Journal: Cell death & disease

Article Title: α1,3-fucosylation of MEST promotes invasion potential of cytotrophoblast cells by activating translation initiation.

doi: 10.1038/s41419-023-06166-4

Figure Lengend Snippet: Fig. 4 α1,3 fucosylated-MEST mediate interaction of MEST and eIF4E2. a Schematic of analysis strategy for identification MEST interacting protein. b GO analysis of the proteins interacted with MEST. c Venn diagrams showing the number of the proteins interact with MEST form NP, MP and GO analysis-based function. d Pull down proteins separated with SDS-PAGE and staining with CBB. e N-glycosylation of MEST at Asn163 enhances the interaction of MEST and eIF4E2. HTR-8/SVneo cells transfected with His-tagged MEST cDNA and His-tagged MEST N163Q mutant plasmid (MEST mut), respectively, then co-immunoprecipitated (co-IP) with anti His antibody and blotted with anti-eIF4E2. f FUT4 regulated interaction between MEST and eIF4E2 by regulating LeY on MEST. HTR-8/SVneo cells transfected with FUT4 cDNA and FUT4 siRNA, respectively, then interaction was detected by co-IP assay. g Confocal immunofluorescence showing the location of MEST and eIF4E2 in cells, lower panel is magnification of up panel of white frame.

Article Snippet: After blocking in 5% non-fat dry milk for 2 h, the membranes were incubated with the primary antibody at 4 °C overnight: FUT4 (19497-1-AP), PCNA (10205-2-AP), Cyclin D1 (26939-1- AP), Cyclin E1 (11554-1-AP), MMP-2 (10373-2-AP), MMP-9 (10375-2-AP), TIMP1 (16644-1-AP), TIMP2 (17353-1-AP), MEST (11118-1-AP), eIF4E2 (12227-1-AP), GIGYF2 (24790-1-AP) and GAPDH (10494-1-AP) (Proteintech, Wuhan, China); 4E-T (sc-134233, Santa Cruz, USA); LeY (ab-3359, Abcam, USA); biotinylated LTL (B-1325-2, Vector Laboratories, California, USA).

Techniques: SDS Page, Staining, Glycoproteomics, Transfection, Mutagenesis, Plasmid Preparation, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Cell death & disease

Article Title: α1,3-fucosylation of MEST promotes invasion potential of cytotrophoblast cells by activating translation initiation.

doi: 10.1038/s41419-023-06166-4

Figure Lengend Snippet: Fig. 5 Genome-wide α1,3 fucosylated MEST preferentially stimulates the translation and expression of implantation relate genes. a Heatmap showing the alternation of mRNAs immunoprecipitated by anti-eIF4E2 antibody in HTR-8/SVneo cells using RIP-seq assay. HTR-8/ SVneo cells transfected with FUT4 siRNA or FUT4 cDNA. b Binding peak density analysis of binding site of mRNA and eIF4E2. c Venn diagrams displays the number of genes in FUT4 cDNA group (blue), FUT siRNA group (yellow), or both (overlapping) group. The changed of 117 overlapping mRNAs is shown in column graphs. d GO analysis of the mRNAs with high affinity to eIF4E2. e Integrative Genomics Viewer (IGV) tracks showing the binding of eIF4E2 at the 5’-UTR regions of the representative genes. f The eIF4E2-MEST/4E-T/GLGYF2 interaction is decreased in HTR-8/SVneo cells transfected with MEST mut, compared with MEST cDNA transfection. eIF4E2 co-IP was performed, and the immunoprecipitated fractions were analyzed by immunoblotting for the indicated proteins. g The eIF4E2-MEST/4E-T/GLGYF2 interaction in HTR-8/SVneo cells transfected with FUT4 cDNA or FUT4 siRNA, compared with scramble group. eIF4E2 co-IP was performed, and the immunoprecipitated fractions were analyzed by immunoblotting for the indicated proteins.

Article Snippet: After blocking in 5% non-fat dry milk for 2 h, the membranes were incubated with the primary antibody at 4 °C overnight: FUT4 (19497-1-AP), PCNA (10205-2-AP), Cyclin D1 (26939-1- AP), Cyclin E1 (11554-1-AP), MMP-2 (10373-2-AP), MMP-9 (10375-2-AP), TIMP1 (16644-1-AP), TIMP2 (17353-1-AP), MEST (11118-1-AP), eIF4E2 (12227-1-AP), GIGYF2 (24790-1-AP) and GAPDH (10494-1-AP) (Proteintech, Wuhan, China); 4E-T (sc-134233, Santa Cruz, USA); LeY (ab-3359, Abcam, USA); biotinylated LTL (B-1325-2, Vector Laboratories, California, USA).

Techniques: Genome Wide, Expressing, Immunoprecipitation, Transfection, Binding Assay, Co-Immunoprecipitation Assay, Western Blot

Journal: Cell death & disease

Article Title: α1,3-fucosylation of MEST promotes invasion potential of cytotrophoblast cells by activating translation initiation.

doi: 10.1038/s41419-023-06166-4

Figure Lengend Snippet: Fig. 8 A summary scheme of LeY on MEST promotes embryonic trophoblast cells migration and invasion by activating translation initiation. The α1,3-fucosylation of glycoproteins in the villi of normal pregnancy and miscarriage was screened by Lectin Chip. Anti-LeY affinity chromatography was used to screen Ley scaffold glycoproteins by anti-LeY antibody. The target MEST was selected, and the specific interaction protein (eIF4E2) was discovered by anti-MEST affinity chromatography. By RIP-seq and polysome profiling, implantation-related mRNAs bound with eIF4E2 were confirmed. Reduced LeY on MEST inhibited binding of eIF4E2 with MEST, migration and invasion of trophoblast cells by inactivating translation initiation.

Article Snippet: After blocking in 5% non-fat dry milk for 2 h, the membranes were incubated with the primary antibody at 4 °C overnight: FUT4 (19497-1-AP), PCNA (10205-2-AP), Cyclin D1 (26939-1- AP), Cyclin E1 (11554-1-AP), MMP-2 (10373-2-AP), MMP-9 (10375-2-AP), TIMP1 (16644-1-AP), TIMP2 (17353-1-AP), MEST (11118-1-AP), eIF4E2 (12227-1-AP), GIGYF2 (24790-1-AP) and GAPDH (10494-1-AP) (Proteintech, Wuhan, China); 4E-T (sc-134233, Santa Cruz, USA); LeY (ab-3359, Abcam, USA); biotinylated LTL (B-1325-2, Vector Laboratories, California, USA).

Techniques: Migration, Chromatography, Binding 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: 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