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METTL13 modulates mRNA translation through methylation of eEF1A at Lys55 and the N-terminus

ABSTRACT: The experiment aimed at identifying lysine methylation dependent interactions for eEF1A. FLAG-tagged wild type eEF1A and a methylation deficient mutant, carrying lysine-to-arginine mutations of the well-established methylation sites (Lys36, Lys55, Lys79, Lys165 and Lys318) were overexpressed in HEK-293 cells and co-purifying proteins were quantified using the MaxLFQ algorithm. FLAG-tagged

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Diploid Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Magnus Jakobsson

LAB HEAD: Jesper Velgard Olsen

PROVIDER: PXD009895 | Pride | 2018-07-04

REPOSITORIES: Pride

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The dual methyltransferase METTL13 targets N terminus and Lys55 of eEF1A and modulates codon-specific translation rates.

Jakobsson Magnus E ME Małecki Jędrzej M JM Halabelian Levon L Nilges Benedikt S BS Pinto Rita R Kudithipudi Srikanth S Munk Stephanie S Davydova Erna E Zuhairi Fawzi R FR Arrowsmith Cheryl H CH Jeltsch Albert A Leidel Sebastian A SA Olsen Jesper V JV Falnes Pål Ø PØ

Nature communications 20180824 1

Eukaryotic elongation factor 1 alpha (eEF1A) delivers aminoacyl-tRNA to the ribosome and thereby plays a key role in protein synthesis. Human eEF1A is subject to extensive post-translational methylation, but several of the responsible enzymes remain unknown. Using a wide range of experimental approaches, we here show that human methyltransferase (MTase)-like protein 13 (METTL13) contains two distinct MTase domains targeting the N terminus and Lys55 of eEF1A, respectively. Our biochemical and str ...[more]

PMID: 30143613

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A novel strategy of profiling protein methylation on lysine and arginine

Project description:Protein methylation is involved in different processes including gene expression regulation, epigenetics, and nucleotide metabolism. Identification of methylated proteins is difficult as methyl groups are small, and do not introduce significant changes in protein hydrophobicity or charge state. The most effective analytical method to date is relative enrichment by pan-specific antibodies and methylation specific binding domains. Here, we present a novel and unbiased chemical strategy to enrich and identify sites of lysine and arginine methylations. This approach makes use of the property that methylation does not alter the charge state of lysine and arginine. This approach revealed over 793 methylation events including 211 arginine and 585 lysine methylation sites in HEK 293 Cells. This strategy proves to be convenient, effective and versatile, with the potential of analyzing other PTMs on both lysine and arginine.

2016-08-16 | PXD001689 | Pride

METTL13 modulates mRNA translation through methylation of eEF1A at Lys55 and the N-terminus, part 2

Project description:The experiment aimed at characterizing the methylation status of eEF1A in cells (HeLa) stressed using a panel of drugs including anisomycin, cycloheximide, 4NQO and adenosine dialdehyde (AdOx).

2018-07-04 | PXD009914 | Pride

A network of DZF proteins controls alternative splicing regulation and fidelity.

Project description:This SuperSeries is composed of the SubSeries listed below. GSE206058: Paired-end total RNA-seq of triplicate biological replicate treatments of HEK-293 cells with negative control siRNAs or siRNAs targeting ZFR, ILF2, or ILF3. GSE206059: eCLIP analysis of FLAG-ZFR binding in HEK-293 Flp-In TREx cell lines, with size-matched input control. GSE206060: RNA Bind-n-Seq of recombinantly expressed and purified ZFR-ILF2 complexes, with PTBP1 as a positive control for successful library generation.

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The dual methyltransferase METTL13 targets N-terminus and Lys55 of eEF1A and regulates codon-specific translation rates

Project description:Eukaryotic elongation factor 1 alpha (eEF1A) delivers aminoacyl-tRNA to the ribosome and thereby plays a key role in protein synthesis. Human eEF1A is post-translationally methylated on several lysine residues as well as on the N-terminus and until recently the responsible methyltransferases were largely elusive. Using mass spectrometry based screens, gene targeted cells and in vitro enzymology we identify the human methyltransferase like proteins 13 (METTL13), which contains two distinct methyltransferase domains, as an enzyme catalyzing both dimethylation of Lys55 and trimethylation of the N-terminus of eEF1A. Moreover, through ribosome profiling we demonstrate that loss of METTL13 function alters translation dynamics and results in changed translation rate of specific codons. In line with the established descriptive nomenclature for this type of enzymes we suggest that METTL13 is redubbed eEF1A dual methyltransferase (eEF1A-DMT / EEF1ADMT).

