FIGURE 1.

Sequence and structure of TRM4 reporter gene and snR81-derived box H/ACA gRNA. (A) TRM4 reporter gene. The carboxy-terminally TAP tagged Trm4 reporter gene is diagrammed (top). Three sites (codons 561, 602 and 646) that were targeted for nonsense mutation (mutated to PTC) are indicated. The sequences surrounding the PTC sites are also shown (bottom). The DNA sequences are depicted in lower case letters. The target codons (that were changed to PTC) are in bold letters. The amino acid sequences are shown below each DNA sequence. (B) Sequence and structure of a box H/ACA RNA. Shown is the designer box H/ACA gRNA derived from snR81 (a naturally occurring yeast box H/ACA RNA). In the designer gRNA, the 5′ pseudouridylation pocket was designed to target the uridine of PTC646 (UAA/UGA), and the 3′ pocket was targeting the uridine of PTC561 (UAG). The two hairpins (5′ and 3′ hairpins) as well as box H and box ACA are indicated. Base-pairing interactions between the guide sequences and the substrate sequences are also depicted. The two arrows indicate the target uridines. The 5′ and 3′ pockets work independently, and they direct U-to-Ψ conversion with essentially the same efficiency.