Chemical secondary structure probing of two highly methylated regions in Xenopus laevis 28S ribosomal RNA.

The large ribosomal subunit (LSU) RNA or 28S rRNA of vertebrates is characterized by two highly conserved and methylated regions towards the 3' end of the molecule that extend from domains IV to V of the molecule. In this report we describe the probing of the secondary structure of these two highly methylated regions in Xenopus laevis LSU RNA by chemical modification using the single-strand nucleotide specific probes; dimethyl sulphate (DMS) and 1-cyclo-hexyl-3-(2-morpholinoethyl-carbodiimide metho-p-toluene sulphonate (CMCT) followed by primer extension. The same regions in in vitro synthesized unmethylated X. laevis 28S rRNA were also probed for comparison. Our results in general tend to support the theoretically determined secondary structure model for the probed domains. From the results obtained, methylated cellular LSU RNA appears to be relatively more reactive than the in vitro transcript to the chemical probes. Accessibility to the probes was found to be similar at most sites for cellular and in vitro transcript LSU RNAs. This implies that structural destabilization due to 2'-O-methylations in cellular LSU RNA is not significant.