Widespread temperature sensitivity and tRNA decay due to mutations in a yeast tRNA

Abstract

Microorganisms have universally adapted their RNAs and proteins to survive at a broad range of temperatures and growth conditions. However, for RNAs, there is little quantitative understanding of the effects of mutations on function at high temperatures. To understand how variant tRNA function is affected by temperature change, we used the tRNA nonsense suppressor of the yeast to perform a high-throughput quantitative screen of tRNA function at two different growth temperatures. This screen yielded comparative values for 9243 single and double variants. Surprisingly, despite the ability of to grow at temperatures as low as 15°C and as high as 39°C, the vast majority of variants that could be scored lost half or more of their function when evaluated at 37°C relative to 28°C. Moreover, temperature sensitivity of a tRNA variant was highly associated with its susceptibility to the rapid tRNA decay (RTD) pathway, implying that RTD is responsible for most of the loss of function of variants at higher temperature. Furthermore, RTD may also operate in aΔ strain, which was previously thought to fully inhibit RTD. Consistent with RTD acting to degrade destabilized tRNAs, the stability of a tRNA molecule can be used to predict temperature sensitivity with high confidence. These findings offer a new perspective on the stability of tRNA molecules and their quality control at high temperature.

Comprehensive sequence-to-function mapping of cofactor-dependent RNA catalysis in the glmS ribozyme

“…Comprehensive functional maps that assess the impact of all possible point and pairwise mutations, for example, can improve our understanding of sequence-structure-function relationships and guide efforts to engineer novel biocatalysts. In addition, comparisons of such maps with other systematic datasets, such those reporting natural sequence conservation 9 , or selection fitness in vitro [32][33][34][35] or in vivo 18,19 , can suggest mechanisms that underlie the higher-order effects of sequence variations. In practice, the sheer number of measurements required to score the kinetics of all single and double mutants surpasses the scale accessible to traditional biochemistry.…”

“…The advent of highly parallelized methods for systematically characterizing RNA function has provided a path forward [18][19][20] . These methods, which rely on high-throughput sequencing techniques, allow characterization of ribozyme function across a diverse sequence space, in some cases including the majority of possible single and double mutations.…”

A new pathway of pre-tRNA surveillance in yeast

“…In this model, the substantial overall RTD of anticodon stem SUP4oc variants is due to a combination of pre-tRNA RTD, triggered by their altered intron-exon structure and presumed inefficient splicing, coupled with the distinct, but relatively inefficient, 5'-3' decay of anticodon stem variants after splicing, resulting in similar overall RTD to that of acceptor stem variants. Subsequent to maturation, tRNAs with destabilizing mutations or lacking specific body modifications are targeted by the RTD pathway (19,20,49,54), particularly at elevated temperatures, which leads to increased instability and attack (48,55). RTD is in competition with cellular proteins that bind charged tRNA, including EF-1A (49, 88) and tRNA synthetases such as ValRS for RTD of tV(AAC) (88).…”

“…With Xrn1, we found that the U2C acceptor stem variant was highly susceptible to decay at 37°C, whereas the A29U and A31U anticodon stem variants were nearly as resistant as the negative control SUP4oc (Figure 2A and B). This assay was done at 37°C, as many RTD substrates are more sensitive to decay in vivo at high temperature (20,55). At 30°C, which is expected to be more stringent for tRNA decay, we still observed substantial sensitivity of the U2C variant to Xrn1, whereas the anticodon stem variants were both highly resistant ( Supplementary Figure 1).…”

“…Although the RTD pathway targets substrates due to 5' end exposure (48), it was surprising to find, from high throughput analysis of variants of the suppressor SUP4oc (tRNA Tyr(GUA) with a UUA anticodon), that the RTD pathway could also target variants with destabilizing mutations in the anticodon stem. Such anticodon stem mutations were not expected to significantly destabilize the 5' end and trigger RTD (54,55).…”