Abstract
In tRNA, dihydrouridine is a conserved modified base generated by the post-transcriptional reduction of uridine. Formation of dihydrouridine 20, located in the D-loop, is catalyzed by dihydrouridine synthase 2 (Dus2). Human Dus2 (HsDus2) expression is upregulated in lung cancers, offering a growth advantage throughout its ability to interact with components of the translation apparatus and inhibit apoptosis. Here, we report the crystal structure of the individual domains of HsDus2 and their functional characterization. HsDus2 is organized into three major modules. The N-terminal catalytic domain contains the flavin cofactor involved in the reduction of uridine. The second module is the conserved α-helical domain known as the tRNA binding domain in HsDus2 homologues. It is connected via a flexible linker to an unusual extended version of a dsRNA binding domain (dsRBD). Enzymatic assays and yeast complementation showed that the catalytic domain binds selectively NADPH but cannot reduce uridine in the absence of the dsRBD. While in Dus enzymes from bacteria, plants and fungi, tRNA binding is essentially achieved by the α-helical domain, we showed that in HsDus2 this function is carried out by the dsRBD. This is the first reported case of a tRNA-modifying enzyme carrying a dsRBD used to bind tRNAs.
Highlights
TRNA maturation requires extensive processing and a large number of chemical modifications [1]. 5,6-dihydrouridine (D) is one of the most abundant modified bases in tRNAs
The UV-visible absorption spectrum of HsDusdusD reporting the presence of flavin was not significantly different from that of full length wild type HsDus2, which indicates that the truncation has not altered the binding of the flavin cofactor (Supplementary Figure S3)
Using biochemical analysis and structural characterization, we here show that HsDus2 uses a unique strategy for binding and modifying its tRNA substrate
Summary
TRNA maturation requires extensive processing and a large number of chemical modifications [1]. 5,6-dihydrouridine (D) is one of the most abundant modified bases in tRNAs. D results from the reduction of the 5,6-uridine double bond, which leads to a non-planar base moiety and the nucleoside does not participate in base stacking [3,4,5,6]. It instead promotes tertiary interactions at the elbow region of tRNAs. A role of D in conformational flexibility is consistent with its high level in tRNAs from psychrophilic bacteria, in which it provides an obvious benefit under conditions where thermal motion, enzymatic reaction kinetics and intermolecular interactions are compromised [7]. Deficiency of D, as well as of other modified bases, results in enhanced tRNA degradation, at rates approaching those seen for mRNA degradation [8]
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