Modulation of RNA editing by functional nucleolar sequestration of ADAR2

Abstract

The adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine (A to I) in primary mRNA transcripts, thereby affecting the splicing pattern or coding potential of mature mRNAs. Although the subnuclear localization of A-to-I editing has not been precisely defined, ADARs have been shown to act before splicing, suggesting that they function near nucleoplasmic sites of transcription. Here we demonstrate that ADAR2, a member of the vertebrate ADAR family, is concentrated in the nucleolus, a subnuclear domain disparate from the sites of mRNA transcription. Selective inhibition of ribosomal RNA synthesis or the introduction of mutations in the double-stranded RNAbinding domains within ADAR2 results in translocation of the protein to the nucleoplasm, suggesting that nucleolar association of ADAR2 depends on its ability to bind to ribosomal RNA. Fluorescence recovery after photobleaching reveals that ADAR2 can shuttle rapidly between subnuclear compartments. Enhanced translocation of endogenous ADAR2 from the nucleolus to the nucleoplasm results in increased editing of endogenous ADAR2 substrates. These observations indicate that the nucleolar localization of ADAR2 represents an important mechanism by which RNA editing can be modulated by the sequestration of enzymatic activity from potential RNA substrates in the nucleoplasm.T he conversion of adenosine to inosine (A to I) by RNA editing was first observed in yeast tRNAs (1) but has since been identified in numerous viral and cellular mRNA transcripts (2-4). A-to-I editing is most often identified as an adenosineto-guanosine (A-to-G) discrepancy between genomic and cDNA sequences due to the similar base-pairing properties of inosine and guanosine during cDNA synthesis. A-to-I conversion is catalyzed via hydrolytic deamination at the C6 position of the adenine ring (5) and requires an extended region of doublestranded RNA (dsRNA) in potential RNA substrates formed by intramolecular base-pairing interactions between exon and intron sequences (2-4). A family of adenosine deaminases that act on RNA (ADARs) have been extensively characterized and are responsible for catalyzing the site-specific A-to-I conversion observed in cellular mRNA transcripts (6-8). ADAR-mediated RNA editing events can change the amino acid coding potential of genomically encoded transcripts to produce protein products with altered functional properties (2, 3). For example, the editing of RNAs encoding mammalian ionotropic glutamate receptor (GluR) subunits can lead to the production of heteromeric glutamate-gated channels with altered ion permeation and agonist-induced desensitization kinetics (9-11), whereas the editing of transcripts encoding the 2C-subtype of serotonin receptor (5-HT 2C R) can generate receptor isoforms that couple to heterotrimeric G proteins with decreased efficiency (12-14). A-to-I editing events have also been described in nontranslated RNA species and noncoding regions of mRNA transcripts (15)(16)(17), suggesting that such modif...

Substrate-dependent Contribution of Double-stranded RNA-binding Motifs to ADAR2 Function

“…Transfection of eGFP alone resulted in a diffuse pattern of fluorescence in the cytoplasm and nucleus, with no preferential concentration in nucleoli, whereas the pattern of eYFP-nucleolin fluorescence was highly restricted to the nucleolar compartment (Figure 2A). The fluorescence pattern for cells cotransfected with both eGFP and eYFP-nucleolin was identical to that observed when each fluorophore was transfected independently, with little observable bleedthrough between the emission channels for these simultaneously excited fluorescent proteins (Figure 2A).Transient expression of wild-type eGFP-ADAR2 in NIH/ 3T3 cells demonstrated the previously observed pattern of nucleolar localization (Fn/Fo ϭ 13.8 Ϯ 2.4), and deletion of both dsRBM domains and the intervening 81-aa linker [⌬dsRBM1/2 (⌬76 -301)] produced the expected pattern of diffuse nuclear fluorescence (Figure 2A), suggesting that dsRNA binding was required to maintain the steady-state localization of ADAR2 in nucleoli (Desterro et al, 2003;Sansam et al, 2003). Expression of a mutant eGFP-ADAR2 fusion protein (K127A, K281A), containing substitutions for highly conserved amino acids in the loop between the ␤3 and ␣2 regions for all dsRBMs (Tian et al, 2004), resulted in a diffuse pattern of nuclear fluorescence nearly identical to the pattern observed when the region containing both dsRBMs was deleted from the fusion protein (Figure 2A).…”

“…These disadvantages include differences in the levels of expression for transfected fusion proteins and minigene-derived RNA substrates and the fact that such cellular RNA processing events do not necessarily occur in the linear range for ADAR2 enzymatic activity. In addition, previous studies have demonstrated that A-to-I conversion takes place in the nucleoplasm (Rueter et al, 1999;Raitskin et al, 2001;Desterro et al, 2003;Sansam et al, 2003) and that translocation of ADAR2 to the nucleoplasm results in increased editing activity (Sansam et al, 2003), making comparisons of editing activity for mutations that simultaneously affect both subnuclear accumulation and site-specific editing difficult to interpret.To circumvent these problems, we used an in vitro editing system using crude nuclear extracts from HEK293 cells transiently transfected with different eGFP-ADAR2 mutants. The relative protein level for wild-type and mutant eGFP-ADAR2 proteins in HEK293 nuclear extracts was determined by quantitative Western blotting analysis using an affinity-purified antiserum directed against amino acids 6 -66 of wild-type ADAR2 (Figure 4) (Sansam et al, 2003;Dawson et al, 2004), and all proteins were diluted to achieve the same final concentration in the in vitro editing reaction.…”

“…For each RNA editing substrate (100 fmol), the amount of the eGFP-ADAR2 protein and the duration of the reaction were empirically determined to assure that all of the editing reactions were performed in the linear range of the assay (Supplemental Figure S1). The extent of total inosine production for the perfect dsRNA was determined by TLC (Rueter et al, 1995), and site-specific editing of the remaining ADAR2 substrates was quantified using modified primerextension analyses (Sansam et al, 2003;Dawson et al, 2004).Using crude nuclear extracts from mock-transfected HEK293 cells, only background levels of editing were observed for any of the RNA substrates ( Figure 5, A and B), consistent with previous observations regarding the low level of endogenous editing activity in this cell line Burns et al, 1997;Rueter et al, 1999;Schaub and Keller, 2002). Deletion of the region containing both dsRBMs and the intervening linker (⌬dsRBM1/2) reduced editing in all RNA targets to near background levels, as did deletion of dsRBM2 alone (⌬dsRBM2), demonstrating that dsRBM2 is essential for the editing of perfect dsRNAs as well as naturally occurring ADAR2 substrates containing imperfect inverted repeats.…”

Editor meets silencer: crosstalk between RNA editing and RNA interference

“…ADAR2 localizes predominantly in the nucleolus 44,46 . The significance of the nucleolar localization of ADAR1S and ADAR2 is not currently clear.…”

The nucleolus: reviewing oldies to have new understandings

“…Thus, many non-traditional functions of the nucleolus have been proposed [5][6][7]9]. These include signal recognition particle assembly, small RNA modification, RNA editing, telomerase maturation, nuclear export, cell cycle control, and stress sensor [17][18][19][20][21][22]. The nucleolar proteins unrelated to ribosome assembly mostly contain an RNA-binding motif or have a chaperone function.…”