eif4a3  (Proteintech)

Journal: Genes & Development

Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons

doi: 10.1101/gad.353081.125

Figure Lengend Snippet: EIF4A3 depletion causes widespread skipping of neighboring exons. ( A ) Western blot showing EIF4A3 and GAPDH levels in SMASh-EIF4A3 HEK293T cells treated with DMSO or danoprevir for 24 h. ( B ) Number of altered splicing events by type after EIF4A3 depletion. ( C ) ΔPSI ( X -axis) versus event probability ( Y -axis; Whippet score) for cassette exons; color reflects local point density (2D Gaussian KDE). Threshold: probability ≥0.9 and ΔPSI ≥10% (mean of three biological replicates). ( D ) Histogram of differentially spliced exons per gene. ( E ) ΔPSI correlation of significantly altered exons from the same gene; the heat map shows point density. ( F ) Same as E but with randomly paired exons. ( G ) Bar chart showing relative positions of similarly affected exons (ΔPSI difference ≤0.02). (Yellow) adjacent, (orange) distal.

Article Snippet: To test EIF4A3 depletion or overexpression, antibodies against EIF4A3 (Proteintech 677401) and GAPDH (Abcam 9485) were used at 1:1000 overnight at 4°C.

Techniques: Western Blot

Journal: Genes & Development

Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons

doi: 10.1101/gad.353081.125

Figure Lengend Snippet: Coordinated inclusion of neighboring exons requires multiple EJC components. ( A ) Western blot showing EIF4A3 overexpression relative to GAPDH in HEK293 Flp-In T-REx cells after induction with doxycycline for 48 h. ( B ) ΔPSI ( X -axis) versus event probability ( Y -axis; Whippet score) for cassette exons upon EIF4A3 overexpression; color reflects local point density (2D Gaussian KDE). Threshold: probability ≥0.9 and ΔPSI ≥10% (mean of three replicates). ( C ) Histogram of differentially spliced exons per gene in siRNA knockdowns of EJC components in HeLa cells. ( D ) ΔPSI correlation of significantly altered exons from the same gene; the heat map shows point density.

Article Snippet: To test EIF4A3 depletion or overexpression, antibodies against EIF4A3 (Proteintech 677401) and GAPDH (Abcam 9485) were used at 1:1000 overnight at 4°C.

Techniques: Western Blot, Over Expression

Journal: Genes & Development

Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons

doi: 10.1101/gad.353081.125

Figure Lengend Snippet: The EJC is required for inclusion of exon blocks at the single transcript level. ( A ) Genome browser shot of individual long reads of mRNAs mapped to the DDX56 gene, showing control ( left ) and EIF4A3 depletion ( right ). Reference (ref.) gene is at the top , with coordinated block exons in orange. Reads are proportionally downsampled to 50 reads (total number of reads is indicated below the reads), with reads where exon blocks are skipped in orange and all other reads in green. The percentage of block-skipping reads is indicated at the right of reads. ( B ) Like A but for the FUS gene. ( C ) Schematic of block-skipping junctions, with the coordinated exon block in yellow (internal introns in orange) and surrounding exons in light blue. ( D ) Violin box plot illustrating the distribution of reads per million (RPM) spanning block-skipping junctions in control and EIF4A3 depletion conditions in mRNA long-read sequencing data sets (mean of three biological replicates). Values >60 were capped to avoid extreme outliers distorting the visualization.

Article Snippet: To test EIF4A3 depletion or overexpression, antibodies against EIF4A3 (Proteintech 677401) and GAPDH (Abcam 9485) were used at 1:1000 overnight at 4°C.

