Comparative studies on the ‘5′-cap’ and in vitro translational activity of cytoplasmic and nuclear poly A(+) RNA

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Abstract
The translational activities of cytoplasmic poly A(+) RNA of normal rat liver and Novikoff hepatoma cells in the wheat germ cell free system were found to be approximately 15–20 times greater than those of the corresponding nuclear poly A(+) RNA. The translationsl activities were 85 and 62 pmoles3H-leucine incorporated/µg cytoplasmic poly A(+) RNA for the liver and tumor respectively and 3–4 pmoles3H-leucine incorporated/µg nuclear poly A(+) RNA. Inasmuch as integrity of the ‘5′-cap’ of mRNA is essential for its translational activity, quantitative comparisons were made of its content in these RNA fractions. Of the total32P incorporated into the tumor cytoplasmic poly A(+) RNA, 0.41% was in the ‘5′-cap’; in nuclear poly A(+) RNA, the ‘5′-cap’ contained 0.11%. After periodate oxidation and labeling with KB3H4, m7 guanosine, the 5′-terminal nucleoside in both liver and Novikoff hepatoma nuclear poly A(+) RNA contained approximately 20% as much isotope as in the cytoplasmic poly A(+) RNA. These results suggest the lower translational activity of nuclear poly A(+) RNA is partly related to its lower content of the ‘5′-cap’. Molecular selection of poly A(+) RNA for transport out of the nucleus or further cytoplasmic processing may account for the higher percentage of the ‘5-cap’ and the greater translational activity of the cytoplasmic poly A(+) RNA. During these studies, it was also found that the m7 guanosine of the ‘5′-cap’ was not removed during translation of the mRNA in the wheat germ system; this result suggests that the ‘5′-cap’ may associate with allosteric binding sites of initiation factor(s).