| RRC ID |
86573
|
| Author |
Yamagami R, Takahashi K, Shingu S, Namba M, Kamizaki K, Hori H.
|
| Title |
tRNA pseudouridine synthase D (TruD) from Thermus thermophilus modifies U13 in tRNAAsp, tRNAGlu, and tRNAGln and U35 in tRNATyr.
|
| Journal |
RNA
|
| Abstract |
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 tRNAAsp in Thermus thermophilus, 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 truD gene disruption mutant. Our data reveal that TruD recognizes tRNAAsp, tRNAGlu, and tRNAGln as substrate tRNAs. In addition, we discover that TruD modifies U35 in tRNATyr, which has previously been reported as a substrate of RluF in Escherichia coli 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 UNUAR sequence in tRNA substrates (N = any nucleotide, R = purine, U = target site). Finally, we identify over 600 mRNA fragments containing this recognition sequence in T. thermophilus 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.
|
| Volume |
31(6)
|
| Pages |
850-867
|
| Published |
2025-5-16
|
| DOI |
10.1261/rna.080405.125
|
| PII |
rna.080405.125
|
| PMID |
40138658
|
| PMC |
PMC12084886
|
| MeSH |
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Hydro-Lyases* / genetics
Hydro-Lyases* / metabolism
Intramolecular Transferases* / genetics
Intramolecular Transferases* / metabolism
Pseudouridine / metabolism
RNA, Transfer / genetics
RNA, Transfer / metabolism
RNA, Transfer, Asp* / chemistry
RNA, Transfer, Asp* / genetics
RNA, Transfer, Asp* / metabolism
RNA, Transfer, Gln* / chemistry
RNA, Transfer, Gln* / genetics
RNA, Transfer, Gln* / metabolism
RNA, Transfer, Glu* / chemistry
RNA, Transfer, Glu* / genetics
RNA, Transfer, Glu* / metabolism
RNA, Transfer, Tyr* / chemistry
RNA, Transfer, Tyr* / genetics
RNA, Transfer, Tyr* / metabolism
Substrate Specificity
Thermus thermophilus* / enzymology
Thermus thermophilus* / genetics
|
| IF |
4.32
|
| Resource |
| DNA material |
Thermus thermophilus HB8 gene disruption plasmid TDs08B04 (THR023228)
Thermus thermophilus expression plasmid TEx09H06 (THR003774) |