2018-07-04 | GSE104033 | GEO

The novel lysine specific methyltransferase METTL21B affects mRNA translation through inducible and dynamic methylation of Lys-165 in human eukaryotic elongation factor 1 alpha (eEF1A)

Project description:Lysine methylation is abundant on histone proteins, representing a dynamic regulator of chromatin state and gene activity, but is also frequent on many nonhistone proteins, including eukaryotic elongation factor 1 alpha (eEF1A). However, the functional significance of eEF1Amethylation remains obscure and it has remained unclear whether eEF1A methylation is dynamic and subject to active regulation. We here demonstrate, using a wide range of in vitro and in vivo approaches, that the previously uncharacterized human methyltransferase METTL21B specifically targets Lys-165 in eEF1A in an aminoacyl-tRNA- and GTP-dependent manner. Interestingly, METTL21B mediated eEF1A methylation showed strong variation across different tissues and cell lines, and was induced by altering growth conditions or by treatment with certain ER-stress-inducing drugs, concomitant with an increase in METTL21B gene expression. Moreover, genetic ablation of METTL21B function in mammalian cells caused substantial alterations in mRNA translation, as measured by ribosome profiling. A non-canonical function for eEF1A in organization of the cellular cytoskeleton has been reported, and interestingly, METTL21B accumulated in centrosomes, in addition to the expected cytosolic localization. In summary, the present study identifies METTL21B as the enzyme responsible for methylation of eEF1A on Lys-165 and shows that this modification is dynamic, inducible and likely of regulatory importance.

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METTL21B is a novel human lysine methyltransferase of translation elongation factor 1A: discovery by CRISPR/Cas9 knock out

Project description:Lysine methylation is widespread on human proteins, however the enzymes that catalyse its addition remain largely unknown. This limits our capacity to study the function and regulation of this modification. Here we report that human METTL21B is a protein methyltransferase, which methylates lysine 165 of eukaryotic translation elongation factor 1A (eEF1A). The CRISPR/Cas9 system was used to knock out putative protein methyltransferases METTL21B and METTL23 in K562 cells. The known eEF1A methyltransferase EEF1AKMT1 was also knocked out as a control. Targeted mass spectrometry revealed the loss of lysine 165 methylation upon knock out of METTL21B, and the expected loss of lysine 79 methylation on knock out of EEF1AKMT1. No loss of eEF1A methylation was seen in the METTL23 knock out. Recombinant METTL21B was then shown to catalyse methylation on lysine 165 in eEF1A1 and eEF1A2 in vitro, confirming it as the methyltransferase responsible for this methylation site. Stable isotope labelling by amino acids in cell culture (SILAC) proteomic analysis revealed dysregulation of processes related to eEF1A function in knock outs of METTL21B and EEF1AKMT1, but specific dysregulation of ribosomal proteins in the knock out of METTL21B. This indicates a specific function for lysine 165 methylation of eEF1A in human. METTL21B is specific to vertebrates, with its target lysine showing similar evolutionary conservation. We suggest METTL21B be renamed eEF1A-KMT3. This is the first study to specifically generate CRISPR/Cas9 knock outs of the genes encoding putative protein methyltransferases, for the purpose of substrate discovery and site mapping. Our approach should prove useful for the discovery of further novel methyltransferases, and more generally for the discovery of sites for other protein-modifying enzymes.

2017-07-03 | PXD005497 | Pride

UPF1 regulatory loop variant RNA-Seq and RIP-Seq

Project description:HEK-293 Trex cells containing stable tetracycline-inducible transgenes driving overexpression of CLIP-tagged UPF1 isoforms 1 and 2 or GFP were used for total RNA-seq. In addition, CLIP-UPF1 isoforms were affinity purified, and bound RNA was subjected to high-throughput sequencing.

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MIR-204 target genes

Project description:The aim of this study is to discover genes regulated by miR-204. Differential gene expression in HEK-293 cells transfected with miR-204-mimic compared to HEK-293 cells transfected with control oligo (HEK-293 control) was analyzed using the Agilent Human Whole Genome 4x44K gene expression array (Agilent Technologies, Santa Clara, CA).

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2024-01-03 | PXD042599 | Pride

Genome-wide identification and DNA methylation of human full-length LINE-1 (L1) retrotransposons in a panel of normal and cancer cell lines.

Project description:We used bs-ATLAS-seq to comprehensively map the genomic location and assess the DNA methylation status of human full-length LINE-1 elements (L1). The approach is focused on the youngest family (L1HS), but it also catches a significant fraction of L1PA2 to L1PA8 elements. This was performed in a panel of 12 human primary or transformed cell lines (BJ, IMR90, MRC5, H1, K562, HCT116, HeLa S3, HepG2, MCF7, HEK-293, HEK-293T, 2102Ep).

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