Base-pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar ribonucleoproteins

In vitro pseudouridylation of 5′ hairpin short substrate variants by the snR34 H/ACA snoRNP. An excess (500 nM) of each [³H-C5] uridine-labeled 5′ substrate RNA was incubated with 50 nM reconstituted snR34 H/ACA snoRNP. The mRNA YRA1 was predicted to be pseudouridylated by the 5′ hairpin of snR34 through the indicated base pairing (Schwartz et al. 2014). Note the difference in the snR34 hairpin base pairing above the pseudouridylation pocket which is needed to accommodate either the wild-type (25S rRNA) substrate or the YRA1 mRNA (top panel). Pseudouridine formation in the wild-type 5′ substrate (black squares) and the YRA1 substrate fragment (gray circles) is depicted in the bottom panel. Mean and standard deviation of three replicates are shown.