Fig 1.
Dihydrouridine-specific chemistry to map dihydrouridine sites in RNA with single-nucleotide resolution.
(A) Bulk nucleoside analysis of detects D in mRNA from WT but not DUS KO yeast. mRNA was purified by selecting for poly(A)+ and tRNAs were removed by size selection. (B) Structures of uridine, dihydrouridine, and tetrahydrouridine. (C) Primer extension analysis of synthetic 4D and 4U RNAs treated with NaBH4 and reverse transcribed with Super Script III RT. D-dependent RT stop positions are highlighted. (D) Schematic of D-seq library preparation. The data underlying this figure can be found in S3 Table. D, dihydrouridine; D-seq, dihydrouridine sequencing; DUS, dihydrouridine synthase; RT, reverse transcriptase; WT, wild-type.
Fig 2.
D-seq identifies known and novel dihydrouridine sites in structured ncRNAs.
(A) Plots of cDNA end positions in Dus2 target tRNA ProAGG and Dus2, Dus4 target tRNA ArgCCG. D Peaks are highlighted. X scale in RPM and Y scale in bp. (B) Summary of known tRNA D positions and corresponding DUS. (C) Plots of cDNA end positions in snR5, snR13, and snR46 snoRNAs. D peaks are highlighted. TSS (transcription start site) of snR5. X scale in RPM and Y scale in bp. (D) snoRNA Ds occur primarily in stem-loop structures that resemble tRNA D loops. Plot of median DMS-induced mutation rate in 25 nt window flanking D site. Red trace is median DMS reactivity flanking D positions. Black dots are median DMS reactivity for randomly selected set of background positions. Blue trace is p-value for difference in DMS reactivity for sequences flanking D or background sites. (E) D sites occur in stem-loop structures of 16 H/ACA and 7 C/D box snoRNAs. The data underlying this figure can be found in S1 and S2 Tables. DMS, dimethyl sulfate; D-seq, dihydrouridine sequencing; ncRNA, non-coding RNA; snoRNA, small nucleolar RNA; TSS, transcription start site.
Fig 3.
D-seq identifies dihydrouridine sites in mRNAs.
(A) Plots of cDNA end positions in ALD6 and SEC63 mRNAs. D peaks are highlighted. Scale in RPM and bp. (B) Distribution of D sites among mRNA features, and background distribution of features for all sites interrogated for D. (C) SDS-PAGE gels showing Top7 protein produced from U and D containing mRNAs with 4 different test codons. Denaturing glyoxal agarose gel showing mRNA integrity. All 4 test constructs showed no significant difference in protein produced per mRNA +/‒ D. Schematic of U/D mRNAs with U/D positions highlighted in red. (D) Plots of cDNA end positions for intronic D in RPL30 mRNA. D peak is highlighted. Scale in RPM and bp. (E) DUS KO strain has increased ratio of RPL30 intron mapping reads to exon mapping reads (p < 0.05, Student’s t test). Model of regulation of RPL30 pre-mRNA splicing by RPL30 protein. (F) mRNA sequences flanking Ds have higher DMS reactivity indicating greater flexibility. Plot of median DMS-induced mutation rate in 25 nt window flanking D site. Red trace is median DMS reactivity surrounding D positions. Black dots are median DMS reactivity for randomly selected set of background positions. Blue is p-value for difference in DMS reactivity for sequences flanking D or background sites. (G) D has multiple impacts on RNA structure. D both promotes loop formation and antagonizes duplex formation. The data underlying this figure can be found in S3 Table. D-seq, dihydrouridine sequencing; DMS, dimethyl sulfate; DUS, dihydrouridine synthase.