Threonylcarbamoyladenosine (t6A) is a modified nucleoside universally conserved in tRNAs in all three kingdoms of life. The recently discovered genes for t6A synthesis, including tsaC and tsaD, are essential in model prokaryotes but not essential in yeast. These genes had been identified as antibacterial targets even before their functions were known. However, the molecular basis for this prokaryotic-specific essentiality has remained a mystery. Here, we show that t6A is a strong positive determinant for aminoacylation of tRNA by bacterial-type but not by eukaryotic-type isoleucyl-tRNA synthetases and might also be a determinant for the essential enzyme tRNAIle-lysidine synthetase. We confirm that t6A is essential in Escherichia coli and a survey of genome-wide essentiality studies shows that genes for t6A synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t6A is dispensable in Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803 and Streptococcus mutans. Proteomic analysis of t6A- D. radiodurans strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t6A in tRNAs. Thus, although t6A is universally conserved in tRNAs, its role in translation might vary greatly between organisms.

Post-transcriptional tRNA modifications are numerous and require a large set of highly conserved enzymes in humans and other organisms. In yeast, the loss of many modifications is tolerated under unstressed conditions; one exception is the N-threonyl-carbamoyl-adenosine (tA) modification, loss of which causes a severe growth phenotype. Here we aimed at a molecular diagnosis in a brother and sister from a consanguineous family who presented with global developmental delay, failure to thrive and a renal defect manifesting in proteinuria and hypomagnesemia. Using exome sequencing, the patients were found to be homozygous for the c.974G>A (p.(Arg325Gln)) variant of the KAE1 gene. KAE1 is a constituent of the KEOPS complex, a five-subunit complex that catalyzes the second biosynthetic step of tA in the cytosol. The yeast KAE1 allele carrying the equivalent mutation did not rescue the tA deficiency of the kae1Δ yeast strain as efficiently as the WT allele; furthermore, tA levels quantified by LC-MS/MS were lower in the kae1Δ strain which was complemented by the mutation than in the kae1Δ strain, which was complemented by the WT allele. We conclude that homozygosity for c.974G>A (p.(Arg325Gln)) in KAE1 likely exerts its pathogenic effect by perturbing tA synthesis, thereby interfering with global protein production. This is the first report of tA biosynthesis defect in human. KAE1 joins the growing list of cytoplasmic tRNA modification enzymes, all associated with severe neurological disorders.

Current next-generation RNA sequencing methods do not provide accurate quantification of small RNAs within a sample due to sequence-dependent biases in capture, ligation, and amplification during library preparation. We present a method, Absolute Quantification (AQ) RNA-seq, that minimizes biases and provides a direct, linear correlation between sequencing read count and copy number for all small RNAs in a sample. Library preparation and data processing were optimized and validated using a 963-member miRNA reference library, oligonucleotide standards of varying lengths, and northern blots. Application of AQRNA-seq to a panel of human cancer cells revealed >800 detectable miRNAs that varied during cancer progression, while application to bacterial tRNA pools, with the challenges of secondary structure and abundant modifications, revealed 80-fold variation in tRNA isoacceptor levels, stress-induced site-specific tRNA fragmentation, quantitative modification maps, and evidence for stress-induced tRNA-driven codon-biased translation. AQRNA-seq thus provides a versatile means to quantitatively map the small RNA landscape in cells.

Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNA in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (tA) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using tA-deficient yeast derivatives, it is shown that tA is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the tA synthesis gene tsaE (brpB) is dispensable and its genome encodes a PrrC toxin. We had previously shown using an HPLC-based assay that the S. mutans tsaE mutant was devoid of tA. However, we describe here a novel and a more sensitive hybridization-based tA detection method (compared to HPLC) that showed tA was still present in the S. mutans ΔtsaE, albeit at greatly reduced levels (93% reduced compared with WT). Moreover, mutants in 2 other S. mutans tA synthesis genes (tsaB and tsaC) were shown to be totally devoid of the modification thus confirming its dispensability in this organism. Furthermore, analysis of tA modification ratios and of tA synthesis genes mRNA levels in S. mutans suggest they may be regulated by growth phase.

