tRNA pseudouridine synthase D (TruD) from<i>Thermus thermophilus</i>modifies U13 in tRNA^Asp, tRNA^Glu, and tRNA^Glnand U35 in tRNA^Tyr

Pseudouridine is a modified nucleoside found in various RNA species, including tRNA, rRNA, mRNA, and other noncoding RNAs. Pseudouridine is synthesized from uridine by pseudouridine synthases. While the landscape of pseudouridines in RNA has been extensively studied, much less is known about substrate RNA recognition mechanisms of pseudouridine synthases. Herein, we investigate the tRNA pseudouridine synthase D (TruD), which catalyzes the formation of pseudouridine at position 13 in tRNA^Asp in <i>Thermus thermophilus</i>, a thermophilic eubacterium. To identify the tRNA substrates of TruD, we compared results of next-generation sequencing experiments combined with bisulfite probing of pseudouridine in tRNAs from both wild-type and a <i>truD</i> gene disruption mutant. Our data reveal that TruD recognizes tRNA^Asp, tRNA^Glu, and tRNA^Gln as substrate tRNAs. In addition, we discover that TruD modifies U35 in tRNA^Tyr, which has previously been reported as a substrate of RluF in <i>Escherichia coli</i> These findings were validated through in vitro assays with recombinant TruD, which further demonstrated that TruD can act on other RNAs, including a CDC8 mRNA fragment, a known substrate of Pus7, the eukaryotic counterpart of TruD. Systematic mutational analysis of CDC8 transcripts reveals that TruD preferentially pseudouridylates the UN<u>U</u>AR sequence in tRNA substrates (N = any nucleotide, R = purine, <u>U</u> = target site). Finally, we identify over 600 mRNA fragments containing this recognition sequence in <i>T. thermophilus</i> ORFs and demonstrate the ability of TruD to act on these potential mRNA substrates. Our findings suggest the possibility that many other RNAs are modified by TruD in vivo.