et al., 1999). This suggests that the premature termination codon triggers the nonsense mediated decay (NMD) pathway, degrading the RNA so that no protein is produced. However, degradation through the NMD pathway has not been proven. 26 The endothelin B receptor The endothelin B receptor (ETB) was found to be a selectively edited substrate when mutations in the endothelin gene were examined from patients with the genetic disorder Hirschsprung disease (HSCR). HSCR is an innate disorder characterized by an absence of ganglion cells in the distal colon. An editing site was found within exon 4 of the ETB, resulting in an amino acid change from glutamine (Q) to arginine (R). Editing at this site has also been found in healthy individuals although a much higher frequency was found in patients with HSCR (Tanoue et al., 2002). However, further studies are required to determine how editing on the ETB is related to the Hirschsprung disease. The tyrosine phosphatase, non-receptor type 6 gene The tyrosine phosphatase, non-receptor type 6 (PTPN6) gene was found to be edited at 7 sites within intron 3 and exon 4. One of these sites is located at the branch point of intron 3. Editing at this site destroys the branch formation site making it unrecognizable to the splicing machinery, which results in intron retention. It is predicted that the intron retention leads to the production of a non-functional protein. An increased editing rate was found in acute myeloid leukemia patients. How RNA editing is implicated in acute myeloid leukemia is not understood (Beghini et al., 2000). The hepatitis delta virus and the polyomavirus The human hepatitis delta virus (HDV) has been shown to be edited at a single adenosine, converting an amber stop codon into a tryptophan (W) codon (Luo et al., 1990; Casey & Gerin, 1995). This amber/W site enables the virus to express both a short and a long form of the viral protein delta antigen (HDAg). The short form (HDAg-S) is used during viral replication while the long form (HDAg-L) is involved in packaging of new virus particles. Editing at this site is essential for the viral life cycle (Polson et al., 1996). Another example of an edited eukaryotic virus is the mouse polyomavirus. The viral expression is divided into an early and a late transcription unit, transcribed from its circular genome in opposite directions. The early transcript unit yields three proteins involved in the inititation of DNA replication and cell transformation whereas the late transcription unit yields three viral structural proteins. The early and late polyadenylation sites were found to overlap, creating a double stranded RNA structure suitable for editing (Gu et al., 2006). Editing of the polyadenylation sites, leads to degradation of the early transcript while the late transcript is stabilized. Late antisense RNA only accumulates after replication initiation. RNA editing will therefore regulate the early-late switch during the polyomavirus infection. 27 Other candidates for A-to-I editing Using comparative genomic approaches, site-selectively edited sites were found and experimentally verified within the Filamin A (FLNA) mRNA, cytoplasmic FMR1 interacting protein (CYFIP2) mRNA and the bladder cancer associated protein (BLCAP) mRNA (Levanon et al., 2005). Editing of these transcripts result in recoding of single amino acids. The functions of these genes have so far not been revealed. miRNAs The RNA interference (RNAi) is a multi-step process that results in gene silencing (reviewed by (Filipowicz et al., 2005)). The RNAi machinery can be triggered by several classes of dsRNA including a class of non-coding RNAs, each about 20-22 nt long, called micro-RNAs (miRNAs). The first published miRNA with detectable amounts of A-to-I editing was found in the human and mouse pri-miRNA22 (Luciano et al., 2004). The function of editing on this miRNA is not known. Moreover, a miRNA promoting T lymphoid-lineage cells named miRNA-142 (Chen et al., 2004), was shown to be edited at several sites (Yang et al., 2006). In vitro studies revealed that both ADAR1 and ADAR2 deaminates adenosines selective and overlapping at the stem-loop of the miRNA-142. Investigating the effect of pri-mRNA142 editing showed that it resulted in suppression of processing by an RNase III endonuclease named Drosha, since the double stranded RNA recognition structure was altered. Moreover, the edited miRNA-142 was shown to be degraded by Tudor-SN (Yang et al., 2006), a known component of RISC (Caudy et al., 2003). A family of six human miR-376 RNAs and three mouse miR-376 RNAs (a-c) were found to have highly similar sequences. These miRNAs were found to be edited by ADAR1 and ADAR2 to different extent. Interestingly, editing in the target sequence of the miRNA was found to redirect where the miRNA hybridizes and thereby silences a different set of genes than the unedited miRNA (Kawahara et al., 2007). The 3’UTR, frequently targeted by the miRNAs is in addition the most common target for hyper editing. This suggests an interplay between RNA editing of the mRNA target and miRNA hybridization. Analyzing 3236 edited positions in the 3’UTR of human transcripts revealed that several evolutionary conserved miRNAs target these sites (Liang & Landweber, 2007). However, statistical analyses of this data set suggested that RNA editing tends to avoid miRNA target sites. 