Secondary structure of heterogeneous nuclear RNA: Two classes of double-stranded RNA in native ribonucleoprotein

Abstract

Heterogeneous nuclear RNA (hmRNA) from HeLa [human cervical cancercells] cells contains intramolecular duplexes. Since hmRNA is associated with protein in vivo, it is possible that the double-stranded regions observed in deproteinized hmRNA form spontaneously on the release of protein from single-stranded but potentially complementary sequences. This is not the case for a class of double-stranded sequences that is defined by resistance to RNases A + T1 at high ionic strength. Exposure of HeLa hmRNA .cntdot. ribonucleoprotein (hmRNP) particles to Escherichia coli RNase III, a double-strand-specific endoribonuclease, destroys most of the sequences resistant to RNases A + T1. This effect is completely blocked when hmRNP is exposed to RNase III in the presence of an excess of purified double-stranded RNA. There exist 2 classes of double-stranded RNA in hmRNP at a salt concentration of 0.13 M. These are distinguished by their relative resistance to RNases A +T1. The more stable double-stranded sequences, which are resistant to RNases A + T1 at 0.13 M, comprise 1.0-1.1% of the nucleotides in hnRNP. The less stable double-stranded sequences comprise an additional 1.5-2.0% of the nucleotides in hnRNP. These are sensitive to RNase III at 0.13 M, but are not resistant to RNases A + T1 unless the salt concentration is raised to 0.63 M. The demonstration that double-stranded sequences resistant to RNases A + T1 exist in native ribonucleoprotein and are not artifacts of deproteinization makes it appropriate to seriously consider their possible functional role in hnRNA metabolism, perhaps as binding sites for regulatory proteins involved in mRNA processing.