Journal: Molecular Autism

Article Title: The eIF4E homolog 4EHP (eIF4E2) regulates hippocampal long-term depression and impacts social behavior

doi: 10.1186/s13229-020-00394-7

Figure Lengend Snippet: Loss of 4EHP in excitatory neurons exaggerates hippocampal mGluR-LTD and impairs social behavior. a , b Confirmation of loss of 4EHP expression in the prefrontal cortex and hippocampus, respectively, of 4EHP-eKO (flx/flx) versus 4EHP-WT (+ / +) mice using western blot. GAPDH was used as a loading control. c , d Confirmation of loss of 4EHP expression in excitatory neurons in the prefrontal cortex and hippocampus, respectively, of 4EHP-eKO versus 4EHP-WT mice using immunofluorescence microscopy. 4EHP expression is colored in red and Hoechst-stained nucleus in blue. Scale bar represents 20 µm. e Schematic representation of stimulating (left) and recording (right) electrode position for measuring DHPG-induced long-term depression (mGluR-LTD) in the CA1 hippocampus. The red fibers represent CA3 pyramidal projections to the CA1 (Schaffer collaterals). f Field excitatory postsynaptic potential (fEPSP) recordings of CA1 pyramidal neurons during mGluR-LTD. Baseline was recorded for 30 min prior to adding mGluR1/5 agonist, DHPG (100 µM), to slices for 10 min. LTD was recorded for 90 min. The inset is the average of all fEPSPs at time a and b for each genotype, n = 8 per group. g Average of the last 10 min of recording. h Schematic representation of the three-chamber social preference and social novelty test. Mice were first habituated to the apparatus for 10 min. Two cages (mouse holding devices) were then placed in opposite corners of opposing chambers; one cage was empty (E) and one contained a conspecific stranger mouse (S1). After 10 min, a novel stranger mouse (S2) was added to E for the social novelty test lasting 10 min. i The amount of time the test mouse spent sniffing either S1 or E. j The amount of time the test mouse spent sniffing either S1 or S2. k Schematic representation of the direct (reciprocal) social interaction test. Test mice were first habituated to a clean home cage for 5 min. A novel stranger mouse was then added, and mice could freely interact for 10 min. l Nose-to-anogenital sniffing time of the stranger mouse by the test mouse. m Total interaction time including nose-to-nose sniffing, nose-to-anogenital sniffing, following, chasing, mounting, and fighting. Reciprocal interaction of the stranger mouse to test mouse was also included. Data are presented as mean ± s.e.m.; *p < 0.05, **p < 0.01, ****p < 0.0001, N.S., not significant; calculated by unpaired t-test or 2-way ANOVA with Bonferroni multiple comparisons test. Sample size is located within bar graphs. Eif4e2 is the mouse gene encoding 4EHP

Article Snippet: Sections were then incubated in the following primary antibodies: eIF4E2 (sc-100731, Santa Cruz), EMX1 (PA5-35373, Thermo), PVALB (195004, Synaptic System), Somatostatin 28 (ab111912, Abcam), Laminin (L9393, Sigma), diluted 1:100 in blocking solution overnight at 4 °C.

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Microscopy, Staining

Journal: Molecular Autism

Article Title: The eIF4E homolog 4EHP (eIF4E2) regulates hippocampal long-term depression and impacts social behavior

doi: 10.1186/s13229-020-00394-7

Figure Lengend Snippet: Proposed model. a 4EHP binds to the mRNA 5′ cap where its stable expression and function is maintained and reciprocated by physical interaction with GIGYF2. b Homozygous deletion of 4EHP in excitatory neurons of the forebrain (4EHP-eKO) results in reduced protein expression of GIGYF2, exaggerated mGluR-LTD, and impaired social behavior (possibly due to translation de-repression of specific mRNAs without affecting global protein synthesis). c Heterozygous deletion of Gigyf2 , Eif4e2 , or both does not result in ASD-like behaviors (possibly due to haplosufficiency). d Proposed model for the development of ASD in patients harboring GIGYF2 mutations

Article Snippet: Sections were then incubated in the following primary antibodies: eIF4E2 (sc-100731, Santa Cruz), EMX1 (PA5-35373, Thermo), PVALB (195004, Synaptic System), Somatostatin 28 (ab111912, Abcam), Laminin (L9393, Sigma), diluted 1:100 in blocking solution overnight at 4 °C.

