Mapping 2'-O-methyl groups in ribosomal RNA.
Ribosomal RNAs (rRNAs) from all sources contain modified nucleosides, whose numbers range from a few in mitochondrial rRNA to more than 200 in the complete rRNAs of some higher eukaryotes. In eukaryotic rRNA the great majority of modified nucleosides are 2'-O-methylated nucleosides or pseudouridines. The locations of most of the 2'-O-methylated nucleosides in rRNA from some representative eukaryotes are known from studies whose aim was full characterization of rRNA methylation. More recently, and particularly in connection with the discovery of methylation guide RNAs, it is often required to check for the presence or absence of 2'-O-methyl nucleosides at specified locations within rRNA. Three methods that can be applied for such "local" objectives are reviewed. Two of the methods are based on primer extension by reverse transcriptase. They exploit, respectively, a tendency of 2'-O-methyl groups to impede reverse transcriptase at low dNTP concentrations, or the resistance of phosphodiester bonds adjacent to 2'-O-methyl groups to alkaline hydrolysis. Examples of these methods are summarized. Although the two methods are relatively straightforward, they suffer from various experimental limitations, as discussed. The third method is technically more sophisticated but is capable of overcoming the limitations of the first two methods. It is based on the resistance of a target 2'-O-methylated site to cleavage by RNase H when the site is hybridized to an appropriate chimeric oligonucleotide. An overview of the approaches and methods now available for the complete mapping of 2'-O-methyl groups in rRNA is presented.
Maden
B E
BE
School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZB, United Kingdom. foulkesb@liv.ac.uk
D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D005821:Genetic Techniques; D006868:Hydrolysis; D011485:Protein Binding; D012335:RNA, Ribosomal / Q000737:chemistry* / Q000648:ultrastructure*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D016914:Ribonuclease H / Q000737:chemistry / Q000494:pharmacology; D017422:Sequence Analysis, DNA
D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H