(A) A pseudouridylation pocket generated in xtSCARNA4 and SNORA28 to make artificial guide RNAs for Xenopus U2 snRNA modification at position 60 (SCARNA4 > U2-Ψ60 and xtSNORA28 > U2-Ψ60). Mutations that shorten the ASE are indicated in blue; a fragment of U2 snRNA inserted in a U87-based artificial substrate RNA is underlined. (B) Mapping pseudouridines in human and Xenopus U2 snRNA. Normally, human U2 snRNA (HeLa, gray trace) is pseudouridylated at position 60; Xenopus U2 snRNA is not modified at this position (XTC, green trace). Expression of SCARNA4 > U2-Ψ60 (red trace, star) but not xtSNORA28>U2-Ψ60 (dark blue trace, arrowhead) induced U2 snRNA pseudouridylation at position 60 in the Xenopus laevis cell line XTC. (C) Pseudouridylation of the artificial substrate RNA containing a fragment of vertebrate U2 snRNA (U87-U2[51–68]) in yeast cells. Even with a short ASE, xtSNORA28 > U2-Ψ60 is functional on U2 snRNA artificial substrate (blue trace, star).
