ABUNDANT ADRENAL-SPECTI IC TRANSCRIPTION OF THE P450c21A PSEUDOGENE—IMPLICATIONS FOR CONGENITAL ADRENAL HYPERPLASIA

Abstract

21-hydroxylase is encoded by the 21B gene in the HLA locus on chromosome 6. The duplicated 21A gene is generally regarded as being a non-functional pseudogene, because it has several mutations that would prevent it from encoding P450c21 protein, and because others have reported that it is not transcribed into RNA. Through the use of cDNA cloning and sequencing, RNA-based PCR, and RNase protection experiments, we now show that the 21A and 21B genes are actively transcribed into a family of four large, stable, previously undescribed adrenal-specific mRNAs. These transcripts, termed “Y” RNAs, do not encode P450c21. YA transcripts use the 21A promoter and cap site, and encompass all of the coding region plus the first two introns of 21 A. The short YA RNA (3.0 kb) is spliced from a point in the 3′ untranslated region of 21A to another three exons beginning 4.5 kb downstream; the long YA RNA (7.5 kb) retains this 4.5 kb, which comprises all of the opposite strand of the XA gene. The short and long forms of YB arise from the 21B promoter in the same fashion. We designed a riboprobe spanning the splices 4.5 kb XA region of the short YA and extending 3′ past the A/B gene duplication junction, permitting the display of the four Y RNAs and 21B RNA as five discrete products in an RNase protection assay showing that the long forms of YA and YB predominate over the short forms, and that these are about 20% of the abundance of 21B mRNA. RNase protection experiments also show that expression of Y RNAs is confined to the adrenal. The 21/X/Y system is the first reported example of three overlapping genes in a eukaryotic organism. Transcription of YA exposes 21A gene sequences to single-stranded nicking, possibly explaining the very high incidence of 21A/21B gene conversions that cause 85% of cases of congenital adrenal hyperplasia.