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

Abstract

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 tRNALysUUU or tRNAGlnUUG, 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 tRNAGlnUUG. 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.

Protein folding and tRNA biology

“…BElongator^is also related to other cellular processes, such as elongation by RNA polymerase II, histone acetylation, telomeric gene silencing, and DNA repair (Chen et al 2011;Karlsborn et al 2014). Noteworthy in yeast, the phenotype produced by a defective U34 modification (morphological and growth alterations) was reverted by the overexpression of the involved tRNA (Klassen et al 2016b). These observations strongly suggest that the defective U34 modification affected the thermodynamics of the interactions at the ribosome.…”

The RNA modification landscape in human disease

“…Although most RNA modifications do not appear to be essential for viability in fungi-but may play important roles in fitness-they have been shown to be critical for maintaining protein homeostasis (Nedialkova and Leidel 2015;Klassen et al 2016), proper cellular signaling (Zinshteyn and Gilbert 2013), and translation fidelity (Patil et al 2012;Agris et al 2017).…”

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.…”

“…18 The Rnq1 protein exhibits an extremely high Gln content (19% Gln) and since the majority of these residues (69%) are encoded by the CAA codon, Rnq1 synthesis was expected to depend strongly on the presence of mcm -GFP protein biosynthesis, a severe defect was observed when both modifications were absent. 1 Thus, the Q-rich prion domain of Rnq1 is inefficiently synthesized in the analyzed tRNA modification double mutants. Whether or not such reduced synthesis affects the prion behavior of Rnq1 is currently under investigation.…”

“…Indeed, when consecutive replicative cycles of phenotypically normal virgin mutant cells were followed by micro-dissection, most mutant cells carried out this phenotypic transition after a number of normal cell divisions. 1 Hence, aged tRNA modification mutant cells are much more likely to accumulate cytological abnormalities compared to virgin or young mother cells. Interestingly, similar cytological phenotypes occur in mutants with inappropriate anticodon loop modification of tRNA Lys UUU or tRNA Gln UUG , suggesting that the defect originates from tRNA malfunction in general, rather than specific malfunction of tRNA Gln UUG .…”