Ubiquitin-related modifier Urm1 acts as a sulphur carrier in thiolation of eukaryotic transfer RNA

Abstract

Covalent attachment of ubiquitin-like proteins (UBLs) can modify a protein's function or localization. In bacteria, UBL homologues act as sulphur carriers. Leidel et al. now show that the oldest UBL protein, Urm1p from yeast, is also a sulphur carrier that modifies transfer RNA. Urm1p is first adenylated by, and then thiolated by, Uba4p, after which the sulphur moiety is transferred to the U34 'wobble' position in cytoplasmic adenylated tRNAs. This finding adds support for the theory that ubiquitin-like systems evolved from bacterial sulphur-carrier systems. The identification of a pathway generating thiolated uridine at the tRNA wobble position might also have therapeutic implications, since HIV reverse transcriptase relies on this modification to initiate reverse transcription of the HIV RNA-genome in vitro. This paper shows that the yeast ubiquitin-like protein Urm1p is a sulphur carrier that modifies tRNA. It identifies a pathway whereby Urm1p is first adenylated by Uba4p, then is subsequently thiolated by Uba4p, after which the sulphur moiety is transferred from the thiolated Urm1p onto U34 of a cytoplasmic adenylated tRNA. Ubiquitin-like proteins (UBLs) can change protein function, localization or turnover by covalent attachment to lysine residues1. Although UBLs achieve this conjugation through an intricate enzymatic cascade, their bacterial counterparts MoaD and ThiS function as sulphur carrier proteins2,3. Here we show that Urm1p, the most ancient UBL3, acts as a sulphur carrier in the process of eukaryotic transfer RNA (tRNA) modification, providing a possible evolutionary link between UBL and sulphur transfer. Moreover, we identify Uba4p, Ncs2p, Ncs6p and Yor251cp as components of this conserved pathway. Using in vitro assays, we show that Ncs6p binds to tRNA, whereas Uba4p first adenylates and then directly transfers sulphur onto Urm1p. Finally, functional analysis reveals that the thiolation function of Urm1p is critical to regulate cellular responses to nutrient starvation and oxidative stress conditions, most likely by increasing translation fidelity.