Synthetic Polynucleotides and the Amino Acid Code, V

A minor fraction of basic fibroblast growth factor mRNA is deaminated in Xenopus stage VI and matured oocytes

“…The significance of the identification of deaminated bFGF mRNA As mentioned (see Introduction), two types of deamination, hypermutation and selective, have been observed among ADAR substrates+ The cellular substrates discovered so far are largely of the selective type, and only a single hypermutated cellular RNA, the Drosophila 4f-rnp transcript, has been reported+ Our studies indicate that bFGF mRNA provides a second example of a cellular RNA that is hypermutated by an ADAR+ By analyzing RNA populations we determined that the deaminated bFGF mRNA molecules comprise a minor population of the steady-state bFGF mRNA+ The results of our analyses emphasize that the identification of a single hypermutated cDNA does not mean that all RNAs of a given sequence are ADAR substrates+ Since only a single cDNA has been analyzed in the case of 4f-rnp (Petschek et al+, 1996), analyses of populations of 4f-rnp cDNAs would be informative+ Our studies say nothing about the function of the inosines within bFGF mRNAs+ It has been hypothesized that inosine could mark an RNA for degradation and previous reports correlated the deamination of bFGF mRNA during oocyte maturation with its subsequent degradation (Kimelman & Kirschner, 1989)+ In our studies the fraction of deaminated molecules observed before and after oocyte maturation was almost identical, suggesting that inosine-containing molecules are stable and persist after maturation+ Still other studies in our laboratory involving the microinjection of synthetic dsRNA indicate that hypermutation does not lead to degradation during oocyte maturation (data not shown)+ Regardless, the recent discovery of a mammalian ribonuclease (I-RNase) that is specific for inosine-containing single-stranded RNA (Scadden & Smith, 1997) suggests that degradation mechanisms are still worthy of further consideration+ This is particularly true since the bFGF antisense transcript has also been implicated in regulating the stability of the sense RNA in other organisms, including rat, chicken, and human (reviewed in Knee & Murphy, 1997)+ Although we observed that only a minor fraction of bFGF mRNA was deaminated, it remains possible that the deamination of this minor fraction causes a significant alteration of bFGF expression+ Inosine is translated as guanosine (Basilio et al+, 1962), and thus, the multiple deamination events we observed within bFGF mRNAs would be predicted to drastically alter the amino acid sequence of the encoded protein+ In cells infected with polyoma virus, complementary viral transcripts are deaminated, and the resulting hypermutated viral RNAs are retained in the nucleus (Kumar & Carmichael, 1997)+ Hypermutated cellular RNAs such as bFGF might also be retained in the nucleus, thus reducing the level of transcript available for translation+ Further studies are clearly required to determine if the ADARs play a role in the posttranscriptional regulation of bFGF expression+ Even if the deamination of bFGF mRNA does not play a role in stage VI oocytes, it could play a role at other times in Xenopus development+ Of course, it is possible that the deamination of bFGF serves no biological role, but merely exemplifies a background reaction that will occur to a minor extent anytime complementary RNAs hybridize+ In this regard, our observation of deaminated bFGF molecules, combined with the previous observation (Kimelman & Kirschner, 1989), emphasizes that the complementary region of the sense and antisense transcripts are hybridized in vivo+ Possibly the hybridization of these RNAs, even in the absence of deamination, is biologically i...…”

dADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing

“…RNA editing involving the conversion of adenosine (A) to inosine (I) in pre-mRNA is a posttranscriptional process catalyzed by proteins of the ADAR (adenosine deaminases that act on RNA) family (Rueter & Emeson, 1998)+ ADARs require duplex RNA secondary structure in their target substrates+ There are two types of A-to-I RNA editing, promiscuous and specific (reviewed in Bass, 1997)+ Viral RNA genomes provided the first reported instance of promiscuous A-to-I RNA editing where extensive conversion of A to G (guanosine) was observed (Cattaneo et al+, 1988)+ Since I base pairs with cytidine, G replaces I when cDNAs are generated+ Mechanistically, cRNA intermediates of the replication process or transcripts base pair with complete complementarity to the viral genomic RNA+ When the substrates for ADARs are extensive duplexes, up to 50% of adenosines are converted to inosine+ Such promiscuous editing has been proposed to have roles in gene regulation, viral defense (Bass & Weintraub, 1988) and in biased hypermutation and persistent infection of certain viruses (Bass, 1997)+ Promiscuous editing is also seen in vivo when naturally occurring antisense transcripts are generated, as in the case of the Xenopus bFGF gene (Saccomanno & Bass, 1999) and in polyoma virus (Kumar & Carmichael, 1997)+ Supporting a role in gene regulation is the finding that extensively modified early transcripts from polyoma are almost exclusively retained in the nucleus at late times in infection, thus preventing translation, a critical feature of the polyoma life cycle (Kumar & Carmichael, 1997)+ Another potential role in mRNA regulation via extensive editing was recently proposed in C. elegans where sev-eral transcripts containing extended 39 noncoding hairpin structures are extensively modified within hairpin regions (Morse & Bass, 1999)+ ADARs can also edit specific adenosines in mRNA precursors (pre-mRNA)+ Surprisingly, most of these examples are restricted to genes encoding ligand-gated ion channels or receptor genes expressed in the nervous system and result in only one or a few adenosines deaminated per transcript (Rueter & Emeson, 1998)+ Because the translation machinery has been shown to interpret I as G (Basilio et al+, 1962), modifications introduced by an ADAR are predicted to change the coding potential of the edited transcripts+ This was first shown for pre-mRNAs encoding subunits of the glutamate-gated ion channel receptors (GluRs)+ The GluR-B transcripts have numerous distinct RNA editing sites that, when edited, have profound consequences for channel function (reviewed in Seeburg et al+, 1998)+ For instance, editing that results in the conversion of a glutamine codon (CAG) to a arginine codon (CGG) at the Q/R site alters the Ca 2ϩ permeability of channels containing the edited subunit (Kohler et al+, 1993)+ The physiological significance of editing at the Q/R site is demonstrated by the neurological dysfunction and postnatal lethality associated with mutant mice expressing an editing incompetent allele of GluR-B (Brusa et al+, ...…”

Down-regulation of RNA Editing in Pediatric Astrocytomas

“…This deamination is the most widespread type of RNA editing in higher eukaryotes. Since the translation machinery reads inosine as guanosine (1), the ADARs can modify protein codons and thus modulate protein sequence and function of several gene products. The editing of a specific adenosine is not usually 100% efficient and as a consequence, the ADARs can generate different protein isoforms in the same cell.…”