28 Figure 5. Predicted secondary structures of substrates for mammalian ADAR enzymes. The substrates are: a) Q/R-site of GluR-B; b) Q/R-site of GluR-6; c) R/G-site of GluR-B; d) A-E sites of 5HT2; e) I/V-site of Kv1.1 and f) -1 site of Adar2. Edited sites are indicated with an A in bold. Exon sequences are in grey while intron sequences are indicated as a thin black line. Numbers represent total number of nucleotides in each loop. Hyper-editing It has been postulated that the main function for A-to-I editing is recoding of mRNAs at single positions. However, greater than 90% of all A-to-I editing substrates identified have been found in Alu repetitive elements (Levanon et al., 2004). Alu repetitive elements are short interspersed elements (SINE) of approximately 300 nt in length and only found in primates. They can be situ29 ated within introns and untranslated regions (UTRs) of mRNAs and represent approximately 10% of the human genome (Batzer & Deininger, 2002). Completely double stranded regions formed by inverted Alu repeats are ideal for hyper-editing. There is no evidence for any specific sequence interactions between ADARs and Alu sequences and the function of hyper-editing is not fully understood. Editing within Alu repeats have been suggested to destroy or create splice sites (Athanasiadis et al., 2004). Hyper-editing might also change the stability of a double stranded RNA so it might not be recognized by double stranded RNA binding proteins, for examples see the miRNA section. Moreover, nuclear retention of inosine containing RNAs might be a result of hyper-editing (Zhang & Carmichael, 2001). However, since the Alu repetitive elements are primate specific, this type of editing is not evolutionary conserved. Why receptors? Only a few site-selective ADAR substrates have been detected in mammals. Most of these substrates are in pre-mRNAs expressed in the central nervous system coding for receptor subunits. Cell signaling takes advantage of ion gradient differences across the cell membrane. To establish an ion gradient, an active transport of ions from a low to a high electrochemical potential across the membrane is needed. Once the ion gradient is established, the electrical signal is relatively stable since the cell membrane is almost completely impermeable to ions. The ion channel regulates the ion gradient across the cell membrane. In neurons, the ion gradient is used when an electrical signal is transported. Ions travel across the cell membrane, like a wave from one side of the neuron to the other. The electrical signal can be transmitted from one cell to another via a region called the synaptic cleft. At the synaptic cleft the electrical signal is converted into a chemical signal, also known as neurotransmission. A receptor is a protein complex where ligands, such as neurotransmitters, bind and initiates a cellular response. There are several types of receptors, for example ionotropic and metabotropic receptors. Ionotropic receptors affect the cell directly, functioning as a ligand-gated ion channels whereas metabotropic receptors function indirectly, being coupled to a G protein that controls the ion channel. Ligand-gated ion channels are composed of an extracellular ligandbinding domain and an ion-channel domain that is integral to the membrane. Neurotransmitters bind to the channel, located in the postsynaptic membrane and the ion channel opens and selected ions flow down their electrochemical gradients. The ion channel determines the ion flow and thereby the nature of the electrical signal. Ligand-gated ion channels are usually closed in the resting state. After activation, the ion channels undergo spontaneous closing, also known as desentization. There are mainly two ways neurotransmitters 30 and their corresponding ion-channels function. It can promote transmembrane transmission of an electrical signal referred to as excitatory or depolarizing. A neurotransmitter can also slow down or stop a transmembrane transmission, referred to as