Journal: Plant Communications

Article Title: Selective recognition of PTRE1 transcripts mediated by protein–protein interaction between the m 6 A reader ECT2 and PTRE1

doi: 10.1016/j.xplc.2024.101043

Figure Lengend Snippet: ECT2 physically interacts with PTRE1 and 20S subunits. (A) Interaction between ECT2 and PTRE1 demonstrated by a split-ubiquitin yeast two-hybrid assay. ECT2 and PTRE1 were fused to the C-terminal (Cub) and N-terminal (NubG) halves of ubiquitin, respectively. Negative controls included the large T antigen (LargeT) and the N-terminally truncated p53 (Δp53). (B) Visualization of the ECT2–PTRE1 interaction through confocal imaging in a BiFC assay conducted by co-expressing ECT2-cEYFP and PTRE1-nEYFP in N. benthamiana leaves. Scale bar, 50 μm. (C) A volcano plot illustrating the distribution of proteins identified by IP–MS, with infinity was set as FC = 25,000, comparing ECT2-mCherry to mCherry . Proteins showing significant upregulation (fold change > 1.5 and p value ≤ 0.05) are highlighted in yellow, whereas non-significant changes are depicted in gray. PTRE1, 20S subunits, ECT2, and mCherry are marked in red. (D) Interactions of ECT2 with various 20S proteasome subunits demonstrated through a split-ubiquitin yeast two-hybrid assay. The large T antigen (LargeT) served as a negative control.

Article Snippet: PTRE1 was detected using custom-made anti-PTRE1 antibodies (Genscript) raised in rabbits against a PTRE1-specific peptide CTRQPPRGPGGGHP. mCherry-tagged ECT2 was detected using commercial mouse monoclonal RFP antibodies (6G6, Chromotek).

Techniques: Ubiquitin Proteomics, Y2H Assay, Imaging, Bimolecular Fluorescence Complementation Assay, Expressing, Protein-Protein interactions, Negative Control

Journal: Plant Communications

Article Title: Selective recognition of PTRE1 transcripts mediated by protein–protein interaction between the m 6 A reader ECT2 and PTRE1

doi: 10.1016/j.xplc.2024.101043

Figure Lengend Snippet: The middle region of ECT2 affect m 6 A mRNA binding. (A) A volcano plot illustrating the distribution of proteins identified by IP–MS, comparing ECT2-mCherry to ECT2Δ156-413-mCherry. Proteins exhibiting significant upregulation (fold change > 1.5 and p value ≤ 0.05) are highlighted in yellow, those downregulated are marked in purple, and those with non-significant changes are shown in gray. PTRE1, 20S subunits, ECT2, and mCherry are marked in red. (B and D) Gene functional clustering analysis of ECT2-IP targets using the DAVID Gene Functional Classification Tool ( https://david.ncifcrf.gov/ ). This analysis specifically focuses on the cellular component (CC) category (GOTERM_CC). Each node represents an enriched GO term, with node size proportional to the total number of genes associated with each term. Detailed lists of target genes for (B) and (D) are provided in Supplemental Tables 8 and , respectively. (C) A Venn diagram depicting the overlap of ECT2-enriched proteins between ECT2-mCherry and ECT2Δ156-413-mCherry compared to free mCherry. Refer to Supplemental Table 9 for more details. (E) Reduction of ECT2 RNA-binding ability in the 35S::ECT2Δ156-413-mCherry transgenic plants. (F) Different binding affinities of ECT2 and ECT2Δ156-413 to PTRE1 revealed by the FA–RIP–qRT–PCR analysis. AT2G07689 served as the internal control gene. Error bars represent s.d. ( n = 3).

Article Snippet: PTRE1 was detected using custom-made anti-PTRE1 antibodies (Genscript) raised in rabbits against a PTRE1-specific peptide CTRQPPRGPGGGHP. mCherry-tagged ECT2 was detected using commercial mouse monoclonal RFP antibodies (6G6, Chromotek).

Techniques: Binding Assay, Protein-Protein interactions, Functional Assay, RNA Binding Assay, Transgenic Assay, Quantitative RT-PCR, Control

