Wobble uridines (U₃₄) are generally modified in all species. U₃₄ modifications can be essential in metazoans but are not required for viability in fungi. In this review, we provide an overview on the types of modifications and how they affect the physico-chemical properties of wobble uridines. We describe the molecular machinery required to introduce these modifications into tRNA posttranscriptionally and discuss how posttranslational regulation may affect the activity of the modifying enzymes. We highlight the activity of anticodon specific RNases that target U₃₄ containing tRNA. Finally, we discuss how defects in wobble uridine modifications lead to phenotypes in different species. Importantly, this review will mainly focus on the cytoplasmic tRNAs of eukaryotes. A recent review has extensively covered their bacterial and mitochondrial counterparts. ¹

Using budding yeast, we investigated a negative interaction network among genes for tRNA modifications previously implicated in anticodon-codon interaction: 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm⁵s²U34: ELP3, URM1), pseudouridine (Ψ38/39: DEG1) and cyclic N6-threonyl-carbamoyl-adenosine (ct⁶A37: TCD1). In line with functional cross talk between these modifications, we find that combined removal of either ct⁶A37 or Ψ38/39 and mcm⁵U34 or s²U34 results in morphologically altered cells with synthetic growth defects. Phenotypic suppression by tRNA overexpression suggests that these defects are caused by malfunction of tRNA^Lys_UUU or tRNA^Gln_UUG, respectively. Indeed, mRNA translation and synthesis of the Gln-rich prion Rnq1 are severely impaired in the absence of Ψ38/39 and mcm⁵U34 or s²U34, and this defect can be rescued by overexpression of tRNA^Gln_UUG. Surprisingly, we find that combined modification defects in the anticodon loops of different tRNAs induce similar cell polarity- and nuclear segregation defects that are accompanied by increased aggregation of cellular proteins. Since conditional expression of an artificial aggregation-prone protein triggered similar cytological aberrancies, protein aggregation is likely responsible for loss of morphogenesis and cytokinesis control in mutants with inappropriate tRNA anticodon loop modifications.

In eukaryotes, wobble uridines in the anticodons of tRNA^Lys_UUU, tRNA^Glu_UUC and tRNA^Gln_UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm⁵s²U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm⁵ side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm⁵s²U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm⁵U and s²U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm⁵s²U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA^Lys_UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm⁵ and s² wobble uridine modification that could account for growth impairment and mainly originates from tRNA^Lys_UUU hypomodification and malfunction.

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. In addition to the positive role of different tRNA modifications in prevention of neurodegeneration, certain cancer types upregulate tRNA modification genes to sustain cancer cell gene expression and metastasis. Multiple associations of defects in genes encoding subunits of the tRNA modifier complex Elongator with human disease highlight the importance of proper anticodon wobble uridine modifications (xm⁵U₃₄) for health. Elongator functionality requires communication with accessory proteins and dynamic phosphorylation, providing regulatory control of its function. Here, we summarized recent insights into molecular functions of the complex and the role of Elongator dependent tRNA modification in human disease.