inhibitory or hyperpolarization. In the mammalian brain, the glutamate receptors are responsible for the majority of excitatory (depolarizing) postsynaptic signals, whereas the gamma-aminobutyric acid type A (GABAA) receptors are responsible for the majority of inhibitory (hyperpolarizing) postsynaptic signals (Cromer et al., 2002). With few exceptions, all currently known A-to-I editing transcripts encode membrane proteins that function as voltage or ligand gated ion channels or as G protein-coupled receptors expressed in nervous system. This is paradoxical since ADARs are expressed in most tissues of the body. The GluR-B Q/R-site is the only known transcript edited to nearly 100% in all developmental stages, meaning that all other edited transcripts are present in both the unedited and the edited form. However, the regulation of ion channels and neurotransmitter receptors is highly complex. Increased protein diversity and different regulations during development makes A-to-I editing an essential fine-tuning player within the complex developmental neurons. ADAR is required for normal life Editing is required for normal life in both vertebrates and invertebrates. ADAR2 knock-out mice (ADAR2-/-) have reduced editing at numerous selective editing sites, including the glutamate receptors, the serotonin receptors and the ADAR2 pre-mRNA. The most significant reduction was at the Q/R-site of the GluR-B, where editing was reduced from almost 100% to 10%. The mice are prone to epilepsy and die shortly after birth (Higuchi et al., 2000). By introducing arginine (R) at the Q/R-site of the GluR-B (GluRBR/R), the ADAR2-/- phenotype was rescued (Higuchi et al., 2000) suggesting that editing of the Q/R site of GluR-B is the most important and the only essential ADAR2-site in mice. ADAR1 has also been shown to be essential in mammals. Remarkably, ADAR1 knock-out mice (ADAR1-/-) die at the embryonic stage between day 11.5 and 12.5 (Hartner et al., 2004; Wang et al., 2004). It is not known if this is a result from lack of editing at one or several selective sites, or lack of editing at non-specific sites. Even though the ADAR enzymes are not essential in all organisms, they are highly important for normal behavior and brain development. For instance, dADAR in Drosophila or adr1-2 in C. elegans are not essential, but absence of either protein affects behavior. Deletion of dADAR in flies results in defects in motor control, mating and flight (Palladino et al., 2000b), and deletion of the adr1-2 in C. elegans results in chemotaxis defects (Tonkin et al., 2002). 31 RNA editing and human disease phenotypes The main role for ADAR might be to fine-tune the transcriptome. Hence, Ato-I editing is frequently used in areas that require increased proteome diversity, like the CNS. As described earlier, editing has been shown to be involved in the regulation of neurotransmission. Dysfunction of A-to-I editing can potentially cause human diseases of learning, memory, language and behavior. During the last years, diseases like depression, epilepsy, schizophrenia and amyotrophic lateral sclerosis (ALS) have been linked to ADAR editing. Overactivity of excitatory neurotransmitters or underactivity of inhibitory neurotransmitters can lead to seizure activity in animals, as a result of an uncoordinated flow of electrical activity in the brain. As mentioned before, mice that are not able to edit the Q/R site of the GluR-B have an overactivity of Ca2+ for the receptor (Brusa et al., 1995). Accordingly, underediting of the Q/R-site has been proposed to be responsible for motor neuron death in sporadic amyotrophic lateral sclerosis (ALS). The ALS disease is associated with the progressive symptoms of muscle weakness, muscle atrophy and spasticity (Kawahara et al., 2004). Several connections between major psychiatric disorders, such as depression, and editing of the serotonin receptor have been investigated during the last years. Two different studies argue that there are altered editing of the serotonin receptors in the prefrontal cortex of suicide victims (Niswender et al., 2001; Gurevich et al., 2002). However, these studies are not consistent with each other. Since editing of the serotonin transcript can differ between different inbred strains of mice (Englander et al., 2005), it is hard to be conclusive about editing differences of the serotonin receptor between different patients. Moreover, ADAR has been linked to a human pigmentary disease called dyschromatosis symmetrica hereditaria (DSH). Patients with DSH have hyperpigmented and hypopigmented macules on the back of their hands and the tops of their feet. A genome wide search was done to determine the responsible