FIGURE 4.

SNORA22 is the EBER2-specific H/ACA snoRNA that guides DKC1. (A) DKC1 binds directly to EBER2 in vivo. CLIP-seq for DKC1 was carried out in EBV-positive BJAB-B1 cells to examine direct binding of EBER2 as a substrate. A footprint region at the 3′ end of EBER2 (nucleotides 128–168), which overlaps with the pseudouridylation site at position U160, was detected. A crosslink-induced mutation site (CIMS), which is indicative of a bona fide direct RNA–protein interaction, was also observed within the footprint region. (B) A predicted RNA duplex between EBER2 and SNORA22 is shown surrounding the pseudouridylation site at U160. SNORA22 nucleotides engaging in RNA–RNA interaction are highlighted, and the conserved Box H is indicated. Please see Supplemental Figure 1A–D for further information. (C) Knockdown of SNORA22 decreases the abundance of EBER2 Ψ160. Left panel shows the experimental outline for depleting EBER2 and SNORA22 prior to CMCT treatment and primer extension assay. Knockdown of SNORA22 with two distinct ASOs results in lowered levels of premature RT stops.