Energy requirement and kineties of transport of poly(A)‐free histone mRNA compared to poly(A)‐rich mRNA from isolated L‐cell nuclei

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Abstract
ATP‐promoted efflux of poly(A)‐rich RNA from isolated nuclei of prelabeled mouse lymphoma L5178y cells has an activation energy of 51.5 kJ/mol, similar to that found for the nuclear envelope nucleoside triphosphatase (48.1 kJ/mol) assumed to be involved in mediating nucleocytoplasmic transport of at least some RNA. Here we show that efflux of two specific poly(A)‐rich mRNAs (actin and β‐tubulin) from isolated L‐cell nuclei is almost totally dependent on the presence of ATP, while efflux of poly(A)‐free histone mRNA (H4, H2B, and H1) also occurs to a marked extent in the absence of this nucleotide. Measurements of temperature dependence of transport rate revealed an activation energy of 56.1 kJ/mol for actin mRNA, while the activation energy for histone‐H4‐mRNA efflux was in the same range as that found for ATP‐induced release of RNA from demembranated nuclei (about 15–20 kJ/mol). Addition of nonhydrolyzable nucleotide analogs of ATP to the in vitro system used for measurement of RNA transport did not result in release of nonhistone mRNA (actin), but enhanced the efflux of H4 mRNA to approximately the same extent as ATP. Although not absolutely required, addition of ATP stimulated the rate of export of histone mRNA about twofold. Only the poly(A)‐rich RNA, but not the poly(A)‐free RNA, released from isolated nuclei was found to compete with poly(A) for the nuclear envelope mRNA‐binding site, indicating the mechanism of transport for both RNA classes to be distinct. Export of both nonhistone and histone mRNA was found to be inhibited by a monoclonal antibody against a p60 nuclear‐pore‐complex antigen. This antibody had no effect on the nucleoside triphosphatase, mediating transport of poly(A)‐rich mRNA.