gene for this disease. Mutation causing DSH was surprisingly mapped to the ADAR1 gene. Four different mutations were found to change amino acids codons within the ADAR1 transcript, where two of these changes result in stop codons (Miyamura et al., 2003). However, if editing is involved in DSH is not known. Methods to detect novel editing sites Most mammalian selective ADAR substrates have been found serendipitously, or have been identified by homology to edited substrates in other species. Nevertheless, several attempts have been performed to find novel sites of A-to-I editing. One experimental method was developed by Morse et al. detecting ADAR candidates by cleaving poly(A)+ RNA specifically after inosine and identifying the cleaved substrates by differential display (Morse 32 & Bass, 1997). The GluR-B transcript was successfully amplified, but no new substrates for selective editing have been identified using this method. Another approach to directly detect and isolate inosine-containing RNAs, a polyclonal antibody against inosine was developed and used to enrich inosine-containing mRNAs. Immunoprecipitated mRNAs from wild type and mutant (dADAR-/-) flies were amplified and hybridized to Drosophila cDNA arrays. About 500 mRNAs were enriched in the wild type array compared to the dADAR-/- array. In addition, by comparing cDNA sequences with their genomic counterparts 800 genes were found to have A/G discrepancy within the coding region. Comparing these two approaches, only 62 genes were common to both groups, and seven of these were experimentally verified as A-to-I editing substrates (Xia et al., 2005). With the availability of an increased number of sequenced genomes, cDNA and EST data, several bioinformatic methods have during the last years been developed to find new ADAR substrates. A phylogenetic method was described, hypothesizing that if RNA editing of a particular site is conserved between species, the duplex formation would be conserved as well (Hoopengardner et al., 2003). By aligning numerous Drosophila species, the exonic sequences neighboring the edited sites were shown to be highly conserved. Related species were screened using this high degree of sequence identity as a potential signature of ADAR editing sites. Sixteen new substrates were found in Drosophila, including multiple editing sites in the K+ channel genes: shaker, ether-a-go-go and slowpoke. The screening strategy was applied to the mammalian shaker genes and editing of the human hKv1.1 gene was found (Hoopengardner et al., 2003). Using different comparative genomics approaches, a high number of edited substrates have been detected. Most of these substrates are hyper-edited in their 5’ or 3’ UTRs within Alu repetitive elements (Athanasiadis et al., 2004; Blow et al., 2004; Levanon et al., 2004). However, some selectively edited sites have been found and experimentally verified, for instance within the Filamin A (FLNA) mRNA, cytoplasmic FMR1 interacting protein (CYFIP2) mRNA and the bladder cancer associated protein (BLCAP) mRNA (Levanon et al., 2005). 33 Present Investigation Aim After reading the literature I believe that the known selectively edited transcripts constitute just the tip of the iceberg. There are several indications that there are many more selectively edited sites yet to be discovered. For instance, known edited sites cannot explain the complex phenotypes revealed by engineering ADAR deficient mouse (Higuchi et al., 2000; Hartner et al., 2004; Wang et al., 2004), worm (Tonkin et al., 2002) and fly (Palladino et al., 2000b). Most of the known selective editing sites have been found serendipitously over the last 16 years and no all-embracing method has been developed to find novel substrates for selective editing that also detects the already known substrates. Moreover, almost all substrates subjected to selective editing have been found in the brain although ADARs are ubiquitously expressed in all mammalian tissues. I am confident that there are more editing substrates to uncover, that has been foreseen using the methods and the genome sequences available today. The aim of my thesis has been to find novel A-to-I editing substrates in mammals. Paper I - the method To find substrates for editing, we developed an experimental approach based on immunoprecipitation (IP) and microarrays. Previous studies in our laboratory have shown that ADAR2 distinguishes between binding to a selectively edited site and a random sequence of double stranded RNA (Klaue et al., 2003). There is a preferred interaction between ADAR2 and a selectively edited site over a long completely base paired region. Moreover, ADAR2 was shown to bind with a similar affinity