Transcriptome-wide mapping reveals a diverse dihydrouridine landscape including mRNA

Abstract

Dihydrouridine is a modified nucleotide universally present in tRNAs, but the complete dihydrouridine landscape is unknown in any organism. We introduce dihydrouridine sequencing (D-seq) for transcriptome-wide mapping of D with single-nucleotide resolution and use it to uncover novel classes of dihydrouridine-containing RNA in yeast which include mRNA and small nucleolar RNA (snoRNA). The novel D sites are concentrated in conserved stem-loop regions consistent with a role for D in folding many functional RNA structures. We demonstrate dihydrouridine synthase (DUS)-dependent changes in splicing of a D-containing pre-mRNA in cells and show that D-modified mRNAs can be efficiently translated by eukaryotic ribosomes in vitro. This work establishes D as a new functional component of the mRNA epitranscriptome and paves the way for identifying the RNA targets of multiple DUS enzymes that are dysregulated in human disease.

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.