Loss of Anticodon Wobble Uridine Modifications Affects tRNALys Function and Protein Levels in Saccharomyces cerevisiae

Abstract

In eukaryotes, wobble uridines in the anticodons of tRNALys UUU, tRNAGlu UUC and tRNAGln 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 tRNALys 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 tRNALys UUU hypomodification and malfunction.

tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast

“…To facilitate deletion of DEG1 in urm1Δ::KanMX4 or elp3Δ::KanMX4 strains, these were first transformed with pFF8 ( ELP3 ; (19)) or pHA-URM1 ( URM1 ; (39)). Subsequently, DEG1 was deleted by using a PCR generated deletion cassette ( deg1Δ::SpHIS5 ) and URA3 plasmids pFF8 or pHA-URM1 eliminated by growth on minimal media containing uracil and 5-fluoro-orotate (1 ml/ml).…”

“…Subsequently, DEG1 was deleted by using a PCR generated deletion cassette ( deg1Δ::SpHIS5 ) and URA3 plasmids pFF8 or pHA-URM1 eliminated by growth on minimal media containing uracil and 5-fluoro-orotate (1 ml/ml). Overexpression of tRNA species involved YEplac195( URA3 )-, YEplac181( LEU2 )-, or pRS423( HIS3 )-based plasmids YEpQ (tRNA Gln UUG, pQ), pSZ16 (tRNA Glu UUC, pE), pDJ82 (tRNA Lys UUU, pK) and pQKE (tRNA Gln UUG, tRNA Glu UUC and tRNA Lys UUU) (19,40,41). The insert from YEplac195-based YEpQ containing the tRNA Gln UUG gene was subcloned into pRS425 using SalI and EcoRI, generating pRK55.…”

“…Simultaneous loss of both U34 modification pathways in yeast results in growth delay, reduced protein levels, increased protein aggregation due to translational slow down or even inviability depending on strain backgrounds (18–22). While phenotypically milder, the removal of either U34 modification pathway (Elongator or Urm1) alone already causes a wide range of detectable phenotypes, including drug and various stress sensitivities and cell cycle delay (19–21). Also, nutrient sensitive regulatory circuits such as target of rapamycin (TOR) or general amino acid control (GAAC) signaling pathways become deregulated in response to mcm 5 /s 2 U modification defects (22–24).…”

Combined tRNA modification defects impair protein homeostasis and synthesis of the yeast prion protein Rnq1

“…[5][6][7][8] Absence of either alone removes the mcm 5 -(Elongator) or the s 2 U part (Urm1) and induces overlapping defects including increased ribosomal frameshifting during mRNA translation, while elimination of both modification pathways severely aggravates these defects or may even cause synthetic lethality. 1,[9][10][11][12][13] Further, complete absence of mcm 5 s 2 U was shown to cause a substantial ribosomal slow down at the A-ending codons for Lys and Gln.…”

The Many Faces of Elongator in Neurodevelopment and Disease

“…tRNA are more than simple adaptor molecules and have a surprising range of functions in the cell, such as tuning translation and protein expression in a tissue-specific manner and stress signaling, whereby these functions are all dependent on its modifications (Giegé, 2008; Thompson and Parker, 2009; Kirchner and Ignatova, 2015). Klassen et al (2015) recently showed that the loss of wobble uridine modifications in the Elongator deficient yeast strain affects tRNA Lys UUU function and results in a reduced total protein level. Moreover, the loss of these modifications in a subset of tRNAs was shown to lead to ribosome pausing at their cognate codons in both, yeast and C. elegans (Nedialkova and Leidel, 2015).…”