FIGURE 2.

“Snorbozyme” mediated knock down of U81 snoRNA. (A) Fluorescent in situ hybridization of U16-RZ2 or U20-RZ1 snorbozymes transfected into HEK-293T cells: snorbozyme expression was detected by employing a Cy3-labeled (red) antisense oligonucleotide. U3 snoRNA was used as a nucleolar marker and was visualized by employing an Oregon Green 488-labeled antisense oligonucleotide; nuclei of HEK-293T cells were stained with DAPI (blue). (B,C) Northern blot analysis of U81 snoRNA and snorbozyme expression levels: upon transfection of increasing amounts (1–10 μg) of U16-RZ2 and U20-RZ1 snorbozyme expressing plasmids, U81 expression levels were reduced by 60% and 30%, respectively. (D,E) Primer extension analysis of nucleotide A₃₉₁ within 28S rRNA investigating the 2′-O-methylation activity of U81 snoRNA within HEK-293T cells, transfected with U16-RZ2 or U20-RZ1 snorbozyme constructs. Methylation of A₃₉₁ is indicated by a black arrow; a second 2′-O-methylation at position A₃₈₉, which is guided by U26 snoRNA, is indicated by a red arrow and serves as an internal loading control. Ø: reverse transcription control reaction at 0.5 mM dNTP lacking a stop signal at A₃₉₁ and A₃₈₉, respectively (see the text).