Hybridization of the complementary mRNAs for P450c21 (steroid 21-hydroxylase) and tenascin-X is prevented by sequence-specific binding of nuclear proteins.

Abstract

The CYP21 gene that encodes the steroid 21-hydroxylase, P450c21, is overlapped on the opposite strand of DNA by the TX-X gene encoding the extracellular matrix protein, tenascin-X. These transcripts contain perfectly complementary segments of 299 bases at their 3'-ends. As these genes are tandemly duplicated and are transcribed in the adrenal cortex, we investigated whether these self-complementary transcripts formed RNA-RNA hybrids in vivo. Formation of heterogeneous nuclear ribonucleoprotein complexes between nascent RNA transcripts and nuclear proteins might modulate such potential RNA-RNA interactions. Using a double RNase protection assay, we found that these RNAs form very small amounts of double-stranded RNA-RNA hybrids in adrenal cells in vivo. To understand why these mRNAs fail to hybridize in vivo, we studied the actions of nuclear proteins on the binding and annealing of their complementary regions in vitro. The nucleation of interstrand annealing was kinetically 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 contribute to discontiguous base pairing. Increasing concentrations of nuclear proteins increased the relative proportion of these RNAs in perfect RNase-resistant duplexes but reached only about 20% of the total available RNA strands at saturating concentrations of nuclear proteins. Preincubation of either of the two single-stranded RNAs with nuclear proteins strongly inhibited the nucleation step of annealing, whereas preincubation 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'-half of each RNA strand. These results indicate that the transcription of complementary, opposite-strand RNAs is not a mechanism for the regulation of the abundance of adrenal P450c21 mRNA and suggest that nuclear proteins strongly interfere with interstrand RNA base pairing in vitro as well as in vivo.