Regulated transport of messenger ribonucleic acid from isolated liver nuclei by nucleic acid binding proteins

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Abstract
Rat liver nucleocytosolic mRNA transport is regulated by proteins with a high affinity for nucleic acids. In the cell-free system described, the energy-dependent transport of all RNA classes [tRNA, mRNA and rRNA] exhibited a dependence upon the availability of discrete minor sets of cytosol proteins. In addition to having a different level of saturation, only the mRNA transport protein activities are increased by cAMP, an effect most likely mediated by a cAMP-dependent protein kinase. The mRNA transport proteins were isolated from cytosol by precipitation with streptomycin sulfate followed by DNA-cellulose affinity chromatography, or from oligo-(thymidylate)-cellulose bound cytoplasmic messenger ribonucleoprotein (mRNP) particles by high-salt extraction. Either method yielded a protein fraction which exhibited a 1000-fold increase in mRNA transport activity as compared to cytosol. Over 1/2 of the mRNA transport activity is associated with the mRNP of the cell. A partial homology between the cytosol and mRNP-derived proteins was demonstrated by polyacrylamide gel electrophoresis. One major (20,000 daltons) and several minor proteins (23,000, 52,000, 54,000 and 72,000 daltons) were in common. Nuclear 4-5S RNA, mRNA and rRNA pulse labeled in vivo for 20 min exited from in vitro incubated nuclei in 3 phases, according to their differential in vivo rates of labeling and intranuclear pool sizes. The amount of nuclear RNA transported in vitro as mRNA (about 1.0%) agrees with the in vivo estimates. Additional evidence for in vivo equivalence was provided by the physicochemical characterization and bioassay of the RNA. The transported mRNA sedimented in urea-sucrose gradients as an 8-18S heterodisperse product. This RNA initiated cell-free translation with the synthesis of precursor peptides as diverse in size as those for albumin and .alpha.2U-globulin. The relative abundance of various transported mRNA was different than the corresponding abundance of liver cytoplasmic mRNA.