Abstract
Dihydrouridine is a highly abundant modified nucleoside found widely in tRNAs of eubacteria, eukaryotes, and some archaea. In cytoplasmic tRNA of Saccharomyces cerevisiae, dihydrouridine occurs exclusively at positions 16, 17, 20, 20A, 20B, and 47. Here we show that the known dihydrouridine synthases Dus1p and Dus2p and two previously uncharacterized homologs, Dus3p (encoded by YLR401c) and Dus4p (YLR405w), are required for all of the dihydrouridine modification of cytoplasmic tRNAs in S. cerevisiae. We have mapped the in vivo position specificity of the four Dus proteins, by three complementary approaches: determination of the molar ratio of dihydrouridine in purified tRNAs from different dus mutants; microarray analysis of a large number of tRNAs based on differential hybridization of uridine and dihydrouridine-containing tRNAs to the complementary oligonucleotides; and the development and use of a novel dihydrouridine mapping technique, employing primer extension. We show that each of the four Dus proteins has a distinct position specificity: Dus1p for U(16) and U(17), Dus2p for U(20), Dus3p for U(47), and Dus4p for U(20a) and U(20b).
Highlights
A ubiquitous feature of tRNAs is the presence of numerous base and ribose modifications [1]
We demonstrate that formation of dihydrouridine in yeast cytoplasmic tRNAs is carried out by a family of four dihydrouridine synthases (Dus1p, Dus2p, Dus3p, and Dus4p), each acting at specific positions in tRNAs
Dus1p modifies U16 and U17, Dus2p modifies U20, Dus3p modifies U47, and Dus4p modifies U20a and U20b. These four proteins are responsible for all dihydrouridine modification of cytoplasmic tRNAs in yeast, since bulk cellular RNA from a dus1-⌬, dus2-⌬, dus3-⌬, dus4-⌬ strain (Fig. 1A) lacks any detectable dihydrouridine and since the specificities of the four proteins account for all known modifications of sequenced yeast cytoplasmic tRNAs
Summary
MATa leu2-⌬0, met15-⌬0, ura3-⌬0 MATa his3-⌬1, leu2-⌬0, met15-⌬0, ura3-⌬0 BY4730 dus1-⌬::kanMX4 BY4741 dus2-⌬::kanMX4 BY4741 dus3-⌬::kanMX4 BY4741 dus4-⌬::kanMX4 BY4730 dus1-⌬::kanMX4, dus2-⌬::LEU2 BY4730 dus1-⌬::kanMX4, dus3-⌬::URA3 BY4730 dus1-⌬::kanMX4, dus4-⌬0 BY4741 dus2-⌬::LEU2, dus3-⌬::kanMX4 BY4741 dus2-⌬::LEU2, dus4-⌬::kanMX4 BY4741 dus3-⌬::kanMX4, dus4-⌬::URA3 BY4730 dus1-⌬::kanMX4, dus2-⌬::LEU2, dus3-⌬0 BY4730 dus1-⌬::kanMX4, dus2-⌬::LEU2, dus4-⌬0 BY4730 dus1-⌬::kanMX4, dus3-⌬::URA3, dus4-⌬0 BY4741 dus2-⌬::LEU2, dus3-⌬::kanMX4, dus4-⌬0 BY4730 dus1-⌬::kanMX4, dus2-⌬::LEU2, dus3-⌬0, dus4-⌬0. This study This study This study This study This study This study This study This study This study This study This study developed a preliminary map to describe the specificity of the Dus proteins. Microarray analysis of individual dus deletion strains provided an independent map of the specificities that showed good agreement with the one generated from calculating total dihydrouridine content. The specificity model was confirmed and extended by the development and use of a new dihydrouridine mapping method suitable for analysis of individual tRNAs in bulk RNA. Our results indicate that each the four yeast Dus proteins is specific for dihydrouridine modification at one or two distinct positions in cytoplasmic tRNAs
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