Intracellular distributions of the low molecular weight RNA species of HeLa cells were determined by a nonaqueous method of cell fractionation, in which lyophilized cells were homogenized and centrifuged in anhydrous glycerol. The nonaqueous method was used to avoid artifactual extraction of weakly bound nuclear RNA during cell fractionation. We found that the mature small RNA species K, A, C, and D were almost entirely (greater than 95%) nuclear, and that mature 4S tRNA was partially (5-10%) nuclear. Our results gave higher nuclear content of the mature species K, A, C, and 4S than was shown previously with conventional aqueous cell fractionation. The nonaqueous method also gave higher nuclear proportions of some short-lived precursors to mature small RNAs. We found that approximately one-half of recently synthesized pre-4S RNA and more than one-half of recently synthesized 5S RNA were nuclear, whereas these species had been thought to be cytoplasmic from previous work. The species C' and D', precursors to the stable nuclear species C and D respectively, were found to be partially nuclear, also in contrast to earlier work. The stable cytoplasmic species L (oncornavirus 7S RNA) was found to be mostly nuclear shortly after synthesis.

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