Techniques: Control, Blocking Assay, Sequencing

Journal: Genes & Development

Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons

doi: 10.1101/gad.353081.125

Figure Lengend Snippet: EJC-dependent block exon inclusion occurs cotranscriptionally. ( A ) Illustration of the cell fractionation steps yielding nascent RNA (nRNA). ( B ) Western blot showing a successful cell fractionation, with tubulin as a cytoplasmic marker and RNA polymerase II (Pol 2) as a chromatin marker. ( C ) Schematic of block-skipping junctions. ( D ) Sashimi plot of nRNA short reads (green) aligned to the ACACA gene. The block exon region in yellow–orange is indicated in the reference gene below . Block-skipping junction read coverage is highlighted in yellow. ( E ) Like D but for the DDX56 gene. ( F ) Violin plot showing normalized read count in reads per million (RPM) in nRNA short reads spanning block-skipping junctions in control and EIF4A3 depletion conditions for three biological replicates (rep). ( G ) Scatter plot showing correlation of RPM for block exon-skipping junctions in mRNA versus nRNA short-read sequencing data sets. Pearson correlation and P -value are indicated in the top left of the plot.

Article Snippet: To test EIF4A3 depletion or overexpression, antibodies against EIF4A3 (Proteintech 677401) and GAPDH (Abcam 9485) were used at 1:1000 overnight at 4°C.

Techniques: Blocking Assay, Cell Fractionation, Western Blot, Marker, Control, Sequencing

Journal: Genes & Development

Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons

doi: 10.1101/gad.353081.125

Figure Lengend Snippet: Introns within exon blocks are spliced out before the whole block is spliced in. ( A ) ΔSPI ( X -axis) versus P -value ( Y -axis); color reflects local point density (2D Gaussian KDE). Threshold: P -value ≤0.01 and ΔSPI ≥10% (mean of three replicates). ( B ) Normalized frequency histogram of SPI values for block internal introns (orange), block-flanking introns (dark green), and other introns (gray) in control ( left ) and EIF4A3 depletion ( right ) conditions. ( C ) Splicing order of introns surrounding the first (orange) and last (green) exons of coordinated exon blocks in control cells. The Y -axis is probability density (kernel density estimate), and the X -axis is fraction of upstream intron spliced first ( F UPFI ).

Article Snippet: To test EIF4A3 depletion or overexpression, antibodies against EIF4A3 (Proteintech 677401) and GAPDH (Abcam 9485) were used at 1:1000 overnight at 4°C.

Techniques: Blocking Assay, Control

Journal: Genes & Development

Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons

doi: 10.1101/gad.353081.125

Figure Lengend Snippet: EIF4A3 depletion causes widespread skipping of neighboring exons. ( A ) Western blot showing EIF4A3 and GAPDH levels in SMASh-EIF4A3 HEK293T cells treated with DMSO or danoprevir for 24 h. ( B ) Number of altered splicing events by type after EIF4A3 depletion. ( C ) ΔPSI ( X -axis) versus event probability ( Y -axis; Whippet score) for cassette exons; color reflects local point density (2D Gaussian KDE). Threshold: probability ≥0.9 and ΔPSI ≥10% (mean of three biological replicates). ( D ) Histogram of differentially spliced exons per gene. ( E ) ΔPSI correlation of significantly altered exons from the same gene; the heat map shows point density. ( F ) Same as E but with randomly paired exons. ( G ) Bar chart showing relative positions of similarly affected exons (ΔPSI difference ≤0.02). (Yellow) adjacent, (orange) distal.

Article Snippet: To test EIF4A3 depletion or overexpression, antibodies against EIF4A3 (Proteintech 677401) and GAPDH (Abcam 9485) were used at 1:1000 overnight at 4°C.

Techniques: Western Blot

Journal: Genes & Development

Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons

doi: 10.1101/gad.353081.125

Figure Lengend Snippet: Coordinated inclusion of neighboring exons requires multiple EJC components. ( A ) Western blot showing EIF4A3 overexpression relative to GAPDH in HEK293 Flp-In T-REx cells after induction with doxycycline for 48 h. ( B ) ΔPSI ( X -axis) versus event probability ( Y -axis; Whippet score) for cassette exons upon EIF4A3 overexpression; color reflects local point density (2D Gaussian KDE). Threshold: probability ≥0.9 and ΔPSI ≥10% (mean of three replicates). ( C ) Histogram of differentially spliced exons per gene in siRNA knockdowns of EJC components in HeLa cells. ( D ) ΔPSI correlation of significantly altered exons from the same gene; the heat map shows point density.

