Structural requirements for RNA editing in glutamate receptor pre-mRNAs by recombinant double-stranded RNA adenosine deaminase.
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.
Maas
S
S
Laboratory of Molecular Neuroendocrinology, Center for Molecular Biology (ZMBH), University of Heidelberg, Federal Republic of Germany.
Melcher
T
T
Herb
A
A
Seeburg
P H
PH
Keller
W
W
Krause
S
S
Higuchi
M
M
O'Connell
M A
MA
D000241:Adenosine / Q000378:metabolism; D000243:Adenosine Deaminase / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D008969:Molecular Sequence Data; D017393:RNA Editing; D012322:RNA Precursors / Q000235:genetics*; D012330:RNA, Double-Stranded / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D017470:Receptors, Glutamate / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D012322:RNA Precursors; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D017470:Receptors, Glutamate; D011994:Recombinant Proteins; D000243:Adenosine Deaminase; D000241:Adenosine