Cryo-EM structures of the human Elongator complex at work.

tRNA modifications affect ribosomal elongation speed and co-translational folding dynamics. The Elongator complex is responsible for introducing 5-carboxymethyl at wobble uridine bases (cmU) in eukaryotic tRNAs. However, the structure and function of human Elongator remain poorly understood.

Fluoropyrimidines trigger decay of hypomodified tRNA in yeast.

Therapeutic fluoropyrimidines 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC) are in long use for treatment of human cancers and severe invasive fungal infections, respectively. 5-Fluorouridine triphosphate represents a bioactive metabolite of both drugs and is incorporated into target cells' RNA. Here we use the model fungus Saccharomyces cerevisiae to define fluorinated tRNA as a key mediator of 5-FU and 5-FC cytotoxicity when specific tRNA methylations are absent.

Evolutionary Conservation in Protein-Protein Interactions and Structures of the Elongator Sub-Complex ELP456 from and Yeast.

The Elongator complex plays a pivotal role in the wobble uridine modification of the tRNA anticodon. Comprising two sets of six distinct subunits, namely, Elongator proteins (ELP1-ELP6) and associated proteins, the holo-Elongator complex demonstrates remarkable functional and structural conservation across eukaryotes. However, the precise details of the evolutionary conservation of the holo-Elongator complex and its individual sub-complexes (i.

Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines.

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif is found next to another ill-defined cysteine that we show is conserved across eukaryotes.

Diphthamide - a conserved modification of eEF2 with clinical relevance.

Diphthamide, a complex modification on eukaryotic translation elongation factor 2 (eEF2), assures reading-frame fidelity during translation. Diphthamide and enzymes for its synthesis are conserved in eukaryotes and archaea. Originally identified as target for diphtheria toxin (DT) in humans, its clinical relevance now proves to be broader than the link to pathogenic bacteria.

Gene Mutations Identify a Candidate SAM Pocket in Radical Enzyme Dph1•Dph2 for Diphthamide Synthesis on EF2.

In eukaryotes, the Dph1•Dph2 dimer is a non-canonical radical SAM enzyme. Using iron-sulfur (FeS) clusters, it cleaves the cosubstrate S-adenosyl-methionine (SAM) to form a 3-amino-3-carboxy-propyl (ACP) radical for the synthesis of diphthamide. The latter decorates a histidine residue on elongation factor 2 (EF2) conserved from archaea to yeast and humans and is important for accurate mRNA translation and protein synthesis.

Experience with German Research Consortia in the Field of Chemical Biology of Native Nucleic Acid Modifications.

The chemical biology of native nucleic acid modifications has seen an intense upswing, first concerning DNA modifications in the field of epigenetics and then concerning RNA modifications in a field that was correspondingly rebaptized epitranscriptomics by analogy. The German Research Foundation (DFG) has funded several consortia with a scientific focus in these fields, strengthening the traditionally well-developed nucleic acid chemistry community and inciting it to team up with colleagues from the life sciences and data science to tackle interdisciplinary challenges. This Perspective focuses on the genesis, scientific outcome, and downstream impact of the DFG priority program SPP1784 and offers insight into how it fecundated further consortia in the field.

Lipidome remodeling in response to nutrient replenishment requires the tRNA modifier Deg1/Pus3 in yeast.

In the yeast Saccharomyces cerevisiae, the absence of the pseudouridine synthase Pus3/Deg1, which modifies tRNA positions 38 and 39, results in increased lipid droplet (LD) content and translational defects. In addition, starvation-like transcriptome alterations and induced protein aggregation were observed. In this study, we show that the deg1 mutant increases specific misreading errors.

DPH1 and DPH2 variants that confer susceptibility to diphthamide deficiency syndrome in human cells and yeast models.

The autosomal-recessive diphthamide deficiency syndrome presents as intellectual disability with developmental abnormalities, seizures, craniofacial and additional morphological phenotypes. It is caused by reduced activity of proteins that synthesize diphthamide on human translation elongation factor 2. Diphthamide synthesis requires seven proteins (DPH1-DPH7), with clinical deficiency described for DPH1, DPH2 and DPH5.

Yeast gene (alias ) operates in the initiation step of diphthamide synthesis on elongation factor 2.

In yeast, Elongator-dependent tRNA modifications are regulated by the Kti11•Kti13 dimer and hijacked for cell killing by zymocin, a tRNase ribotoxin. Kti11 (alias Dph3) also controls modification of elongation factor 2 (EF2) with diphthamide, the target for lethal ADP-ribosylation by diphtheria toxin (DT). Diphthamide formation on EF2 involves four biosynthetic steps encoded by the network and an ill-defined function.

Cryo-EM structure of the fully assembled Elongator complex.

Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional addition of chemical groups. Modifications in the tRNA anticodons are directly influencing ribosome decoding and dynamics during translation elongation and are crucial for maintaining proteome integrity.

AtELP4 a subunit of the Elongator complex in , mediates cell proliferation and dorsoventral polarity during leaf morphogenesis.

The Elongator complex in eukaryotes has conserved tRNA modification functions and contributes to various physiological processes such as transcriptional control, DNA replication and repair, and chromatin accessibility. ELONGATOR PROTEIN 4 (AtELP4) is one of the six subunits (AtELP1-AtELP6) in Elongator. In addition, there is an Elongator-associated protein, DEFORMED ROOTS AND LEAVES 1 (DRL1), whose homolog in yeast (Kti12) binds tRNAs.

E2/E3-independent ubiquitin-like protein conjugation by Urm1 is directly coupled to cysteine persulfidation.

