FIGURE 5.

U84 and HBII295 box C/D orphan snoRNA guide site-specific ribose methylation of artificial RNA in human cells. (A) Schematic structure of the ph-pol1 expression construct used for transfection of human cells. The RNA polymerase I promoter and terminator, and a lacZ gene portion with a multiple cloning site (MCS) are indicated. The synthetic DNA fragment was inserted into the MCS. The artificial transcript includes three potential methylation sites (shown by white letters) targeted by HBII295, U76, and U24. The potential base pairing between the guide sequence of HBII295 and the artificial transcript is shown. The HBII295 boxD sequence is underlined. (B) An RPA confirmed the specific degradation of snoRNAs with ASOs for GFP control, HBII295, and U84 (GFP, ΔHBII295, and ΔU84, respectively, shown above the lanes). (C) Primer extension mapping of 2′-O-methylated nucleotides. A 5′-end-labeled oligonucleotide primer was annealed with RNA extracted from GFP, ΔHBII295, and ΔU84 cells (shown above the lanes), and extended with AMV reverse transcriptase in the presence of 1, 0.04, or 0.004 mM dNTPs (indicated as black triangles above the lanes). Lanes C, T, A, and G are dideoxy sequencing reactions performed on the ph-pol1 expression construct.