Techniques: Expressing

Journal: Molecular Autism

Article Title: The eIF4E homolog 4EHP (eIF4E2) regulates hippocampal long-term depression and impacts social behavior

doi: 10.1186/s13229-020-00394-7

Figure Lengend Snippet: Heterozygous deletion of Gigyf2 , Eif4e2 , or both in mice does not result in ASD-like behavioral deficits. a , e and i The amount of time the test mouse of the specified genotype spent sniffing either S1 or E. b , f , j The amount of time the test mouse of the specified genotype spent sniffing either S1 or S2. d, h , l The number of marbles buried by the specified genotypes in 20 min. c , g , k Distance travelled over time during the 10 min habituation phase of the three-chamber social interaction test by the specified genotypes. Data are presented as mean ± s.e.m.; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS not significant; calculated by unpaired t-test or 2-way ANOVA with Bonferroni multiple comparisons test. Sample size is located within bar graphs

Article Snippet: Sections were then incubated in the following primary antibodies: eIF4E2 (sc-100731, Santa Cruz), EMX1 (PA5-35373, Thermo), PVALB (195004, Synaptic System), Somatostatin 28 (ab111912, Abcam), Laminin (L9393, Sigma), diluted 1:100 in blocking solution overnight at 4 °C.

Techniques:

Journal: eLife

Article Title: Ribosome collisions trigger cis-acting feedback inhibition of translation initiation

doi: 10.7554/eLife.60038

Figure Lengend Snippet: ( A ) Schematic representation of the reporter construct containing GFP followed by a viral 2A sequence and either the (K AAA ) 21 stall sequence or a ( K ) 0 control insert that does not stall. ( B ) Flp-In T-REx 293 cells containing the indicated reporter construct stably integrated at a doxycycline-inducible site were treated for 5 days with either non-targeting control siRNAs (gray shaded) or siRNAs targeting GIGYF2 (red traces). The reporter construct was then induced with doxycycline for 20 hr prior to analysis by flow cytometry. GFP-PrP was used as an irrelevant fluorescent protein control to exclude non-specific effects on translation. ( C–E ) Flp-In T-REx 293 cells with stably intergrated (K AAA ) 21 reporter were treated with siRNAs targeting GIGYF2 (red traces), 4EHP (blue traces) or control (gray shaded) as in panel A, induced with doxycycline for 20 hr, and analysed by flow cytometry. Panels D and E used cells that were knocked out for GIGYF2 or ZNF598, respectively. GFP expression in all graphs is plotted on a log scale as a histogram and represents around 20,000 cells.

Article Snippet: First, a pcDNA3.1-based plasmid containing Twin-Strep-GIGYF2 was prepared, after which the 4EHP ORF [PCR amplified from a commercially available plasmid (Origene cat. RC201391)] was inserted 5’ of a P2A sequence that preceded GIGYF2.

Techniques: Construct, Sequencing, Stable Transfection, Flow Cytometry, Expressing

Journal: eLife

Article Title: Ribosome collisions trigger cis-acting feedback inhibition of translation initiation

doi: 10.7554/eLife.60038

Figure Lengend Snippet: ( A ) Flp-In T-REx 293 with stably integrated (K AAA ) 21 stalling reporter were treated with siRNAs targeting the indicated genes for 72 hr before induction of the reporter with doxycycline for another 6 hr. Total RNA was isolated from each sample and analysed by quantititative RT-PCR. Two biological replicates, each containing three technical replicates, were analysed using two sets of primers targeting the same transcript. Relative mRNA levels were normalised to the standard curve and against reference genes and plotted for each condition (mean ± SD of four measurements). Levels of mRNA between the control and each of the other samples were compared using the non-parametric Mann-Whitney test and each was found to be non-significant (n.s., indicating a p-value greater than 0.05). ( B ) Total lysates from WT or ∆GIGYF2 cells treated for 5 days with siRNA targeting GIGYF2, 4EHP and control siRNA were analysed by western blotting for the expression of indicated proteins.