Journal: Plant Communications

Article Title: Selective recognition of PTRE1 transcripts mediated by protein–protein interaction between the m 6 A reader ECT2 and PTRE1

doi: 10.1016/j.xplc.2024.101043

Figure Lengend Snippet: m 6 A-binding site of ECT2 is not essential for PTRE1 binding, and the interaction occurs on ribosomes. (A) A split-ubiquitin yeast two-hybrid assay showing that the m 6 A-binding site mutant form of ECT2 (ECT2M) retains the ability to interact with PTRE1. (B) Confocal imaging of a BiFC assay involving co-expression of ECT2M-cEYFP and PTRE1-nEYFP in N. benthamiana leaves. Scale bar, 50μm. (C) Confocal imaging of a BiFC assay involving co-expression of ECT2-cEYFP, PTRE1-nEYFP, and either the ribosomal marker eS21B-mCherry or uS5B-mCherry in N. benthamiana leaves. Yellow fluorescence in the top panel indicates the interaction between ECT2-cEYFP and PTRE1-nEYFP. Red fluorescence in the middle panel indicates the ribosomal markers. The co-localization of these signals is shown in the merged image (bottom panel). Scale bar, 5 μm. (D) Co-localization (bottom panel) of the yellow fluorescence from the interaction between ECT2M-cEYFP and PTRE1-nEYFP (top panel) and the red fluorescence from ribosomal markers (middle panel). Images were taken 1 day after transformation. Scale bar, 5μm.

Article Snippet: PTRE1 was detected using custom-made anti-PTRE1 antibodies (Genscript) raised in rabbits against a PTRE1-specific peptide CTRQPPRGPGGGHP. mCherry-tagged ECT2 was detected using commercial mouse monoclonal RFP antibodies (6G6, Chromotek).

Techniques: Binding Assay, Ubiquitin Proteomics, Y2H Assay, Mutagenesis, Imaging, Bimolecular Fluorescence Complementation Assay, Expressing, Marker, Fluorescence, Transformation Assay

Journal: Plant Communications

Article Title: Selective recognition of PTRE1 transcripts mediated by protein–protein interaction between the m 6 A reader ECT2 and PTRE1

doi: 10.1016/j.xplc.2024.101043

Figure Lengend Snippet: Role of the middle region of ECT2 in mediating protein interactions and regulating PTRE1 transcript. (A) A split-ubiquitin yeast two-hybrid assay showing the interaction between ECT2 and the N-terminal of PTRE1 (amino acids 1–165 out of the 302 full length). This interaction requires the presence of the middle region of ECT2 (amino acids 156–413). The endogenous ER protein Alg5 served as the negative control. (B) Schematic representation of different ECT2 fragments used in yeast two-hybrid assays. The YTH domain (YT521-B-like domain) of ECT2 is highlighted in orange, and the YPQ domain in blue. (C) Plants overexpressing a mutant form of ECT2 ( ECT2Δ156-413) lacking protein–protein interaction capabilities did not exhibit reduced leaf size phenotype observed in plants overexpressing the wild-type ECT2 ( 35S::ECT2-mCherry ). Scale bar, 2 cm. (D) qRT–PCR analysis showing increased accumulation of ECT2 transcripts in the transgenic lines. ACTIN2 served as the reference gene. (E) Increase in PTRE1 transcript levels in plants overexpressing ECT2 but not in those overexpressing interaction-deficient mutant ECT2Δ156-413 . ACTIN2 served as the reference gene. Error bars represent standard deviation (s.d., n = 3). Different letters above the bars indicate statistically significant differences as determined by one-way analysis of variance (ANOVA; Duncan’s multiple range test, p = 0.01).

Article Snippet: PTRE1 was detected using custom-made anti-PTRE1 antibodies (Genscript) raised in rabbits against a PTRE1-specific peptide CTRQPPRGPGGGHP. mCherry-tagged ECT2 was detected using commercial mouse monoclonal RFP antibodies (6G6, Chromotek).

Techniques: Ubiquitin Proteomics, Y2H Assay, Negative Control, Mutagenesis, Quantitative RT-PCR, Transgenic Assay, Standard Deviation

Journal: Plant Communications

Article Title: Selective recognition of PTRE1 transcripts mediated by protein–protein interaction between the m 6 A reader ECT2 and PTRE1

doi: 10.1016/j.xplc.2024.101043

Figure Lengend Snippet: Proposed working model of protein–protein interactions mediating ECT2 target selection. This model demonstrates how ECT2 binds to newly synthesized 20S proteasome subunits and the PTRE1 protein on the ribosome. Protein–protein interactions position ECT2 close to the m 6 A-modified 3′ UTR of the mRNA being translated. This proximity enhances the protein–RNA interaction, which in turn leads to increased mRNA accumulation and enhanced proteasome activity.

Article Snippet: PTRE1 was detected using custom-made anti-PTRE1 antibodies (Genscript) raised in rabbits against a PTRE1-specific peptide CTRQPPRGPGGGHP. mCherry-tagged ECT2 was detected using commercial mouse monoclonal RFP antibodies (6G6, Chromotek).

Techniques: Protein-Protein interactions, Selection, Synthesized, Modification, Activity Assay