Studies on ribosomal RNA and ribosomal precursor RNA in mammalian cells

HeLa cell rRNA and its nucleolar precursors were examined by techniques for RNA fingerprinting and sequence analysis, using RNA which had been labelled in vivo with 14C methyl methionine or 32P phosphate. The following observations were made:- 1. Fingerprints obtained by digesting methyl labelled 18S RNA with T1 ribonuclease or with a combination of T1 ribonuclease plus alkaline phosphatase showed about 40 spots. 2. Fingerprints obtained by digesting 28S methyl labelled RNA in the same way showed about 54 spots. 3. The fingerprints of 28S RNA were widely different from those of 18S RNA, there being only 9 spots which are common to both RNA species. 4. 32S fingerprints were almost identical to 28S fingerprints in all respects. 5. The fingerprints of 45S RNA contain most of the 18S spots as well as all the 28S characteristic spots. 6. A close examination of 45S RNA showed that there are two 18S spots which are not present in 45S RNA and one 18S spot which is only weakly labelled in 45S RNA. These 18S products were found to contain methylated bases. They represent secondary methylation of the RNA. All of the other methylated products characterized in this study contain 2'-0'methyl ribose groups. This suggested that most if not all of the early methylation of the precursor molecule is 2'-O-methylation while the secondary methylation which occurs late in the maturation process is base methylation, 7. A wide variety of sequences have been found to be methylated. Most of these sequences are present only once in the RNA molecule. 8. The methylation patterns of rRNA from three different mammalian species (man, HeLa; C13, hamster and L929 cells, mouse) were compared and found to show remarkable similarities