Role of Pseudouridine Formation by Deg1 for Functionality of Two Glutamine Isoacceptor tRNAs.

Raffael Schaffrath,Roland Klassen

doi:10.3390/biom7010008

Abstract

Loss of Deg1/Pus3 and concomitant elimination of pseudouridine in tRNA at positions 38 and 39 (ψ38/39) was shown to specifically impair the function of tRNAGlnUUG under conditions of temperature-induced down-regulation of wobble uridine thiolation in budding yeast and is linked to intellectual disability in humans. To further characterize the differential importance of the frequent ψ38/39 modification for tRNAs in yeast, we analyzed the in vivo function of non-sense suppressor tRNAs SUP4 and sup70-65 in the absence of the modifier. In the tRNATyrGψA variant SUP4, UAA read-through is enabled due to an anticodon mutation (UψA), whereas sup70-65 is a mutant form of tRNAGlnCUG (SUP70) that mediates UAG decoding due to a mutation of the anticodon-loop closing base pair (G31:C39 to A31:C39). While SUP4 function is unaltered in deg1/pus3 mutants, the ability of sup70-65 to mediate non-sense suppression and to complement a genomic deletion of the essential SUP70 gene is severely compromised. These results and the differential suppression of growth defects in deg1 mutants by multi-copy SUP70 or tQ(UUG) are consistent with the interpretation that ψ38 is most important for tRNAGlnUUG function under heat stress but becomes crucial for tRNAGlnCUG as well when the anticodon loop is destabilized by the sup70-65 mutation. Thus, ψ38/39 may protect the anticodon loop configuration from disturbances by loss of other modifications or base changes.

Highlights

In addition to standard nucleosides, tRNA is known to contain a large variety of modified nucleosides, formed by the addition of chemical groups or isomerization
The thermosensitive growth phenotype of deg1 mutants was shown to be suppressible by overexpression of tRNAGln UUG but not tRNAGln CUG or any other tRNA carrying U38 or U39, which is modified by Deg1 to ψ [8]
These results suggest that under heat stress conditions, growth defect of yeast cells lacking ψ38 results almost exclusively from malfunction of tRNAGln UUG rather than tRNAGln CUG, despite containing almost identical anticodon stem loop (ASL)

Summary

Introduction

In addition to standard nucleosides, tRNA is known to contain a large variety of modified nucleosides, formed by the addition of chemical groups or isomerization. Pseudouridine (ψ) is an isomer of uridine (5-ribosyl-uracil) and represents the initially discovered and most abundant modified nucleoside present in all three domains of life [1,2]. The model organism Saccharomyces cerevisiae has ten characterized pseudouridine synthases (Pus and Cbf5) that modify cytoplasmic and mitochondrial tRNAs and/or other types of RNA, including rRNA, small nuclear RNA (snRNA) and mRNA [2]. Despite its ubiquitous presence in tRNA, ψ appears to affect tRNA function rather subtly, since it is not generally required for cell viability, as demonstrated by the non-essential nature of all of the PUS genes in the yeast system [3]. The absence of PUS3/DEG1 in yeast uniquely causes slow growth, in particular at elevated temperature [4]. The general effects of ψ on tRNA function are likely mediated by increasing the rigidity of the sugar phosphate backbone and base stacking [2,5]

Methods

Results

Conclusion