Active transport of messenger ribonucleoprotein particles in a reconstituted cell-free system

The ability of a reconstituted cell-free system to transport mRNA as a ribonucleoprotein particle has been examined. Poly(A) messenger ribonucleoproteins (mRNPs), UV cross-linked after release from isolated liver nuclei in a cell-free system, exhibited a buoyant density of 1.33 g/cm3 in cesium sulfate and 1.47 g/cm3 in cesium chloride, values identical to those of poly(A) mRNP isolated directly from liver polysomes. Furthermore, the in vivo and in vitro transported mRNP showed a similar degree of resistance to RNase digestion and had sedimentation coefficients approximately 2.5 times that of the isolated mRNA. Release of both total mRNA and alpha 2 mu-globulin mRNA was proportional to the concentration of a specific cytoplasmic protein. Removal of the transport proteins from the cytosol with streptomycin sulfate provided a basal system incapable of supporting the active transport of alpha 2 mu-globulin mRNA. Hybridization of released RNA with a recombinant probe specific for intron 6 of alpha 2 mu-globulin showed that intron sequences were retained within the nucleus under optimal alpha 2 mu-globulin mRNA transport conditions and that the transported alpha 2 mu-globulin mRNA was of mature size.