Article Snippet: First, a pcDNA3.1-based plasmid containing Twin-Strep-GIGYF2 was prepared, after which the 4EHP ORF [PCR amplified from a commercially available plasmid (Origene cat. RC201391)] was inserted 5’ of a P2A sequence that preceded GIGYF2.

Techniques: Stable Transfection, Isolation, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY, Western Blot, Expressing

Journal: eLife

Article Title: Ribosome collisions trigger cis-acting feedback inhibition of translation initiation

doi: 10.7554/eLife.60038

Figure Lengend Snippet: ( A ) WT HEK293 cells were transfected with constructs encoding indicated proteins (Z- ZNF598-3xFLAG; G - 4EHP-P2A-TST-GIGYF2; E - EDF1-GFP) and allowed to express for 20 hr. Cytosol was depleted of ribosomes by sedimentation and subjected to immunoprecipitation (IP) of 3xFLAG-ZNF598 (left panel) or EDF1-GFP (right panel). When indicated, transfected cells were additionally pre-treated with 1.8 μM emetine for 30 min prior to lysis. Total lysate as well as eluates after IP were analysed by western blotting. The stained blot verifies that the immunoprecipitated proteins were recovered. The IP lanes for each antigen (and the stained blot) are from the same gel and exposure as the lysate lanes, with intervening lanes removed digitally. The relative amounts of lysate and IP samples are indicated on the figure. ( B ) Cells lacking EDF1 (∆EDF1) or GIGYF2 (∆GIGYF2) were transfected with indicated plasmids and analysed as in panel A. ( C ) WT (top panel) or ∆EDF1 (bottom panel) cells were transfected with ZNF598-3xFLAG and 4EHP-P2A-TST-GIGYF2 encoding plasmids. After 20 hr of expression and, when indicated, pre-treatment with 1.8 μM emetine, cell lysates were fractionated on a sucrose gradient. Ribosome free fractions 1–4 and heavy polysome fractions 7–10 were pooled and each was analysed by FLAG-IP and western blotting. ( D ) WT or ∆EDF1 cells were transfected with indicated plasmids and IP was performed as in panel B. Note that EDF1 tagged with 3xFLAG does not recover GIGYF2 (lane 2), unlike ZNF598 containing the same tag (lanes 4 and 6). ( E ) ZNF598-GIGYF2-4EHP form a complex independent of the ribosome. This complex does not include or require EDF1. However, both EDF1 and the ZNF598-GIGYF2-4EHP engage collided ribosomes. Without EDF1, the ZNF598-GIGYF2-4EHP complex is less stably bound to collided ribosomes.

Article Snippet: First, a pcDNA3.1-based plasmid containing Twin-Strep-GIGYF2 was prepared, after which the 4EHP ORF [PCR amplified from a commercially available plasmid (Origene cat. RC201391)] was inserted 5’ of a P2A sequence that preceded GIGYF2.

Techniques: Transfection, Construct, Sedimentation, Immunoprecipitation, Lysis, Western Blot, Staining, Expressing, Stable Transfection

Journal: eLife

Article Title: Ribosome collisions trigger cis-acting feedback inhibition of translation initiation

doi: 10.7554/eLife.60038

Figure Lengend Snippet: WT (upper panel), ∆GIGYF2 (middle panel) or ∆ZNF598 (bottom panel) cells with stably integrated (K AAA ) 21 reporter were co-transfected with either ZNF598 or 4EHP-P2A-GIGYF2 together with BFP encoding plasmids. After 8 hr of recombinant protein expression, reporter expression was induced for another 15 hr and cells were analysed by flow cytometry. Cells with high expression of BFP (i.e., transfected cells) were gated and analysed for the expression of GFP (left panels), RFP (middle panels) and the RFP:GFP ratio (right panels).

Article Snippet: First, a pcDNA3.1-based plasmid containing Twin-Strep-GIGYF2 was prepared, after which the 4EHP ORF [PCR amplified from a commercially available plasmid (Origene cat. RC201391)] was inserted 5’ of a P2A sequence that preceded GIGYF2.