to the substrate as to the edited RNA product (Öhman et al., 2000). Based on this knowledge we developed an immunoprecipitation assay to pull down intrinsic ADAR2-RNA substrate complexes using an affinity purified anti-ADAR2 antibody. To analyze the enriched RNA from the ADAR2 co-immunoprecipitation, we used genomic microarrays. The advantage of using microarrays to detect the enriched RNAs, over cloning and sequence determination, is that a higher level of 34 background can be accepted and that the array detects products of a relatively low abundance. The IP method is illustrated in figure 6. We chose mouse brain tissue in our experiments. One reason for this is that the editing levels, within the repetitive elements, are at least an order of magnitude lower in mouse than in humans (Eisenberg et al., 2005). The hyper-edited, primate specific Alu repeats are not present in the mouse genome. However, the mouse genome contains four other SINEs called B1, B2, B4 and ID. The divergence level of the repetitive elements is higher in mouse SINEs than in the human Alus, which makes the mouse repeats less double stranded and hence less attractive structures for editing (Neeman et al., 2006). Mouse is therefore an advantageous model organism to avoid a high background of A-to-I hyper-editing in non-coding sequence. Since Ato-I editing has been found almost exclusively in the nervous system, total brain was our initial choice of tissue. Figure 6. Illustration of the IP-array method to find novel substrates for A-to-I editing. Cell lysis extract is prepared from mouse brain. The extract is immunoprecipitated using an ADAR2 specific polyclonal antibody. Target RNA is then extracted from the mRNP complexes upon protein removal. The RNA is amplified, labeled and further hybridized to a mouse genomic oligo array. 35 Paper II - finding novel substrates for A-to-I editing Using the IP-array method, we obtained a list of about 200 candidate genes that were enriched by the anti-ADAR2 antibody. Table I lists some of the previously known selectively edited substrates and two novel substrates (in bold) showing the mean enriched value using this method. Although many candidate genes for A-to-I editing were found, the positions of the edited sites within these genes were not revealed by this method. Bioinformatic analyses were used to find the potential sites of editing within the candidate genes. In collaboration with Jacob Pedersen and David Haussler we combined the data from our IP-array method with candidates extracted from the program called EvoFold, developed to predict evolutionary conserved short stem-loop structures in RNA (Pedersen et al., 2006). Several double stranded RNA structures known to be substrates for A-to-I editing were detected using EvoFold. Gene Gabra-3 5HT2C CTN-RNA Endothelin B GluR-B ADAR2 GluR-C GluR-D BLCAP GluR-5 FLNA CYFIP2 KCNA1 GluR-6 Mean enriched 7.2 3.0 2.9 2.9 2.6 1.9 1.2 1.1 1.0 0.9 0.8 0.8 0.7 0.7 Table 1. Enriched known editing targets by an anti-ADAR2 antibody compared to a non-specific IP verified by microarray. The following abbreviations are used: Gabra3, gamma-aminobutyric acid type A (GABA A) receptor subunit α3; 5-HT2C, serotonin receptor subtype 2C; CTN-RNA, CAT2 Transcribed Nuclear-RNA; Ednrb, Endothelin receptor type B; GluR-B, Glutamate receptor subunit B; ADAR2, adenosine deaminase that acts on RNA 2; GluR-C, Glutamate receptor subunit C; GluR-D, Glutamate receptor subunit D; Blcap, bladder cancer associated protein; GluR-5, Glutamate receptor subunit 5; Flna, Filamin A mRNA; Cyfip2 cytoplasmic FMR1 interacting protein mRNA 2; Kcna1, potassium voltage-gated channel, shakerrelated subfamily, member 1; GluR-6, Glutamate receptor subunit 6. One of the top scored genes found by combing our IP-array method with the stem-loop prediction program EvoFold, was a transcript coding for the gamma-aminobutyric acid type A (GABAA) receptor subunit α3 (Gabra-3). 36 Gabra-3 was enriched sevenfold in the IP-array method (table I) and had a promising predicted structure suitable for A-to-I editing within exon 9 (figure 7). Figure 7. The predicted structure of the stem-loop within exon 9 of Gabra-3. The edited A is printed in bold and circled in grey. To examine if the Gabra-3 transcript was edited, we compared the genomic sequence with the cDNA sequence from mouse brain. An adenosine was found in the genomic sequences whereas a guanosine was found at the equivalent position in the cDNA sequence. The edited site was named I/M since an isoleucine (I) AUA codon was changed to an AUI read as a methionine (M) at this position. Moreover, when looking at the cDNA sequence from an ADAR2-/- mouse, editing of the I/M-site was highly reduced. We further analyzed if a gabra-3 reporter gene containing exon 9, including the I/M site, could be edited by different combinations of ADAR expression vectors during transient transfections of human embryonic kidney cells (HEK 293). This experiments revealed that the Gabra-3 transcript is efficiently edited by both ADAR1 and ADAR2. The editing complementary sequence is located 15 bases upstream of the edited site in a short predicted stem loop of 54 nucleotides within exon 9. 