Conserved 5-methyluridine tRNA modification modulates ribosome translocation

Abstract

While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA inE. coli, Trm2 inS. cerevisiae) exhibit altered tRNA modifications patterns. Furthermore, m5U54 deficient tRNAs are desensitized to small molecules that prevent translocationin vitro.This finding is consistent with our observations that, relative to wild-type cells,trm2Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.

Beyond the Anticodon: tRNA Core Modifications and Their Impact on Structure, Translation and Stress Adaptation

“… Modification circuits involving tRNA core modifications: ( a ) modification circuits identified in T. thermophilus in response to temperature changes represented on a cloverleaf scheme [ 94 , 95 , 107 ]; ( b ) T-arm modification circuits identified in yeast [ 69 , 70 ]; ( c , d ) modification circuits identified in E. coli [ 109 , 110 , 111 ]; and ( e ) modification circuits identified in E. coli [ 111 ]. Modifications are represented by colored circles (see the legend in the box for the corresponding colors).…”

“…Recently, several reports have shed light on modification cross-talks among tRNA core modifications in E. coli . They reported that the introduction of U47 is globally enhanced by the prior presence of m 7 G46 [ 109 ] and also stimulated by m 5 U54 in E. coli , , , and [ 110 , 111 ] ( Figure 3 c). 55 seems to also participate in this network, in which it may either slightly stimulate or inhibit the introduction of U47 in E. coli and , respectively [ 111 ].…”

“… 55 seems to also participate in this network, in which it may either slightly stimulate or inhibit the introduction of U47 in E. coli and , respectively [ 111 ]. In addition, the formation of ms 2 A37 in the ACL of E. coli is activated by m 5 U54 [ 110 , 111 ] ( Figure 3 d), and the introduction of s 4 U8 is enhanced in the presence of 55 and m 5 U54 in E. coli [ 111 ] ( Figure 3 e). These interdependencies in E. coli tRNA core modifications were identified using a combination of nucleotide quantification and sequencing techniques, such as HPLC, liquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS), multiplex small RNA-sequencing (MSR-seq) and primer extension analysis.…”

“…Several recent studies have shed new light on the mechanisms by which modifications in the tRNA core impact fitness and translation [ 110 , 111 , 132 ]. Effects of certain tRNA core modifications on fitness have been known for many years.…”

“…In a recent report that conducted in vitro kinetic studies of translation with E. coli tRNAs prepared from trmA cells, the m 5 U54 modification was shown to play a role in the translocation process [ 110 ]. The use of hygromycin B—an antibiotic known to block the translocation of ribosomes—led to the proposal that m 5 U54 affects the translocation step and could reduce the translation speed by restraining the translocation of tRNAs [ 110 ].…”