Minor form of human 5.8s ribosomal RNA

Part One: The Minor Form of Human 5.8S rRNA An elongated form of 5.8S rRNA has been found in a wide range of eukaryotes from yeast to rodents. This minor form of 5.8S rRNA is about six nucleotides longer than the major form and composes from 10% to 30% of the total 5.8S rRNA found in yeast and rodents respectively. The minor form of 5.8S rRNA (pCCGAUA-) found in mice and rats may be generated by the formation of a secondary cleavage site caused by heterogeneity in the rRNA genes. The insertion of an adenylic acid residue in the precursor rRNA generates this additional cleavage site, i.e. -ACGA- or -ACCGA for the major and minor forms respectively. There is also heterogeneity with respect to the degree of methylation in rodent 5.8s rRNA. The conformation of the two chain length isomers is influenced by 2'-O-methylation of the uridylic acid residue at position 14, i.e. the most compact conformation is not ribose methylated in that position. The molecules which are methylated in the 14th position cannot adopt the most compact conformation. In the present study I have discovered a minor form of 5.8S rRNA in human placenta and I have determined its sequence; it differs from the major form of human 5.8S rRNA in having an additional sequence (CUCGUA) on the 5'-terminus. The sequence of the major rodent 5.8S rRNA is completely conserved in the major human 5.8S rRNA but the elongation on the 5'-ends of the minor 5.8S rRNAs from the two species are only 50% conserved. Human minor 5.8S rRNA was completely methylated at the uridylic acid residue at 14 making it the first 5.8S rRNA found to be completely methylated.

Part Two: Purification and Characterization of a Ribose Transmethylase from Ehrlich Ascites Cells A filter binding assay was developed for measuring ribose transmethylase activity in cell extracts and was used to quantify ribose and base transmethylase in Ehrlich ascites cells and normal mouse liver. Ribose and base transmethylase activities were elevated two-fold in Ehrlich ascites cells compared to normal mouse liver when methyl-deficient mouse tRNA was used as substrate but base transmethylase activity was elevated tenfold in Ehrlich ascites cells when E. coli tRNA was used as substrate. E. coli tRNA did not serve as a methyl acceptor for ribose transmethylases.

The ribose transmethylase was purified 910-fold from Ehrlich ascites cell extracts and complete elimination of base transmethylase was achieved in one experiment. This purified ribose transmethylase was found to have an apparent Km_tRNA of 20uM tRNA and an apparent Km_SAM of 12.8uM SAM. The apparent molecular weight of the ribose transmethylase, as determined by gel filtration chromatography, was 240, 000 daltons. SDS-PAGE of the purified ribose transmethylase showed a predominant protein band of approximately 60,000 daltons.