Modifications found in the Anticodon Stem Loop (ASL) of tRNAs play important roles in regulating translational speed and accuracy. Threonylcarbamoyl adenosine (t6A37) and 5-methoxycarbonyl methyl-2-thiouridine (mcm5s2U34) are critical ASL modifications that have been linked to several human diseases. The model yeast Saccharomyces cerevisiae is viable despite the absence of both modifications, growth is however greatly impaired. The major observed consequence is a subsequent increase in protein aggregates and aberrant morphology. Proteomic analysis of the t6A-deficient strain (sua5 mutant) revealed a global mistranslation leading to protein aggregation without regard to physicochemical properties or t6A-dependent or biased codon usage in parent genes. However, loss of sua5 led to increased expression of soluble proteins for mitochondrial function, protein quality processing/trafficking, oxidative stress response, and energy homeostasis. These results point to a global function for t6A in protein homeostasis very similar to mcm5/s2U modifications.

N 6 -threonylcarbamoyl adenosine (t 6 A) is a nucleoside modification found in all kingdoms of life at position 37 of tRNAs decoding ANN codons, which functions in part to restrict translation initiation to AUG and suppress frameshifting at tandem ANN codons. In Bacteria the proteins TsaB, TsaC (or C2), TsaD, and TsaE, comprise the biosynthetic apparatus responsible for t 6 A formation. TsaC(C2) and TsaD harbor the relevant active sites, with TsaC(C2) catalyzing the formation of the intermediate threonylcarbamoyladenosine monophosphate (TC-AMP) from ATP, threonine, and CO 2 , and TsaD catalyzing the transfer of the threonylcarbamoyl moiety from TC-AMP to A 37 of substrate tRNAs. Several related modified nucleosides, including hydroxynorvalylcarbamoyl adenosine (hn 6 A), have been identified in select organisms, but nothing is known about their biosynthesis. To better understand the mechanism and structural constraints on t 6 A formation, and to determine if related modified nucleosides are formed via parallel biosynthetic pathways or the t 6 A pathway, we carried out biochemical and biophysical investigations of the t 6 A systems from E. coli and T. maritima to address these questions. Using kinetic assays of TsaC(C2), tRNA modification assays, and NMR, our data demonstrate that TsaC(C2) exhibit relaxed substrate specificity, producing a variety of TC-AMP analogs that can differ in both the identity of the amino acid and nucleotide component, whereas TsaD displays more stringent specificity, but efficiently produces hn 6 A in E. coli and T. maritima tRNA. Thus, in organisms that contain modifications such as hn 6 A in their tRNA, we conclude that their origin is due to formation via the t 6 A pathway.

Accuracy in protein biosynthesis is maintained through multiple pathways, with a critical checkpoint occurring at the tRNA aminoacylation step catalyzed by aminoacyl-tRNA synthetases (ARSs). In addition to the editing functions inherent to some synthetases, single-domain trans-editing factors, which are structurally homologous to ARS editing domains, have evolved as alternative mechanisms to correct mistakes in aminoacyl-tRNA synthesis. To date, ARS-like trans-editing domains have been shown to act on specific tRNAs that are mischarged with genetically encoded amino acids. However, structurally related non-protein amino acids are ubiquitous in cells and threaten the proteome. Here, we show that a previously uncharacterized homolog of the bacterial prolyl-tRNA synthetase (ProRS) editing domain edits a known ProRS aminoacylation error, Ala-tRNA, but displays even more robust editing of tRNAs misaminoacylated with the non-protein amino acid α-aminobutyrate (2-aminobutyrate, Abu) in vitro and in vivo. Our results indicate that editing by trans-editing domains such as ProXp-x studied here may offer advantages to cells, especially under environmental conditions where concentrations of non-protein amino acids may challenge the substrate specificity of ARSs.

regulating translational speed and accuracy. Threonylcarbamoyl adenosine (t6A37) and 5-methoxycarbonylmethyl-thiouridine (mcm5s2U34) are critical ASL modifications that have been linked to several human diseases. The model yeast Saccharomyces cerevisiae is viable despite the absence of both modifications, growth is however greatly impaired. The major observed consequence is a subsequent increase in protein aggregates and aberrant morphology. Proteomic analysis of the t6A-deficient strain revealed a global mistranslation leading to protein aggregation without regard to physicochemical properties or t6A-dependent or biased codon usage in parent genes. However, loss of sua5 led to increased expression of soluble proteins for mitochondrial function, protein quality processing/trafficking, oxidative stress response, and energy homeostasis. These results point to a global function for t6A in protein homeostasis very similar to mcm5/s2U modifications.