Transcriptome-wide mapping reveals a diverse dihydrouridine landscape including mRNA

Area of Science:

• Molecular Biology
• RNA Biology
• Biochemistry

Background:

• Dihydrouridine (D) is a crucial modified nucleotide found in transfer RNAs (tRNAs).
• The complete distribution and functional significance of dihydrouridine across the entire transcriptome remain largely uncharacterized in most organisms.

Purpose of the Study:

• To develop a method for transcriptome-wide mapping of dihydrouridine with single-nucleotide resolution.
• To identify novel RNA species containing dihydrouridine and investigate its functional implications in yeast.

Main Methods:

• Development and application of dihydrouridine sequencing (D-seq) for high-resolution mapping.
• Bioinformatic analysis to identify D-modified RNA species, including messenger RNA (mRNA) and small nucleolar RNA (snoRNA).
• Functional assays to assess the impact of D on pre-mRNA splicing and mRNA translation.

Main Results:

• D-seq successfully mapped dihydrouridine sites across the yeast transcriptome.
• Novel classes of dihydrouridine-containing RNAs, including mRNA and snoRNA, were discovered.
• D-modified sites were frequently located in conserved stem-loop regions, suggesting a role in RNA structure.
• Dihydrouridine synthase (DUS)-dependent changes in pre-mRNA splicing were observed.
• D-modified mRNAs were efficiently translated by eukaryotic ribosomes in vitro.

Conclusions:

• Dihydrouridine is a significant component of the mRNA epitranscriptome, extending beyond its known role in tRNAs.
• The findings establish dihydrouridine as a functional modification influencing RNA structure, splicing, and translation.
• This work provides a foundation for exploring the roles of DUS enzymes in human diseases associated with epitranscriptomic dysregulation.