Post-transcriptional modification of the tRNA anticodon loop is critical for translation. Yeast Trm7 is required for 29-O-methylation of C 32 and N 34 of tRNA Phe , tRNA Trp , and tRNA Leu(UAA) to form Cm 32 and Nm 34 , and trm7-D mutants have severe growth and translation defects, but the reasons for these defects are not known. We show here that overproduction of tRNA Phe suppresses the growth defect of trm7-D mutants, suggesting that the crucial biological role of Trm7 is the modification of tRNA Phe . We also provide in vivo and in vitro evidence that Trm7 interacts with ORF YMR259c (now named Trm732) for 29-O-methylation of C 32 , and with Rtt10 (named Trm734) for 29-O-methylation of N 34 of substrate tRNAs and provide evidence for a complex circuitry of anticodon loop modification of tRNA Phe , in which formation of Cm 32 and Gm 34 drives modification of m 1 G 37 (1-methylguanosine) to yW (wyebutosine). Further genetic analysis shows that the slow growth of trm7-D mutants is due to the lack of both Cm 32 and Nm 34 , and the accompanying loss of yW, because trm732-D trm734-D mutants phenocopy trm7-D mutants, whereas each single mutant is healthy; nonetheless, TRM732 and TRM734 each have distinct roles, since mutations in these genes have different genetic interactions with trm1-D mutants, which lack m 2,2 G 26 in their tRNAs. We speculate that 29-O-methylation of the anticodon loop may be important throughout eukaryotes because of the widespread conservation of Trm7, Trm732, and Trm734 proteins, and the corresponding modifications, and because the putative human TRM7 ortholog FTSJ1 is implicated in nonsyndromic X-linked mental retardation.

tRNA modifications are crucial for efficient and accurate protein synthesis, and modification defects are frequently associated with disease. Yeast trm7Δ mutants grow poorly due to lack of 2'-O-methylated C32 (Cm32) and Gm34 on tRNAPhe, catalyzed by Trm7-Trm732 and Trm7-Trm734 respectively, which in turn results in loss of wybutosine at G37. Mutations in human FTSJ1, the likely TRM7 homolog, cause non-syndromic X-linked intellectual disability (NSXLID), but the role of FTSJ1 in tRNA modification is unknown. Here we report that tRNAPhe from two genetically independent cell lines of NSXLID patients with loss of function FTSJ1 mutations nearly completely lacks Cm32 and Gm34, and has reduced peroxywybutosine (o2yW37). Additionally, tRNAPhe from an NSXLID patient with a novel FTSJ1-p.A26P missense allele specifically lacks Gm34, but has normal levels of Cm32 and o2yW37. tRNAPhe from the corresponding Saccharomyces cerevisiae trm7-A26P mutant also specifically lacks Gm34, and the reduced Gm34 is not due to weaker Trm734 binding. These results directly link defective 2'-O-methylation of the tRNA anticodon loop to FTSJ1 mutations, suggest that the modification defects cause NSXLID, and may implicate Gm34 of tRNAPhe as the critical modification. These results also underscore the widespread conservation of the circuitry for Trm7-dependent anticodon loop modification of eukaryotic tRNAPhe.

Sequence variation in tRNA genes influences the structure, modification, and stability of tRNA; affects translation fidelity; impacts the activity of numerous isodecoders in metazoans; and leads to human diseases. To comprehensively define the effects of sequence variation on tRNA function, we developed a high-throughput in vivo screen to quantify the activity of a model tRNA, the nonsense suppressor SUP4 oc of Saccharomyces cerevisiae. Using a highly sensitive fluorescent reporter gene with an ochre mutation, fluorescence-activated cell sorting of a library of SUP4 oc mutant yeast strains, and deep sequencing, we scored 25,491 variants. Unexpectedly, SUP4 oc tolerates numerous sequence variations, accommodates slippage in tertiary and secondary interactions, and exhibits genetic interactions that suggest an alternative functional tRNA conformation. Furthermore, we used this methodology to define tRNA variants subject to rapid tRNA decay (RTD). Even though RTD normally degrades tRNAs with exposed 59 ends, mutations that sensitize SUP4 oc to RTD were found to be located throughout the sequence, including the anti-codon stem. Thus, the integrity of the entire tRNA molecule is under surveillance by cellular quality control machinery. This approach to assess activity at high throughput is widely applicable to many problems in tRNA biology.

BackgroundPrimordial dwarfism is a state of extreme prenatal and postnatal growth deficiency, and is characterized by marked clinical and genetic heterogeneity.ResultsTwo presumably unrelated consanguineous families presented with an apparently novel form of primordial dwarfism in which severe growth deficiency is accompanied by distinct facial dysmorphism, brain malformation (microcephaly, agenesis of corpus callosum, and simplified gyration), and severe encephalopathy with seizures. Combined autozygome/exome analysis revealed a novel missense mutation in WDR4 as the likely causal variant. WDR4 is the human ortholog of the yeast Trm82, an essential component of the Trm8/Trm82 holoenzyme that effects a highly conserved and specific (m7G46) methylation of tRNA. The human mutation and the corresponding yeast mutation result in a significant reduction of m7G46 methylation of specific tRNA species, which provides a potential mechanism for primordial dwarfism associated with this lesion, since reduced m7G46 modification causes a growth deficiency phenotype in yeast.ConclusionOur study expands the number of biological pathways underlying primordial dwarfism and adds to a growing list of human diseases linked to abnormal tRNA modification.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0779-x) contains supplementary material, which is available to authorized users.