37 With a loop of 4 nucleotides, this is the shortest stem known to be efficiently edited by ADAR (figure 7). The edited site in the α3 subunit of the GABAA receptor was found to be located in transmembrane (TM) domain 3. Paper III - the function of GABA receptor editing The I/M-site editing was found to be conserved in a variety of organisms, from mouse to chicken. Interestingly, frog and pufferfish were found to have a genomically encoded methionine at the equivalent position. Moreover, the extent of editing was shown to be low at birth but increases with age, reaching close to 100% in the adult brain. Detailed developmental analyzes were done in mouse and chicken. The GABAA receptor is responsible for most inhibitory postsynaptic signals in the mammalian brain. However, until post natal day (P) 8-12, GABA mediates the excitatory rather than inhibitory postsynaptic potentials through the GABAA receptors (Ben-Ari, 2002). The most abundantly expressed GABAA receptor in the adult mammalian brain is composed of two α, two β, and one γ subunits (Chou, 2004). In rat, the α3 subunit is the major αsubunit at birth but declines around P10, whereas the α1 subunit is relatively low at birth but increases around P10 (Liu & Wong-Riley, 2006). Interestingly, editing at the I/M-site of α3 occurs concurrent with the dramatic change in the function of the GABAA receptor. To investigate if the mRNA levels of the GABAA receptor subunits change during development quantitative-PCR was used. The transcripts coding for α1, α3, γ2 and ADAR2 were analyzed at different developmental stages in the chicken retina. The mRNA levels for α1, α3 and γ2 were expressed in a similar way, with low expression during stage 21 to 45, a small peak for Gabra-3 at stage 42 and a dramatic increase for all three subunits in the 6 months old chicken retina mRNA. Surprisingly, the mRNA level for ADAR was evenly expressed throughout development with only two small peaks at stage 35 and 6 months old chicken retina. However, developmental differences for ADAR protein levels may still exist and Western blot experiments are required to answer this question. Thus, our initial results suggest that additional cofactors or other regulatory mechanisms are involved in determining the level of editing, since it cannot be explained by a simple regulation of the Gabra-3 mRNA concentration and the expression level of ADAR2. To better understand the function of the I/M-site editing, we examined the role of editing in the receptor subunit assembly. An earlier study showed that editing of the glutamate receptor subunit B diminished its capability to selfassemble and thereby decreased the concentration of the subunit B at the synapse (Greger et al., 2006). Plasmid expressing the wild type α3 subunit was transfected along with the other required subunits β3 and γ2, with or 38 without an ADAR expression vector into HEK 293 cells. By using confocal microscopy and an anti-α3 antibody binding to the extracellular part of the protein, receptor assembly and presentation on the cell surface was visualized. In absence of the ADAR2 expression vector the α3 subunit was assembled into the receptor and present in the cell membrane. In the presence of ADAR2, Gabra-3 was edited and the amount of the α3 subunit on the cell surface was vastly decreased. These results indicate that editing at the I/M site of Gabra-3 has a negative effect on receptor assembly and membrane presentation. It is possible that the switch in the GABA behavior from excitatory to inhibitory postsynaptic potentials during development is an effect of editing at the I/M site of Gabra-3. In a previous study the A322D mutation in the α1 subunit of the GABAA receptor was shown to reduce expression of the subunit post translationally but prior to receptor assembly. The wild type α1 subunit was present in the membrane whereas the mutated α1 subunit was degraded in ER (Gallagher et al., 2004; Gallagher et al., 2007). This mutation is situated in TM3 of the α1 subunit, and this position is in close proximity to the corresponding I/M site in the α3 subunit. It is possible that editing at the I/M site of Gabra-3 has a negative effect on assembly in a similar