Differentiation of chick embryo cerebral hemispheres. III. Incorporation of [ 3H-methyl]methionine into 29 S, 18 S and 4 S RNAs

Abstract

(1) The methylation of cytoplasmic 29 S, 18 S and 4 S RNAs in the developing cerebral hemispheres of chick embryos was studied during the period of transformation of morphologically undifferentiated cells into neuroblasts and spongioblasts. [ 3H-methyl]methionine was used as a donor of methyl groups, and RNAs were analyzed by polyacrylamide gel electrophoresis. (2) The changes in the amount and of the specific activity in vivo of the methyl donor, S-adenosylmethionine (SAMe), were also determined. The amount of SAMe decreased 50% between 6 and 14 days. Its specific activity decreased 40%. (3) Ribosomal RNAs were methylated at the level of the precursors in cerebral hemispheres at all ages, as shown by the similarity of the radioactivity profiles of cytoplasmic uridine- and methyl-labelled RNAs after 0.5, 1, 2 and 4 h of incubation. There were 1.4–1.5 times more methyl groups incorporated into 18 S than into 29 S RNA. The incorporation of methyl groups into transfer RNA was rapid at all ages. Thus, in developing cerebral hemispheres, the molecular events leading to RNA methylation were similar to those in mammalian cells. (4) The relative specific activities (specific activity of RNA/specific activity of SAMe) of ribosomal RNAs increased about 70–80% between 6 and 14 days. The ratio of the rate of methylation to the rate of synthesis increased similarly. This could reflect an increase in the number of methyl groups attached to ribosomal RNAs due to the expression of new rRNA methylases during cell differentiation. (5) The relative specific activity of transfer RNA did not change significantly with age, but the ratio of the rate of methylation to the rate of synthesis doubled between 6 and 10 days. This increase could be due to a change of the population of tRNA during the period of cell differentiation, and to the expression of new tRNA methylases. (6) An alternative interpretation of the results of determination of relative specific activities is suggested: the SAMe pool could be compartmentalized and the specific activity of the pool used by the RNA methylases could be different from that of the total pool that we have measured. Therefore, the apparent absence of coupling between rRNA methylation and synthesis remains to be proved.