FIGURE 1.

(A) Secondary structure of the U81 snoRNA. Snorbozyme targeting sites (indicated in blue, RZ1-4), shRNA targeting sites (indicated in red, shU81-1 and shU81-2), and the ASO target site (indicated by a dashed orange line) are shown. (B,C) Analysis of U81 snoRNA expression and methylation activity in HEK-293T cells, transfected with increasing amounts (1–10 μg) of shRNA-expressing plasmids. (B,C) Northern blot analysis: two shRNAs, both targeting U81 snoRNA (shU81-1 and shU81-2, respectively), do not reduce U81 snoRNA expression levels. (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 increasing amounts (1–10 μg) of shRNA-expressing plasmids (see above). 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 text). (F,G) Top: Western blot analysis showing reduction in the expression levels of VNP fluorescent protein; target sites for shU81-1 or shU81-2 were inserted in the VPN gene as described in Materials and Methods. Bottom: FACS analysis: upon transfection of increasing amounts of shRNA expressing plasmids (see above), VNP expression in HEK-293T cells is reduced by about 80% (shU81-1) or 50% (shU81-2), respectively.