way as this mutation. Paper IV – CTN-RNA, a novel substrate for ADAR editing Another highly enriched transcript from the ADAR2 specific IP-array was the mouse specific, nuclear retained CAT2 Transcribed Nuclear-RNA (CTNRNA). The CTN-RNA is transcribed from the mouse Cationic Amino acid Transporter 2 (mCAT2) gene, involved in the cellular uptake of cationic amino acids (reviewed by (MacLeod, 1996)). The CTN-RNA and the mCAT2 mRNA differ in their 3’UTR, where an additional 4.5 kb sequence is present within the CTN-RNA. Both transcripts contain a 100 nt short interspersed nucleotide element (SINE) in their 3’UTR, called forward repeat (FwR). The CTN-RNA, but not the mCAT2 mRNA, contains three complementary inverted repeats (IR I-III) situated in the unique 4.5 kb 3’UTR. Interestingly, several A-to-G differences were observed between the sequenced mouse liver cDNA and the genomic DNA of the CTN-RNA (Prasanth et al., 2005). Moreover, the unique 3’ UTR of the CTN-RNA was found to induce nuclear retention, and was suggested to be a result of the inosines created by editing of the 3’UTR (Prasanth et al., 2005). Under stress conditions the CTN-RNA is cleaved to a mCAT2-like transcript and transported to the cytoplasm for translation. We examined if the ADAR enzymes are able to edit the CTN-RNA. A CTN-RNA minigene, containing the FwR and IR I-III, was co-transfected 39 with different combinations of ADAR expression vectors into HEK 293 cells. Our work revealed that ADAR2, but not ADAR1, can efficiently edit the FwR of the CTN-RNA. Moreover, by deleting IR II in the CTN-RNA, the amount of editing was dramatically reduced. Our results suggest that ADAR2 is responsible for editing of the CTN-RNA and the substrate for editing is a double stranded RNA structure of the CTN-RNA formed between FwR and IR II. Thus, our hypothesis is that the CTN-RNA is edited by ADAR2 in its unique 3’UTR and hence nuclear retained. Upon stress, the CTN-RNA is cleaved and transported to the cytoplasm for translation to a mCAT2 like protein (figure 8). However, if A-to-I editing is involved in the nuclear retention and/or the cleavage of the CTN-RNA is currently not known. Figure 8. In unstressed cells, the mCAT2 gene transcribes CTN-RNA and mCAT2 mRNA. The CTN-RNA is edited and nuclear retained, whereas the mCAT2 mRNA is transported to the cytoplasm for translation. Upon stress, the CTN-RNA is posttranscriptionally cleaved and the released mRNA is transported to the cytoplasm for translation into a mCAT2-like protein. 40 Future studies Detecting novel sites of selective editing Even though we have been successful using the EvoFold program, it is not all-embracing since it only detects double stranded RNA structures with relatively short loops. Therefore, we are in the process of developing a more comprehensive computational package to identify the position of edited sites within the detected candidate genes. Different criteria will be used to get a high score on editing probability: i) a stem loop structure with acceptance of bulges and internal loops that are phylogenetically conserved, ii) A/G mismatches comparing genomes and cDNA sequences within the stem loop structure, iii) an amino acid change as a result of the A-to-I editing, iv) conserved amino acids within related species and v) sequence specific analysis where a G downstream of the adenosine and an A, T or C upstream of the adenosine result in a positive score. The potential edited sites with high scores will be verified using the Genome Sequencer 20 system (Roche) based on the 454 technology (Margulies et al., 2005). The 454 technology makes it possible to sequence several thousand individual clones derived from each PCR amplified candidate. Using this approach we will hopefully elucidate additional sites of adenosine deamination in the mammalian transcriptome that has previously been ignored or impossible to detect using other methods. Knockin mice Is editing of the GABAA receptor essential for the organism? Conditional point mutant knockin mice will be made with a permanently edited receptor α3 subunit as well as animals that have lost their capability to edit the I/M site of Gabra-3. Using the method by Skvorak et al. (2006) knockin mice can be made where the endogenous wild type gene is replaced with a mutant at a specific time point or in a specific region of the mouse by the use of Cremediated recombination (Skvorak et al., 2006). Therefore, the role of Gabra3 editing in early development as well as the importance of editing in different tissues can be determined. 