hap-1 eef1akmt4 gene ko cells  (Haplogen Inc)

Journal: Nucleic Acids Research

Article Title: Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

doi: 10.1093/nar/gkx432

Figure Lengend Snippet: Gene structure and targeting strategy for the endothelin converting enzyme 2 ( ECE2 ) /EEF1AKMT4 locus. ( A ) Topology diagram of the 7BS MTase fold. α-helices and β-strands are depicted as grey boxes (denoted Z and A–E) and black arrows (denoted 1–7), respectively, and annotated according to previously established nomenclature . ( B ) Organization and annotation of the human ECE2 locus. Top, current annotation of the ECE2 locus. Regions in the genomic DNA corresponding to annotated exons of ECE2 (located on the forward stand) and CAMK2N2 (located on the reverse strand) are indicated in blue and green, respectively (based on the annotated gene structure according to Ensembl (assembly GRCh37)). Middle, tracks representing spliced expressed sequence tags (ESTs) exported from the UCSC genome browser. Bottom, alternative gene model supported by the EST data. ( C ) Gene targeting strategy and disruption of the EEF1AKMT4 locus by CRISPR/Cas9 technology. Top, schematic representation of the EEF1AKMT4 gene. Exons and introns are represented by boxes (gray, coding region; white, untranslated regions) and lines, respectively. An arrow indicates the region targeted by CRISPR. Bottom, DNA and protein sequence of targeted region of EEF1AKMT4 locus in HAP-1 wild-type (WT) and EEF1AKMT4 knockout (KO) cells. The conserved motif Post I is indicated by a rectangle and the last residue of the motif, D88, is shown in red. ( D ) Structural support for an involvement of D88 in coordinating AdoMet binding. The figure is based on a previously published structure (PDB ID: 2PXX) of eEF1A-KMT4 in complex with S -adenosylhomocysteine (AdoHcy; the unmethylated counterpart of AdoMet) . AdoHcy and D88 are represented as sticks and the second β-strand (β2) is represented as a cartoon. Possible hydrogen bonds between the carboxyl group of D88 and the ribose moiety of AdoHcy are shown (dashed lines). The figure was generated using the PyMOL Molecular Graphics System, Version 1.3 (Schrodinger, LLC).

Article Snippet: HAP-1 EEF1AKMT4 gene KO cells were generated using the CRISPR/Cas9 system as a custom project performed by Horizon Genomics (formerly Haplogen).

Techniques: Sequencing, CRISPR, Knock-Out, Binding Assay, Generated

Journal: Nucleic Acids Research

Article Title: Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

doi: 10.1093/nar/gkx432

Figure Lengend Snippet: Identification of eEF1A proteins as candidate substrates for eEF1A-KMT4. ( A ) eEF1A-KMT4-mediated methylation in protein extracts. Protein extracts from HAP-1 wild-type (WT) or EEF1AKMT4 KO cells were incubated with [ 3 H]-AdoMet and with recombinant eEF1A-KMT4 as indicated. Top panel, Ponceau S-stained membrane of sodium dodecyl sulphate (SDS-PAGE)-separated methylation reactions. Bottom, visualization of methylation by fluorography of membrane in upper panel. ( B ) Mutation of D88 abrogates the enzymatic activity of eEF1A-KMT4. Protein extract from HAP-1 EEF1AKMT4 KO cells incubated with [ 3 H]-AdoMet in the presence of either recombinant WT eEF1A-KMT4 or the corresponding D88A mutant protein. Reactions were analyzed as in (A). ( C ) Outline of chromatography based enrichment strategy for eEF1A-KMT4 substrates. In vitro methylated protein extract was loaded onto an anion exchange (Q) column and, in turn, the flow-through (FTQ) was loaded onto a cation exchange (S) column. Proteins were eluted from both columns with a step gradient of increasing NaCl. ( D ) Enrichment of the ∼50 kDa eEF1A-KMT4 substrate. Proteins were eluted from ion exchange columns with a step gradient ramped from 150 mM (0.15) to 1 M (1.0) NaCl according to the scheme depicted in (C). Collected fractions were analyzed by fluorography as in (A). ( E ) SDS-PAGE analysis of the main substrate-enriched fraction (0.3S). The fraction containing the bulk of methylated substrate in (D) eluted at 300 mM NaCl from the S column (0.3S). The most prominent proteins in the major ∼50 kDaband were identified as eEF1A1 and eEF1A2 by mass spectrometry (MS) (See Table ).

Article Snippet: HAP-1 EEF1AKMT4 gene KO cells were generated using the CRISPR/Cas9 system as a custom project performed by Horizon Genomics (formerly Haplogen).

Techniques: Methylation, Incubation, Recombinant, Staining, SDS Page, Mutagenesis, Activity Assay, Chromatography, In Vitro, Mass Spectrometry

Journal: Nucleic Acids Research

Article Title: Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

doi: 10.1093/nar/gkx432

Figure Lengend Snippet: eEF1A-KMT4-mediated methylation of K36 in eEF1A in vivo . ( A ) EEF1AKMT4 KO abrogates eEF1A-K36 methylation in human cells. Ion chromatograms representing peptides of the different methylation states of K36 in eEF1A proteins from HAP-1 WT and EEF1AKMT4 knock out (KO) cells are shown. The retention time for analyzed peptides is indicated (arrow) and their relative abundances are indicated (obtained by integration of the area of the relevant peaks; expressed as the percentage of the sum of all peaks). Tandem mass spectra supporting identity of analyzed peptides are shown in Supplementary Figure S2. ( B ) eEF1A1 and eEF1A2 are both methylated at K36 in vivo . Chromatograms represent the methylation status of K36 in TAP-tagged eEF1A that was affinity-purified from HEK293-derived cells ectopically expressing TAP-tagged eEF1A1 or eEF1A2. ( C ) Methylation status of eEF1A-K36 in mammalian organs. Chromatograms representing the methylation status of K36 in eEF1A obtained from rat brain, liver, intestine and kidney are shown.

Article Snippet: HAP-1 EEF1AKMT4 gene KO cells were generated using the CRISPR/Cas9 system as a custom project performed by Horizon Genomics (formerly Haplogen).

Techniques: Methylation, In Vivo, Knock-Out, Affinity Purification, Derivative Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

doi: 10.1093/nar/gkx432

Figure Lengend Snippet: The effect of EEF1AKMT4 KO on the translational landscape. ( A ) Differential expression plot showing the log 2 -fold changes on the mRNA and ribosome footprint level in EEF1AKMT4 KO relative to WT cells. Indicated are: not significantly changed genes (DEseq2 padj >0.05) (gray), genes differentially expressed at the mRNA level only (green), genes with differential ribosome footprinting only (blue) and genes that were changed at both the mRNA and ribosome footprinting levels (red). ( B ) GO analysis (Cellular Process) for genes significantly upregulated on the footprint level only. n = 322, numbers in brackets indicate genes matching this category. ( C) GO analysis (Cellular Process) for genes significantly downregulated on the footprint level only. n = 501, numbers in brackets indicate genes matching this category. ( D ) Ribosome occupancy in EEF1AKMT4 KO cells compared with WT cells. A-site occupancy and +1 occupancy are normalized to untranslated downstream codons (mean ± SD, n = 3). Symbol size indicates the relative frequency of codons.

Article Snippet: HAP-1 EEF1AKMT4 gene KO cells were generated using the CRISPR/Cas9 system as a custom project performed by Horizon Genomics (formerly Haplogen).

Techniques: Expressing, Footprinting