Evidence for a role of RNA in eukaryotic chromosome structure. Metabolically stable, small nuclear RNA species are covalently linked to chromosomal DNA in HeLa cells

Abstract

An investigation of metabolically stable, chromatin-associated RNA in HeLa cells has revealed that three small RNA species, 193, 171 and 127 nucleotides in length, are covalently linked to double-stranded chromosomal DNA through phosphodiester bonds. These DNA-linked RNAs appear to be members of the small nuclear RNA species that have been identified in a wide variety of eukaryotic cells, and they are tentatively identified as species C, D and G′, in the nomenclature system currently employed for HeLa cell small nuclear RNAs. These DNA-linked RNAs do not appear to be involved in priming DNA replication, since they are of relatively high metabolic stability ( t 1 2 = 19 hours in HeLa cells with a 21·5-hour cell generation time) and since their covalently contiguous DNA stretches are not enriched in newly replicated material. They lack saturated pyrimidine bases (level of detection = 0·15 mol %) and are therefore not “chromosomal RNA”, as defined by its proponents. The covalent linkage of these small RNA species with chromosomal DNA was discovered by virtue of the fact that when highly purified HeLa cell chromatin is dissociated by chaotropic solutes, these RNAs are released in association with small pieces of double-stranded DNA (approx. 475 nucleotide pairs). These DNA-RNA complexes can then be purified by removing the bulk, high molecular weight DNA by ultra-centrifugation. The resulting DNA-RNA complexes are shown to be covalently joined by several criteria, including equilibrium density-gradient centrifugation in either Cs 2SO 4/dimethylsulfoxide or aqueous Cs 2SO 4/formaldehyde after thermal denaturation (90 °C in 50% formamide, which is 55 deg. C above the melting temperature of this DNA), by the chromat ographicbehavior of the complexes on hydroxylapatite before and after thermal denaturation, and by the demonstration of alkali-resistant ribonucleotides flanking the 3′ hydroxyl termini of the DNA, the latter criterion providing evidence for 3′ to 5′ DNA-RNA phosphodiester bonds. Reconstruction experiments involving addition of the purified RNAs to nuclei or chromatin demonstrate that the covalent DNA-RNA linkages do not arise by ligation events during cell fractionation. Further experiments indicate the existence of a dynamic equilibrium of these small nuclear RNA species between chromosomal and nucleoplasmic loci in vivo, and other considerations suggest that this equilibrium may be cell cycle-dependent. The DNA adjacent to these covalently linked RNAs has the same melting temperature as total HeLa chromosomal DNA and its reassociation kinetics reveal the presence of both repeated and non-repeated sequences, implying that the DNA-linked RNAs are widely distributed throughout the HeLa cell genome. It is proposed that these DNA-linked RNAs are involved in the tertiary structure of chromatin, particularly in relation to the cell cycle.