Fig. 2.

ASO-mediated differential methylation of 18S rRNA A484. (A) Schematic representation of the interaction of regRNP17 with snord16. The guide RNA for the methylation of A484 within 18S rRNA is snord16. Extra base pairings of snord16 with 18S rRNA, described by van Nues et al. (2011), are indicated by orange nucleotides. These extra base pairings of snord16 with 18S rRNA form a loop in snord16, and this snord16 loop contains the regRNP17 interacting region (blue nucleotides). (B) Primer extension assay to detect methylated sites within RNA. A digoxigenin labeled primer designed to interrogate 18S rRNA A484 and A468 was used in reactions with reverse transcriptase and RNA. Decreasing dNTP concentrations (indicated) were used for each reaction. The location of the primer and methylation stops for A484 and A468 are indicated. (C) ASO-mediated alteration of A484 methylation. Cells were treated for 48 h with control, snord16 or scaRNA17 ASOs. Isolated RNA was subjected to primer extension with low (2.5 uM) levels of dNTP. The location of the A484 and A468 methylation sites are shown. Quantification of this and other data are shown in D. For this quantification the signal for A484 was divided by the A468 signal and this ratio was normalized to that obtained with control ASO. Treatment with snord16 ASO results in an 82% decrease in the methylation of A484 (n=3) and treatment with scaRNA17 ASO results in a 1.6-fold increase in the methylation of A484 (n=3), compared to control. Asterisks indicate P<0.05.