41 Other objectives using the IP-array method In the extension of this project we plan to use this method for other applications. Such as detecting new editing substrates in different tissues. Both ADAR1 and ADAR2 are expressed ubiquitously in many tissues although edited substrates have only been found in brain and liver. Moreover, an antibody in the immunoprecipitation against ADAR1 or ADAR3 could be used to detect substrates specifically targeted by these proteins. 42 Concluding remarks The focus in this thesis work has been to find novel A-to-I editing substrates in mammals. We successfully developed an experimental approach and uncovered two novel editing substrates. We found the Gabra-3 transcript to be edited at a single site, recoding an isoleucine to a methionine. Further, this site was shown to be evolutionary conserved and edited in a developmental manner, concurrent with the dramatic excitatory to inhibitory change of the GABAA receptor. About fifteen years ago it was shown that the glutamate receptors, that are responsible for most excitatory postsynaptic signals in the mammalian brain, are regulating the calcium influx by A-to-I editing. In the adult brain an equilibrium between excitation and inhibition is essential. The GABAA receptor is responsible for most inhibitory postsynaptic signals in the mammalian brain. In this thesis work we show that GABAA receptor is a substrate for A-to-I editing. By studying the receptor assembly we revealed that editing of Gabra-3 has a negative effect on the assembly of the protein into the receptor. Our data add additional evidence that RNA editing is of principal significance in the regulation of neurotransmission in eukaryotes. We have shown that ADAR2 efficiently edit the CTN-RNA. The CTNRNA is the only known example of a nuclear retained RNA that is cleaved upon stress and transported to the cytoplasm for translation. The editing sites are within the region required for nuclear retention and in the vicinity of the predicted cleavage site. It is therefore possible that ADAR editing is involved in these processes. Interestingly, editing of the CTN-RNA situated within the mouse specific SINE element must have arose relatively late in evolution. It is noteworthy that such a basic process takes advantage of ADAR editing that is unique for this organism. It is possible that this is a general mechanism for rapid creation of critical proteins. Although several questions remain to be answered our data will help unravel the complex regulation of the rapid mCAT2 protein production upon stress. A future challenge will be to find additional selective editing sites and understand how the ADAR proteins regulate the essential processes in the mammalian nervous system. 43 Acknowledgement I would like to thank the following for their contribution to this work; Marie, you have been an amazing supervisor. You have always been there for me when I needed your support. I am appreciative of the excellent work environment that you provided. I am grateful for being given the opportunity to attend many conferences. They have led to interesting and fruitful collaboration opportunities and have been developing on a personal level. Traveling with you has been a pleasure! It has been a privilege to be a student in your lab. Bitte, my co-supervisor, when I first contacted the department you showed an amazing enthusiasm, which made a huge impact in my decision to apply for a PhD position at the department. Thank you for all help! Lasse, over the years we have had many great discussions regarding anything from science to Dalecarlia. It has been a real pleasure working at the same department as you. Marie Öhman laboratory, Mats, my former roommate, thank you for all the fun years we spent together. Helene and Chammiran, thank you for all great days we have had in the lab, it has been a pleasure! Also thanks to Kicki, Annika, Jurga, Eva and Nadja. Viktoria & Sara, thank you for always being there, I will never forget our many coffee breaks and laughs at the department. Everyone at the Department Pat, for all the fun times. Pär, for the spontaneous (and much needed) beers. Hanna, you have always thrown the best parties. Petra, for great collaboration. Also thanks to Anu, Gawa, Shiva, Inger, Kerstin, Gunnel and all other great people at the department. Department of Zoophysiology, thank you for all help in the animal house during these years. 44 Thanks to all people we have collaborated with such as Jacob Pedersen, Prasanth Kannanganattu, David Spector, Henrik Boije and Finn Hallböök. It has been very inspiring working with you. During my early years at the university I had the pleasure of getting to know Johan & Gustaf. Our occasional dinners together have always been very fun and refreshing. 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