Molecular basis for thiocarboxylation and release of Urm1 by its E1-activating enzyme Uba4.

Ubiquitin-related modifier 1 (Urm1) is a highly conserved member of the ubiquitin-like (UBL) family of proteins. Urm1 is a key component of the eukaryotic transfer RNA (tRNA) thiolation cascade, responsible for introducing sulfur at wobble uridine (U34) in several eukaryotic tRNAs. Urm1 must be thiocarboxylated (Urm1-SH) by its E1 activating enzyme UBL protein activator 4 (Uba4).

E2/E3-independent ubiquitin-like protein conjugation by Urm1 is directly coupled to cysteine persulfidation.

Post-translational modifications by ubiquitin-like proteins (UBLs) are essential for nearly all cellular processes. Ubiquitin-related modifier 1 (Urm1) is a unique UBL, which plays a key role in tRNA anticodon thiolation as a sulfur carrier protein (SCP) and is linked to the noncanonical E1 enzyme Uba4 (ubiquitin-like protein activator 4). While Urm1 has also been observed to conjugate to target proteins like other UBLs, the molecular mechanism of its attachment remains unknown.

Urm1, not quite a ubiquitin-like modifier?

Ubiquitin related modifier 1 (Urm1) is a unique eukaryotic member of the ubiquitin-fold (UbF) protein family and conserved from yeast to humans. Urm1 is dual-functional, acting both as a sulfur carrier for thiolation of tRNA anticodons and as a protein modifier in a lysine-directed Ub-like conjugation also known as urmylation. Although Urm1 conjugation coincides with oxidative stress and targets proteins like 2-Cys peroxiredoxins from yeast (Ahp1) and fly (Prx5), it was unclear how urmylation proceeds molecularly and whether it is affected by the activity of these antioxidant enzymes.

Molecular basis for the bifunctional Uba4-Urm1 sulfur-relay system in tRNA thiolation and ubiquitin-like conjugation.

The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin-related modifier 1 (Urm1) pathway is responsible for the synthesis of 2-thiolated wobble uridine (U ). During the key step of the modification cascade, the E1-like activating enzyme ubiquitin-like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C-terminus of its substrate Urm1.

Redox requirements for ubiquitin-like urmylation of Ahp1, a 2-Cys peroxiredoxin from yeast.

The yeast peroxiredoxin Ahp1, like related anti-oxidant enzymes in other species, undergoes urmylation, a lysine-directed conjugation to ubiquitin-like modifier Urm1. Ahp1 assembles into a homodimer that detoxifies peroxides via forming intersubunit disulfides between peroxidatic and resolving cysteines that are subsequently reduced by the thioredoxin system. Although urmylation coincides with oxidative stress, it is unclear how this modification happens on a molecular level and whether it affects peroxiredoxin activity.

Roles of Elongator Dependent tRNA Modification Pathways in Neurodegeneration and Cancer.

Transfer RNA (tRNA) is subject to a multitude of posttranscriptional modifications which can profoundly impact its functionality as the essential adaptor molecule in messenger RNA (mRNA) translation. Therefore, dynamic regulation of tRNA modification in response to environmental changes can tune the efficiency of gene expression in concert with the emerging epitranscriptomic mRNA regulators. Several of the tRNA modifications are required to prevent human diseases and are particularly important for proper development and generation of neurons.