Classical and novel approaches to the detection and localization of the numerous modified nucleotides in eukaryotic...

Human ribosomes contain more than 200 modified nucleotides. These are made up as follows: more than 100 2'-O-methyl groups, 10 methylated bases, about 95 pseudouridines and at least one other modification. Other mammalian sources that have been examined, as well as the lower vertebrate Xenopus laevis, show very similar patterns of nucleotide modifications, especially as revealed by oligonucleotide fingerprinting for methyl groups. Most of the methyl groups have been located along the rRNA primary structure by matching oligonucleotide sequence data to the complete sequences derived from rDNA. Nearly all of the methyls are in conserved core regions. Saccharomyces carlsbergensis ribosomes contain about 55% as many methyls as vertebrate ribosomes. The locations of most of the S carlsbergensis methyls are also known. However, of the numerous other eukaryotes whose rRNA sequences have been determined indirectly from rDNA, few have yielded detailed data on modified nucleotides. This is in part because the methods applied to vertebrate and yeast ribosomes are highly laborious and are not universally applicable. Therefore in the final part of this paper we briefly review other methods that have been applied to the detection and localization of modified nucleotides in rRNA. In particular, we outline progress towards developing a method whereby reverse transcription shows characteristic pausing at most of the 2'-O-methylation sites in human and Xenopus 18S rRNA. 2'-O-Methylation pauses are distinguishable from most other interruptions; the 2'-O-methyl pauses occur more strongly at low than at high dNTP concentration, whereas most other interruptions are independent of dNTP concentration.