Techniques: Stable Transfection, Transfection, Recombinant, Expressing, Flow Cytometry

Journal: eLife

Article Title: Ribosome collisions trigger cis-acting feedback inhibition of translation initiation

doi: 10.7554/eLife.60038

Figure Lengend Snippet: When two ribosomes collide, EDF1 is most likely to engage the collision first due to its high abundance. Because downstream responses require recruitment of additional factors, no action is taken if the collision resolves quickly by elongation of the lead ribosome. This allows for random bumping of ribosomes on a crowded message. However, if resolution is not immediate, EDF1 stabilises the ZNF598-GIGYF2-4EHP complex. 4EHP inhibits initiation via sequestration of the 5’ cap to avoid further increase in ribosome density. This inhibition is relieved in one of two ways. If the collision is incidental (left limb), as might occur at sites of physiologic ribosome pausing, elongation resumes through the action of eEF1A in complex with aminoacyl-tRNA (aa-tRNA). If the collision is persistent (right limb), as would occur at sites of pathologic stalling, ZNF598 ubiquitinates the ribosome, which signals the ASC-1 complex (ASCC) to disassemble the lead ribosome. The 60S complex of the disassembled lead ribosome engages the ribosome quality control (RQC) complex. Because the stalled ribosome and ensuing collision has been resolved, elongation of trailing ribosomes resumes, causing dissociation of EDF1 and the ZNF598-GIGYF2-4EHP complex. Loss of 4EHP permits initiation to resume. Ub is ubiquitin.

Article Snippet: First, a pcDNA3.1-based plasmid containing Twin-Strep-GIGYF2 was prepared, after which the 4EHP ORF [PCR amplified from a commercially available plasmid (Origene cat. RC201391)] was inserted 5’ of a P2A sequence that preceded GIGYF2.

Techniques: Inhibition

Journal: eLife

Article Title: Ribosome collisions trigger cis-acting feedback inhibition of translation initiation

doi: 10.7554/eLife.60038

Figure Lengend Snippet:

Article Snippet: First, a pcDNA3.1-based plasmid containing Twin-Strep-GIGYF2 was prepared, after which the 4EHP ORF [PCR amplified from a commercially available plasmid (Origene cat. RC201391)] was inserted 5’ of a P2A sequence that preceded GIGYF2.

Techniques: CRISPR, Selection, Recombinant, Expressing, Mutagenesis, In Vitro, Sequencing, Purification, Software, Protease Inhibitor

Journal: iScience

Article Title: Pseudouridine synthase 1 regulates erythropoiesis via transfer RNAs pseudouridylation and cytoplasmic translation

doi: 10.1016/j.isci.2024.109265

Figure Lengend Snippet:

Article Snippet: EIF4E2 Polyclonal Antibody , ABclonal , Cat# A4305; RRID: AB_2765612.

Techniques: Recombinant, Virus, SYBR Green Assay, Cell Culture, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Isolation, Iron Assay, Activity Assay, Dehydrogenase Assay, Cytochrome c Oxidase Assay, Viability Assay, RNA Sequencing, Transgenic Assay, Primer Extension Assay, Software

Journal: Plant Biotechnology Journal

Article Title: Edited eukaryotic translation initiation factors confer resistance against maize lethal necrosis

doi: 10.1111/pbi.14472

Figure Lengend Snippet: Effect of knocking out eif4e genes on resistance against maize lethal necrosis (MLN) in an elite maize line and Mini Maize. (a) Response of CKL05022 knocked out ( KO ) for eif4e1 to MLN 45 days after inoculation (dai). (b) Response of Mini Maize with eif4e1‐KO or eif4e2‐KO to MLN (40 dai). (c) SCMV and MCMV quantification by ELISA in MLN‐inoculated Mini Maize plants. (d) Ears of self‐pollinated Mini Maize.