Article Snippet: To test EIF4A3 depletion or overexpression, antibodies against EIF4A3 (Proteintech 677401) and GAPDH (Abcam 9485) were used at 1:1000 overnight at 4°C.

Techniques: Western Blot, Over Expression

Journal: Genes & Development

Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons

doi: 10.1101/gad.353081.125

Figure Lengend Snippet: The EJC is required for inclusion of exon blocks at the single transcript level. ( A ) Genome browser shot of individual long reads of mRNAs mapped to the DDX56 gene, showing control ( left ) and EIF4A3 depletion ( right ). Reference (ref.) gene is at the top , with coordinated block exons in orange. Reads are proportionally downsampled to 50 reads (total number of reads is indicated below the reads), with reads where exon blocks are skipped in orange and all other reads in green. The percentage of block-skipping reads is indicated at the right of reads. ( B ) Like A but for the FUS gene. ( C ) Schematic of block-skipping junctions, with the coordinated exon block in yellow (internal introns in orange) and surrounding exons in light blue. ( D ) Violin box plot illustrating the distribution of reads per million (RPM) spanning block-skipping junctions in control and EIF4A3 depletion conditions in mRNA long-read sequencing data sets (mean of three biological replicates). Values >60 were capped to avoid extreme outliers distorting the visualization.

Article Snippet: To test EIF4A3 depletion or overexpression, antibodies against EIF4A3 (Proteintech 677401) and GAPDH (Abcam 9485) were used at 1:1000 overnight at 4°C.

Techniques: Control, Blocking Assay, Sequencing

Journal: Genes & Development

Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons

doi: 10.1101/gad.353081.125

Figure Lengend Snippet: EJC-dependent block exon inclusion occurs cotranscriptionally. ( A ) Illustration of the cell fractionation steps yielding nascent RNA (nRNA). ( B ) Western blot showing a successful cell fractionation, with tubulin as a cytoplasmic marker and RNA polymerase II (Pol 2) as a chromatin marker. ( C ) Schematic of block-skipping junctions. ( D ) Sashimi plot of nRNA short reads (green) aligned to the ACACA gene. The block exon region in yellow–orange is indicated in the reference gene below . Block-skipping junction read coverage is highlighted in yellow. ( E ) Like D but for the DDX56 gene. ( F ) Violin plot showing normalized read count in reads per million (RPM) in nRNA short reads spanning block-skipping junctions in control and EIF4A3 depletion conditions for three biological replicates (rep). ( G ) Scatter plot showing correlation of RPM for block exon-skipping junctions in mRNA versus nRNA short-read sequencing data sets. Pearson correlation and P -value are indicated in the top left of the plot.

Article Snippet: To test EIF4A3 depletion or overexpression, antibodies against EIF4A3 (Proteintech 677401) and GAPDH (Abcam 9485) were used at 1:1000 overnight at 4°C.

Techniques: Blocking Assay, Cell Fractionation, Western Blot, Marker, Control, Sequencing

Journal: Genes & Development

Article Title: The exon junction complex coordinates the cotranscriptional inclusion of blocks of neighboring exons

doi: 10.1101/gad.353081.125

Figure Lengend Snippet: Introns within exon blocks are spliced out before the whole block is spliced in. ( A ) ΔSPI ( X -axis) versus P -value ( Y -axis); color reflects local point density (2D Gaussian KDE). Threshold: P -value ≤0.01 and ΔSPI ≥10% (mean of three replicates). ( B ) Normalized frequency histogram of SPI values for block internal introns (orange), block-flanking introns (dark green), and other introns (gray) in control ( left ) and EIF4A3 depletion ( right ) conditions. ( C ) Splicing order of introns surrounding the first (orange) and last (green) exons of coordinated exon blocks in control cells. The Y -axis is probability density (kernel density estimate), and the X -axis is fraction of upstream intron spliced first ( F UPFI ).

Article Snippet: To test EIF4A3 depletion or overexpression, antibodies against EIF4A3 (Proteintech 677401) and GAPDH (Abcam 9485) were used at 1:1000 overnight at 4°C.

Techniques: Blocking Assay, Control