Sub-nuclear fractionation: III. Sub-nuclear distribution of poly(A)-rich RNA

Abstract

Using a mild procedure for sub-nuclear fractionation, recently developed by us [10], rat liver nuclei were resolved into 8 fractions representing nuclear sap, euchromatin, nucleoli, membrane-attached chromatin, and heterochromatin with interchromatin granules. The proportion of poly(A)-containing molecules in newly synthesized RNA in each of the sub-nuclear fractions was determined by ( a) adsorption to cellulose nitrate; ( b) binding to poly(U) immobilised on glass-fibre filters and ( c) chromatography on oligo(dT)-cellulose. The fraction of total nucleotides in unlabelled RNA represented as poly(A) tracts was estimated by hybridization of RNA with 3H-poly(U). The sub-nuclear fractions could be divided into two major classes according to the poly(A) content of their RNA: ( a) fractions with poly(A)-rich RNA (20–25% of RNA), which included euchromatin (f1, f2, f3), a pellet of heterochromatin containing interchromatin granules (fP) and nuclear sap (fS); ( b) fractions poor in poly(A) (2–5% of RNA) comprising nucleoli (f4, f5) and membrane-bound heterochromatin (f6). Hybridization with 3H-poly(U) showed that 0.20–0.25% of total RNA consisted of poly(A) tracts in the first group of sub-nuclear fractions whereas it was 0.03–0.07% in the other group. About l μg of poly(A) associated with RNA was recovered from 1 g equivalent of rat liver nuclei, of which 50% was associated with euchromatinrich fractions, 25% with a ‘pellet’ fraction (presumably in interchromatin granules) and only 15% in the nuclear sap. Much of the poly(A)-rich RNA in the first two fractions was of high molecular weight (>30S) with poly(A) ‘tails’ of 100–150 nucleotides at the 3′-terminus. Over half of the poly(A)-containing RNA in the ‘nucleoplasm’ was of low molecular weight (<6S), presumably representing degraded HnRNA. Taking into account our earlier results on the intranuclear compartmentation of transcription [11], it is concluded that a large part of poly(A)-rich HnRNA in rat liver nuclei is located near its site of synthesis (euchromatin) and in interchromatin granules but very little in the nuclear sap.