Article Snippet: For overexpression of the full‐length eIF4E2 gene, a construct containing a full‐length eIF4E2 cDNA corresponding to the CML536 gene was synthesised (GenScript) and transformed into the eif4e1‐KO Mini Maize under the control of the ZmUbi1‐intron promoter.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Plant Biotechnology Journal

Article Title: Edited eukaryotic translation initiation factors confer resistance against maize lethal necrosis

doi: 10.1111/pbi.14472

Figure Lengend Snippet: Sequence comparison of Mini Maize eIF4E2 against CML536 and CKL05022. (a) Comparison of genomic sequence of eIF4E2 among Mini Maize, CKL05022 and CML536. The deletion across the intron/exon junction in Mini Maize is marked by dashes. The regulatory motif for RNA splicing between the 3rd intron and 4th exon is highlighted in blue and purple. (b) RT‐PCR of eIF4E2 . (c) Sanger sequences of eIF4E2 cDNA with the missing nucleotides in Mini Maize marked by dashes.

Article Snippet: For overexpression of the full‐length eIF4E2 gene, a construct containing a full‐length eIF4E2 cDNA corresponding to the CML536 gene was synthesised (GenScript) and transformed into the eif4e1‐KO Mini Maize under the control of the ZmUbi1‐intron promoter.

Techniques: Sequencing, Comparison, Reverse Transcription Polymerase Chain Reaction

Journal: Plant Biotechnology Journal

Article Title: Edited eukaryotic translation initiation factors confer resistance against maize lethal necrosis

doi: 10.1111/pbi.14472

Figure Lengend Snippet: Effect of overexpression of full‐length (FL) eIF4E2 cDNA from CML536 in Mini Maize knocked‐out for eif4e1 on susceptibility to MLN. (a) Response to MLN 40 days after inoculation of wild‐type, eif4e1‐KO and FL‐ eIF4E2 cDNA overexpressing (OX) events. (b) RT‐PCR of young leaf tissue from wild‐type Mini Maize with eif4e1‐KO and three eIF4E2 (OX) events. The absence of the native, truncated cDNA in the overexpressing events suggests disproportionate expression of the full‐length cDNA under the control of an ectopic promoter. (c) Comparison of Sanger sequence of eIF4E2 cDNA from wild‐type Mini Maize with eif4e1‐KO and an event overexpressing the full‐length cDNA from CML536.

Article Snippet: For overexpression of the full‐length eIF4E2 gene, a construct containing a full‐length eIF4E2 cDNA corresponding to the CML536 gene was synthesised (GenScript) and transformed into the eif4e1‐KO Mini Maize under the control of the ZmUbi1‐intron promoter.

Techniques: Over Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Comparison, Sequencing

Journal: Plant Biotechnology Journal

Article Title: Edited eukaryotic translation initiation factors confer resistance against maize lethal necrosis

doi: 10.1111/pbi.14472

Figure Lengend Snippet: Sequences of the edited alleles of eIF4E2 in CKL05022 pre‐edited for eif4e1‐KO. (a) Sequences of the edited exon 4 from eif4e1‐KO/eIF4E2‐exon‐4ED double edits. A vertical dotted line is drawn at the intron/exon junction. (b) Sanger sequencing of the cDNA of eIF4E2‐exon‐4 edited events from (a). These double‐edited events were generated by transforming the eIF4E2‐exon‐4 guide RNA into the construct‐free CKL05022 eif4e1‐KO event from Figure . An insertion caused a shift in the open reading frame starting with amino acid 185 (event 1), an in‐frame deletion (marked with red dashes) shortened the ORF by four amino acids (event 2) and an insertion of 27 nucleotides, 18 of which were identical to the gene itself starting at 10 nucleotides downstream of the insertion site (green font), shifted the frame starting with the amino acid 185 (event 3). In events 1 and 3, the ORF was prematurely terminated because of the stop codons that resulted from the frameshift (asterisks in black background).

Article Snippet: For overexpression of the full‐length eIF4E2 gene, a construct containing a full‐length eIF4E2 cDNA corresponding to the CML536 gene was synthesised (GenScript) and transformed into the eif4e1‐KO Mini Maize under the control of the ZmUbi1‐intron promoter.

