Abstract
Pre-mRNAs for brain-expressed ionotropic glutamate receptor subunits undergo RNA editing by site-specific adenosine deamination, which alters codons for molecular determinants of channel function. This nuclear process requires double-stranded RNA structures formed by exonic and intronic sequences in the pre-mRNA and is likely to be catalyzed by an adenosine deaminase that recognizes these structures as a substrate. DRADA, a double-stranded RNA adenosine deaminase, is a candidate enzyme for L-glutamate-activated receptor channel (GluR) pre-mRNA editing. We show here that DRADA indeed edits GluR pre-mRNAs, but that it displays selectivity for certain editing sites. Recombinantly expressed DRADA, both in its full-length form and in an N-terminally truncated version, edited the Q/R site in GluR6 pre-mRNA and the R/G site but not the Q/R site of GluR-B pre-mRNA. This substrate selectivity correlated with the base pairing status and sequence environment of the editing-targeted adenosines. The Q/R site of GluR-B pre-mRNA was edited by an activity partially purified from HeLa cells and thus differently structured editing sites in GluR pre-mRNAs appear to be substrates for different enzymatic activities.
Highlights
The alteration of codons by RNA editing, leading to changes in protein structure and function, represents a newly recognized type of posttranscriptional modification in mammalian nuclear transcripts and occurs by site-specific base modification (1, 2)
Different from apolipoprotein B (apoB) RNA editing (1), the site-specific adenosine deamination in glutamategated receptor channels (GluR) transcripts requires a doublestrandedRNA structure formed by the exonic sequence around the editing site and an intronic editing site complementary sequence (ECS) (3, 8), predicting that this type of RNA editing is catalyzed by an adenosine deaminase that operates on dsRNA
At no point during purification could the DRADA is a candidate enzyme for mammalian nuclear transcript editing by adenosine deamination (2, 8, 14)
Summary
The alteration of codons by RNA editing, leading to changes in protein structure and function, represents a newly recognized type of posttranscriptional modification in mammalian nuclear transcripts and occurs by site-specific base modification (1, 2). DsRAD (2, 13), termed DRADA (14), is a dsRNA-specific adenosine deaminase that is widely expressed, both with respect to species and tissue. This enzyme lacks site-selective activity on extended dsRNAs but displays a sequence-dependent modification of specific adenosines in short synthetic dsRNAs (15). Indicating substrate selectivity for certain editing sites, the recombinantly expressed deaminase edited in synthetic pre-mRNAs the R/G site of GluR-B and the Q/R site of GluR6, but not the Q/R site of GluR-B This latter site appears to be the substrate for a different activity, as indicated by fractionation of nuclear extract from HeLa cells. A comparison of the dsRNA structures for the different sites suggests that the structural environment of the to-be-edited adenosine may be one determinant for the substrate selectivity by different editing activities
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