Post-translational modifications by ubiquitin-like proteins (UBLs) are essential for nearly all cellular processes. Ubiquitin-related modifier 1 (Urm1) is a unique UBL, which plays a key role in tRNA anticodon thiolation as a sulfur carrier protein (SCP) and is linked to the noncanonical E1 enzyme Uba4 (ubiquitin-like protein activator 4). While Urm1 has also been observed to conjugate to target proteins like other UBLs, the molecular mechanism of its attachment remains unknown.

Translational fidelity and growth of Arabidopsis require stress-sensitive diphthamide biosynthesis.

Diphthamide, a post-translationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR2 (eEF2), is the human host cell-sensitizing target of diphtheria toxin. Diphthamide biosynthesis depends on the 4Fe-4S-cluster protein Dph1 catalyzing the first committed step, as well as Dph2 to Dph7, in yeast and mammals. Here we show that diphthamide modification of eEF2 is conserved in Arabidopsis thaliana and requires AtDPH1.

A novel DPH5-related diphthamide-deficiency syndrome causing embryonic lethality or profound neurodevelopmental disorder.

Purpose: Diphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs).

Methods: Molecular testing was performed using exome or genome sequencing.

Identifying Interaction Partners of Yeast Protein Disulfide Isomerases Using a Small Thiol-Reactive Cross-Linker: Implications for Secretory Pathway Proteostasis.

Protein disulfide isomerases (PDIs) function in forming the correct disulfide bonds in client proteins, thereby aiding the folding of proteins that enter the secretory pathway. Recently, several PDIs have been identified as targets of organic electrophiles, yet the client proteins of specific PDIs remain largely undefined. Here, we report that PDIs expressed in are targets of divinyl sulfone (DVSF) and other thiol-reactive protein cross-linkers.

Urm1, not quite a ubiquitin-like modifier?

Ubiquitin related modifier 1 (Urm1) is a unique eukaryotic member of the ubiquitin-fold (UbF) protein family and conserved from yeast to humans. Urm1 is dual-functional, acting both as a sulfur carrier for thiolation of tRNA anticodons and as a protein modifier in a lysine-directed Ub-like conjugation also known as urmylation. Although Urm1 conjugation coincides with oxidative stress and targets proteins like 2-Cys peroxiredoxins from yeast (Ahp1) and fly (Prx5), it was unclear how urmylation proceeds molecularly and whether it is affected by the activity of these antioxidant enzymes.

Role of in Phenotypic Variation of Strains Lacking -Dependent Pseudouridylation.

Yeast phenotypes associated with the lack of wobble uridine (U) modifications in tRNA were shown to be modulated by an allelic variation of , a gene encoding an mRNA-binding protein. We demonstrate that phenotypes caused by the loss of Deg1-dependent tRNA pseudouridylation are similarly affected by allelic status. Temperature sensitivity and protein aggregation are elevated in mutants and further increased in the presence of the allele, which encodes a truncated form of Ssd1.

Induction of protein aggregation and starvation response by tRNA modification defects.

Posttranscriptional modifications of anticodon loops contribute to the decoding efficiency of tRNAs by supporting codon recognition and loop stability. Consistently, strong synthetic growth defects are observed in yeast strains simultaneously lacking distinct anticodon loop modifications. These phenotypes are accompanied by translational inefficiency of certain mRNAs and disturbed protein homeostasis resulting in accumulation of protein aggregates.

Eukaryotic life without tQCUG: the role of Elongator-dependent tRNA modifications in Dictyostelium discoideum.

In the Elongator-dependent modification pathway, chemical modifications are introduced at the wobble uridines at position 34 in transfer RNAs (tRNAs), which serve to optimize codon translation rates. Here, we show that this three-step modification pathway exists in Dictyostelium discoideum, model of the evolutionary superfamily Amoebozoa. Not only are previously established modifications observable by mass spectrometry in strains with the most conserved genes of each step deleted, but also additional modifications are detected, indicating a certain plasticity of the pathway in the amoeba.

Diphthamide-deficiency syndrome: a novel human developmental disorder and ribosomopathy.

We describe a novel type of ribosomopathy that is defined by deficiency in diphthamidylation of translation elongation factor 2. The ribosomopathy was identified by correlating phenotypes and biochemical properties of previously described patients with diphthamide biosynthesis gene 1 (DPH1) deficiencies with a new patient that carried inactivating mutations in both alleles of the human diphthamide biosynthesis gene 2 (DPH2). The human DPH1 syndrome is an autosomal recessive disorder associated with developmental delay, abnormal head circumference (microcephaly or macrocephaly), short stature, and congenital heart disease.

Misactivation of multiple starvation responses in yeast by loss of tRNA modifications.

Previously, combined loss of different anticodon loop modifications was shown to impair the function of distinct tRNAs in Saccharomyces cerevisiae. Surprisingly, each scenario resulted in shared cellular phenotypes, the basis of which is unclear. Since loss of tRNA modification may evoke transcriptional responses, we characterized global transcription patterns of modification mutants with defects in either tRNAGlnUUG or tRNALysUUU function.

Loss of Elongator- and KEOPS-Dependent tRNA Modifications Leads to Severe Growth Phenotypes and Protein Aggregation in Yeast.

Modifications found in the Anticodon Stem Loop (ASL) of tRNAs play important roles in regulating translational speed and accuracy. Threonylcarbamoyl adenosine (tA37) and 5-methoxycarbonyl methyl-2-thiouridine (mcmsU34) are critical ASL modifications that have been linked to several human diseases. The model yeast is viable despite the absence of both modifications, growth is however greatly impaired.