Techniques: Sequencing, Generated, Construct

Journal: Plant Biotechnology Journal

Article Title: Edited eukaryotic translation initiation factors confer resistance against maize lethal necrosis

doi: 10.1111/pbi.14472

Figure Lengend Snippet: Effect of double edits of eif4e1‐KO and eIF4E2‐exon‐4ED on MLN resistance in the elite CIMMYT inbred line CKL05022. ED , edited. (a) Response of eif4e1‐KO/eIF4E2‐exon‐4ED double edits to MLN 45 days after inoculation. (b) Self‐pollinated ears of eif4e1‐KO/eIF4E2‐exon‐4ED double edits and control plants. (c) Quantification of SCMV and MCMV 20 days after MLN inoculation. ED, edits 3' of the beginning of the 4th exon.

Article Snippet: For overexpression of the full‐length eIF4E2 gene, a construct containing a full‐length eIF4E2 cDNA corresponding to the CML536 gene was synthesised (GenScript) and transformed into the eif4e1‐KO Mini Maize under the control of the ZmUbi1‐intron promoter.

Techniques: Control

Journal: Plant Biotechnology Journal

Article Title: Edited eukaryotic translation initiation factors confer resistance against maize lethal necrosis

doi: 10.1111/pbi.14472

Figure Lengend Snippet: Effect of single ( eif4e‐KO ) or double ( eif4e1‐KO/eIF4E2 ‐ exon‐4ED) gene edits on response to MLN in CML536. The double‐edited events were generated by transforming the eIF4E2‐exon‐4 guide into a construct‐free CML536‐ eif4e1‐KO event from Figure . Events 1 and 2 in double edits were single‐base indel mutants, shifting the frame starting with the 3rd amino acid of the 4th exon. Event 3 was an in‐frame mutant with the first six amino acids corresponding to the 4th exon deleted (see Figure for details). All three events were equally resistant to MLN.

Article Snippet: For overexpression of the full‐length eIF4E2 gene, a construct containing a full‐length eIF4E2 cDNA corresponding to the CML536 gene was synthesised (GenScript) and transformed into the eif4e1‐KO Mini Maize under the control of the ZmUbi1‐intron promoter.

Techniques: Generated, Construct, Mutagenesis

Journal: Plant Biotechnology Journal

Article Title: Edited eukaryotic translation initiation factors confer resistance against maize lethal necrosis

doi: 10.1111/pbi.14472

Figure Lengend Snippet: Wild‐type eIF4E2 protein and its natural and edited variants in maize (a), and the effect of different combinations of eif4e1‐KO and eIF4E2ED variants on viral replication and plant growth. KO, knockout; X, no growth; − no host; √, normal growth. The numbers in the respective horizonal bars are for the amino acids. (a) The wild‐type eIF4E2 in maize is 220 amino acids long. A naturally occurring variant in Mini Maize has an in‐frame deletion of 22 amino acids corresponding to the 4th exon. Some of the edited variants in eIF4E2 from an elite line, CKL05022, are shorter by nearly the entire stretch of C‐terminal 38 amino acids. (b) Effect of combinations of a knocked‐out eIF4E1 (218 amino acids long) and different variants of eIF4E2 on MLN resistance. Aside from the naturally occurring variant of eIF4E2 in Mini Maize, any frameshift mutation in the 4th exon resulted in complete MLN resistance. In addition to frameshift mutants, we obtained at least two in‐frame mutants that lacked either the first four amino acids (181–184) in CKL05022 (Figure , event 2) or the first six amino acids (183‐188) in CML536 (Figure , event 3) corresponding to the 4th exon that were completely resistant to MLN. The C‐terminal 38 amino acids appear to constitute a domain that is required for recognition by the viruses to translate their proteins but is not necessary for the translation of the maize proteins. The two eIF4E proteins differ only at two positions in the C‐terminal 38‐aa stretch (Figure ). Designing a single guide RNA around the junction of the 3rd intron/4th exon for both the eIF4E1 and eIF4E2 genes would expedite the introduction of MLN resistance in susceptible elite lines. These findings could be extrapolated to other plant species to develop broad‐spectrum resistance against viruses.

Article Snippet: For overexpression of the full‐length eIF4E2 gene, a construct containing a full‐length eIF4E2 cDNA corresponding to the CML536 gene was synthesised (GenScript) and transformed into the eif4e1‐KO Mini Maize under the control of the ZmUbi1‐intron promoter.

Techniques: Knock-Out, Variant Assay, Mutagenesis