Editing of glutamate receptor subunit B pre-mRNA by splice-site variants of interferon-inducible double-stranded RNA-specific adenosine deaminase ADAR1

The interferon-inducible RNA-specific adenosine deaminase (ADAR1) is an RNA -editing enzyme that catalyzes the deamination of adenosine in double-stran ded RNA structures. Three alternative splice-site variants of ADAR1 (ADAR1- a, -b, and -c) occur that possess functionally distinct double-stranded RNA -binding motifs as measured with synthetic double-stranded RNA substrates, The pre-mRNA transcript encoding the B subunit of glutamate receptor (GluR- B) has two functionally important editing sites (Q/R and R/G sites) that un dergo selective A-to-I conversions. We have examined the ability of the thr ee ADAR1 splice-site variants to catalyze the editing of GluR-B pre-mRNA at the Q/R and R/G sites as well as an intron hotspot (+60) of unknown functi on. Measurement of GluR-B pre-mRNA editing in vitro revealed different site -specific deamination catalyzed by the three ADAR1 variants. The ADAR1-a, - b, and -c splice variants all efficiently edited the R/G site and the intro n +60 hotspot but exhibited little editing activity at the Q/R site, ADAR1- b and -c showed higher editing activity than ADAR1-a for the R/G site, wher eas the intron +60 site was edited with comparable efficiency by all three ADAR1 splice variants. Mutational analysis revealed that the functional imp ortance of each of the three RNA-binding motifs of ADAR1 varied with the sp ecific target editing site in GluR-B RNA. Quantitative reverse transcriptio n-polymerase chain reaction analyses of GluR-B RNA from dissected regions o f rat brain showed significant expression and editing at the R/G site in al l brain regions examined except the choroid plexus. The relative levels of the alternatively spliced flip and flop isoforms of GluR-B RNA varied among the choroid plexus, cortex, hippocampus, olfactory bulb, and striatum, but in all regions of rat brain the editing of the flip isoform was greater th an that of the flop isoform.