Isolation and purification of rat hepatoma nuclei active in the transport of messenger RNA in vitro.

Abstract

1 A variety of procedures for obtaining metabolically active nuclei from rat hepatoma tissue-culture (HTC) cells has been explored. All gave preparations which were, to a greater or lesser extent, contaminated with cytoplasmic RNA, which appeared to be absorbed non-specifically during the lysis of the cells. 2 A medium containing spermidine, monovalent and divalent cations, and cytosol has been defined in which the integrity of the isolated nuclei was maintained during incubation in virro. 3 The release of steady-state-labelled and pulse-labelled RNA from HTC cell nuclei during incubation in this medium has been studied. The transport of both was dependent on ATP and cytosol, but was distinguishable by the kinetics and by the fact that only the transport of pulse-labelled RNA appeared to involve hydrolysis of ATP. Transported RNA included ribosomal RNA and messenger-likc RNA as judged by criteria of size, oligo(dT)-cellulose binding, and organisation into ribonucleoproteins. A large proportion of the cytoplasmic RNA which adhered to the nuclei was also released during incubation in vitro. 4 Although the poly(A)-rich RNA transported in vitro was contaminated with poly(A)-rich RNA sequences arising from non-specific leakage of nuclear RNA and from the release of nucleus-adherent cytoplasmic RNA, the extent of this contamination was comparatively small (10% and 10–15% by weight respcctively). The transportcd poly(A)-rich RNA has been shown to consist of a complex mixture of sequences at heterogeneous abundances, resembling polysomal poly(A)-rich mRNA from HTC cells. 5 The pattern of relative abundanccs of the sequences in transported poly(A)-rich RNA was intermediate between that of polysomal and nuclear poly(A)-rich RNAs, and indicated that some degree of sequence-specific selection of processing, or transport, or both, can be maintained in isolated nuclei. Sequence-specific selection in vitro at the level of individual poly(A)-rich RNA sequences was detected using cloned cDNAs for poly(A)-rich polysomal RNA sequences.