The Elongator subunit Elp3 is a non-canonical tRNA acetyltransferase

Lin, Ting‐Yu; Abbassi, Nour El Hana; Zakrzewski, K.; Chramiec‐Głąbik, Andrzej; Jemioła-Rzemińska, Małgorzata; Różycki, Jan; Glatt, Sebastian

doi:10.1038/s41467-019-08579-2

Cited by 57 publications

(73 citation statements)

References 69 publications

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“…In zebrafish, we found diminished levels of this modification in morphant zebrafish. Also, recent structural evidence strongly support the Elongator complex function is tRNA modification [36,37]. Altogether, this data strongly indicates that the primary and evolutionarily-conserved function of the…”

Section: Discussionsupporting

confidence: 56%

Elongator Subunit 3 (Elp3) Is Required for Zebrafish Trunk Development

Rojas-Benítez

Allende

2020

IJMS

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Transfer RNAs (tRNAs) are the most post-transcriptionally modified RNA species. Some of these modifications, especially the ones located in the anti-codon loop, are required for decoding capabilities of tRNAs. Such is the case for 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U), synthetized by the Elongator complex. Mutants for its sub-units display pleiotropic phenotypes. In this paper, we analyze the role of elp3 (Elongator catalytic sub-unit) in zebrafish development. We found that it is required for trunk development; elp3 knock-down animals presented diminished levels of mcm5s2U and sonic hedgehog (Shh) signaling activity. Activation of this pathway was sufficient to revert the phenotype caused by elp3 knockdown, indicating a functional relationship between Elongator and Shh through a yet unknown molecular mechanism.

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Section: Discussionsupporting

confidence: 56%

Elongator Subunit 3 (Elp3) Is Required for Zebrafish Trunk Development

Rojas-Benítez

Allende

2020

IJMS

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“…S4d-e). 34 The observation that ac4C and mcm5s2U are sensitive to the metabolic state of the cell is consistent with the findings of Balasubramanian and coworkers, who reported that starvation conditions reduced NAT10-dependent ac4C levels in transfer RNA. 35 The ability of ACLY perturbation to influence the acetylation of poly(A)RNA, but not total RNA, suggests inhibitory CoA/acyl-CoAs may interact in a distinct manner with different functional forms of NAT10.…”

Section: Evaluating the Dynamic Activity Of Acetyltransferases In Ressupporting

confidence: 90%

A systems chemoproteomic analysis of acyl-CoA/protein interaction networks

Levy

Montgomery

Sardiu

et al. 2019

Preprint

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CoA metabolite interactions, enabling the identification of biological processes susceptible to altered acetyl-CoA levels. Finally, we utilize systems-level analyses to assess the features of novel protein networks that may interact with acyl-CoAs and demonstrate a strategy for high-confidence annotation of direct acetyl-CoA binding proteins and AT enzymes in human proteomes. Overall our studies illustrate the power of integrating chemoproteomics and systems biology analysis methods and provide a novel resource for understanding the diverse signaling roles of acyl-CoAs in biology and disease. Results Validation of CATNIP for the global study of acyl-CoA/protein interactionsIn order to deeply sample acyl-CoA/protein interactions on a proteome-wide scale, we initially set out to integrate CoA-based protein capture methods with LC-MS/MS ( Fig. 2a). In this workflow, whole cell extracts are first incubated with Lys-CoA Sepharose. This affinity matrix enables active site-dependent enrichment of many different classes of CoA-binding proteins, 8 making it ideal for broad profiling studies. Next, enriched proteins are subjected to tryptic digest and analyzed using MudPIT (multidimensional protein identification technology), a proteomics platform that combines strong cation exchange and C18 reverse phase chromatography to pre-fractionate tryptic peptides, followed by ionization and data-dependent MS/MS. 9 The separation afforded by this approach significantly decreases sample complexity, allowing the identification of rare, low abundance peptides from complex proteomic mixtures. To facilitate the identification of acyl-CoA/protein interactions, competition experiments are performed in which proteomes are pre-incubated with a CoA metabolite prior to capture. 10 Decreased enrichment in competition samples compared to controls (as assessed by quantitative spectral counting) signifies that the CoA metabolite interacts with a protein of interest.These interacting proteins can then be further classified into pharmacological or biological networks using either conventional metrics (fold-change, gene ontology, etc) or systems-based analysis tools.As an initial model, we explored the utility of CATNIP to globally profile acetyl-CoA/protein interactions in unfractionated HeLa cell proteomes. Proteomes were pre-incubated with acetyl-CoA or vehicle (buffer) control, followed by enrichment using Lys-CoA Sepharose. These experiments assessed competition at 3, 30, and 300 µM acetyl-CoA, which spans the physiological concentration range of acetyl-CoA in the cytosol and mitochondria. Protein capture in each condition was quantified using distributed normalized spectral abundance factor (dNSAF), a label-free metric that normalizes spectral counts relative to overall protein length ( Fig. S1a-c , Table S1). 11 Each condition was analyzed in triplicate, constituting 12 experiments, >144 hours of instrument time, and over 1.1 million non-redundant peptide spectra collected. We limited our analysis to highconfidence protein identifications (>4...

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“…However, total protein concentration in the soluble fractions was similar for the three strains (Supplementary Figure S3B). Coupled with similar [ 35 were generated and analyzed by SDS PAGE before (total protein) and after enrichment of aggregates, as described previously [4,29]. Aggregates were isolated twice from each strain.…”

Section: The Absence Of Both Asl Modifications Drastically Increasesmentioning

confidence: 99%

“…These complex modifications are synthesized in multi-step pathways [32] with key enzymes in multi-subunit complexes: the KEOPS complex for t 6 A synthesis [33] and the Elongator complex (Elp1-Elp6) for mcm 5 U [28,34]. While the mechanistic aspects of their synthesis are still being elucidated, most genes involved have been identified [32] and the pathways have been reconstituted fully [23] or partially [35,36] in vitro. This has allowed researchers to dissect the role of these modifications in vivo, where it has become apparent that defects in mcm 5 s 2 U34 and t 6 A37 synthesis in yeast gives rise to very similar phenotypes, including activation of the Gcn4 General Amino Acid Control (GAAC) response independently of Gcn2 and sensitivity to similar stresses [3,29,37].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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The Elongator subunit Elp3 is a non-canonical tRNA acetyltransferase

Cited by 57 publications

References 69 publications

Elongator Subunit 3 (Elp3) Is Required for Zebrafish Trunk Development

Elongator Subunit 3 (Elp3) Is Required for Zebrafish Trunk Development

A systems chemoproteomic analysis of acyl-CoA/protein interaction networks

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

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