HYBRIDIZATION OF THE COMPLEMENTARY MESSENGER-RNAS FOR P450C21 (STEROID 21-HYDROXYLASE) AND TENASCIN-X IS PREVENTED BY SEQUENCE-SPECIFIC BINDING OF NUCLEAR PROTEINS

The CYP21 gene that encodes the steroid 21-hydroxylase, P450c21, is ov erlapped on the opposite strand of DNA by the TN-X gene encoding the e xtracellular matrix protein, tenascinn-X. These transcripts contain pe rfectly complementary segments of 299 bases at their 3'-ends. As these genes are tandemly duplicated and are transcribed in the adrenal cort ex, we investigated whether these self-complementary transcripts forme d RNA-RNA hybrids in vivo. Formation of heterogeneous nuclear ribonucl eoprotein complexes between nascent RNA transcripts and nuclear protei ns might modulate such potential RNA-RNA interactions. Using a double RNase protection assay, we found that these RNAs form very small amoun ts of double-stranded RNA-RNA hybrids in adrenal cells in vivo. To und erstand why these mRNAs fail to hybridize in vivo, we studied the acti ons of nuclear proteins on the binding and annealing of their compleme ntary regions in vitro. The nucleation of interstrand annealing was ki netically favored over binding and was efficiently promoted by nuclear extracts. However, RNA-RNA strand zippering was inhibited, suggesting that protein binding and/or stable RNA secondary structures contribut e to discontiguous base pairing. Increasing concentrations of nuclear proteins increased the relative proportion of these RNAs in perfect RN ase-resistant duplexes but reached only about 20% of the total availab le RNA strands at saturating concentrations of nuclear proteins. Prein cubation of either of the two single-stranded RNAs with nuclear protei ns strongly inhibited the nucleation step of annealing, whereas preinc ubation of both strands abolished the annealing. RNase footprinting of the wild type and mutagenized overlapping transcripts suggested that sequence-specific binding of nuclear proteins is limited to the 5'-hal f of each RNA strand. These results indicate that the transcription of complementary, opposite-strand RNAs is not a mechanism for the regula tion of the abundance of adrenal P450c21 mRNA and suggest that nuclear proteins strongly interfere with interstrand RNA base pairing in vitr o as well as in vivo.