Journal: bioRxiv

Article Title: Correction of RBFOX1 deficit rescues Huntington’s disease mis-splicing and pathology

doi: 10.1101/2024.11.06.622223

Figure Lengend Snippet: ( A ) Normalized counts of the three RBFOX genes in striatal RNA from 3.5 month-old WT (n=3) and R6/1 mice (n=3) according to RNA-seq datasets in Elorza et al . 15 ( B ) Quantification of Rbfox1 transcript levels by RT-qPCR in striatal RNA from 3.5 month-old WT (n=7) and R6/1 mice (n=7) (Student’s t-test; * P < 0.05). ( C ) Rbfox1 immunohistochemistry in striatum of 3.5 month-old WT and R6/1 mice. ( E ) Representative RBFOX1 immunohistochemistry staining in striatum of control and HD subjects.

Article Snippet: Antibodies: rabbit anti-RBFOX1 (1:5000, Novus, NBP1-90304), rabbit anti-β-Gal (1:2000, Invitrogen, A-11132), rabbit DARPP32 (1:3000, BD, 611520) and rabbit Cleaved caspase-3 (1:100, Cell Signaling, 9661) for mouse samples.

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Immunohistochemistry, Staining, Control

Journal: bioRxiv

Article Title: Correction of RBFOX1 deficit rescues Huntington’s disease mis-splicing and pathology

doi: 10.1101/2024.11.06.622223

Figure Lengend Snippet: ( A ) Mice expressing tTA under control of the CamKII promoter (CamKII-tTA mice) were bred with mice carrying the β-Gal-BiTetO-RBFOX1 or β-Gal-BiTetO-U2AF2 construct to yield TgRBFOX1 (CamKII-tTA:β-Gal-BiTetO-RBFOX1) mice or TgU2AF2 (CamKII-tTA:β-Gal-BiTetO-U2AF2) . ( B ) Immunohistochemistry with anti-RBFOX1 or anti-U2AF2 antibody in sagittal sections from 1.5 month-old WT, TgRBFOX1 or TgU2AF2 mice.

Article Snippet: Antibodies: rabbit anti-RBFOX1 (1:5000, Novus, NBP1-90304), rabbit anti-β-Gal (1:2000, Invitrogen, A-11132), rabbit DARPP32 (1:3000, BD, 611520) and rabbit Cleaved caspase-3 (1:100, Cell Signaling, 9661) for mouse samples.

Techniques: Expressing, Control, Construct, Immunohistochemistry

Journal: bioRxiv

Article Title: Correction of RBFOX1 deficit rescues Huntington’s disease mis-splicing and pathology

doi: 10.1101/2024.11.06.622223

Figure Lengend Snippet: (A) Gel shows RT-PCR amplification of Tg-RBFOX1 or TgU2AF2 mRNA in wild-type, MildTgRBFOX1 or MildTgU2AF2 and StrongTgRBFOX1 or StrongTgU2AF2 mice. (B) Histogram shows brain weight of WT (n=5/n=8), MildTgRBFOX1 (n=7) or MildTgU2AF2 (n=5) and Strong TgRBFOX1 (n=7) or Strong TgU2AF2 (n=6) mice (ANOVA, followed by Tukey’s post hoc test; *P < 0.05;**P < 0.01; ***P < 0.001).

Article Snippet: Antibodies: rabbit anti-RBFOX1 (1:5000, Novus, NBP1-90304), rabbit anti-β-Gal (1:2000, Invitrogen, A-11132), rabbit DARPP32 (1:3000, BD, 611520) and rabbit Cleaved caspase-3 (1:100, Cell Signaling, 9661) for mouse samples.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: bioRxiv

Article Title: Correction of RBFOX1 deficit rescues Huntington’s disease mis-splicing and pathology

doi: 10.1101/2024.11.06.622223

Figure Lengend Snippet: ( A ) Venn diagram showing the 83 genes in the intersection between the 245 genes with exons mis-spliced in Huntington’s disease 15 and the 543 genes with RBFOX-direct target exons 25 ( B ) Venn diagram showing the 76 genes in the intersection between the 245 genes with exons mis-spliced in Huntington’s disease 15 and the 966 genes that are functional targets of Rbfox1 according to Supplementary Table 1. Representation factor (RF) was determined with Two-sided Fisher’s Exact test, using as background genes the human-mouse orthologous genes coincidentally detected in the human and mouse RNA-seq datasets used to define the Huntington’s disease mis-splicing signature 15 ( n = 12,882).

Article Snippet: Antibodies: rabbit anti-RBFOX1 (1:5000, Novus, NBP1-90304), rabbit anti-β-Gal (1:2000, Invitrogen, A-11132), rabbit DARPP32 (1:3000, BD, 611520) and rabbit Cleaved caspase-3 (1:100, Cell Signaling, 9661) for mouse samples.

Techniques: Functional Assay, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Correction of RBFOX1 deficit rescues Huntington’s disease mis-splicing and pathology

doi: 10.1101/2024.11.06.622223

Figure Lengend Snippet: ( A ) Normalized counts of the three RBFOX genes in striatal RNA from 3.5 month-old WT (n=3) and R6/1 mice (n=3) according to RNA-seq datasets in Elorza et al . 15 ( B ) Quantification of Rbfox1 transcript levels by RT-qPCR in striatal RNA from 3.5 month-old WT (n=7) and R6/1 mice (n=7) (Student’s t-test; * P < 0.05). ( C ) Rbfox1 immunohistochemistry in striatum of 3.5 month-old WT and R6/1 mice. ( E ) Representative RBFOX1 immunohistochemistry staining in striatum of control and HD subjects.

Article Snippet: Human RBFOX1 cDNA (from pENTR-A2BP1, Addgene, 16176) or human U2AF2 cDNA (from pCDNA3-U2AF65-DYK, GenScript, OHu17874) were independently cloned into a plasmid containing a bidirectional TetO sequence that also harbors the β-galactosidase (β-Gal) reporter with a NLS (pBI-G, Clontech, 631004).

Techniques: RNA Sequencing, Quantitative RT-PCR, Immunohistochemistry, Staining, Control

Journal: bioRxiv

Article Title: Correction of RBFOX1 deficit rescues Huntington’s disease mis-splicing and pathology

doi: 10.1101/2024.11.06.622223

Figure Lengend Snippet: ( A ) Mice expressing tTA under control of the CamKII promoter (CamKII-tTA mice) were bred with mice carrying the β-Gal-BiTetO-RBFOX1 or β-Gal-BiTetO-U2AF2 construct to yield TgRBFOX1 (CamKII-tTA:β-Gal-BiTetO-RBFOX1) mice or TgU2AF2 (CamKII-tTA:β-Gal-BiTetO-U2AF2) . ( B ) Immunohistochemistry with anti-RBFOX1 or anti-U2AF2 antibody in sagittal sections from 1.5 month-old WT, TgRBFOX1 or TgU2AF2 mice.

Article Snippet: Human RBFOX1 cDNA (from pENTR-A2BP1, Addgene, 16176) or human U2AF2 cDNA (from pCDNA3-U2AF65-DYK, GenScript, OHu17874) were independently cloned into a plasmid containing a bidirectional TetO sequence that also harbors the β-galactosidase (β-Gal) reporter with a NLS (pBI-G, Clontech, 631004).

Techniques: Expressing, Control, Construct, Immunohistochemistry

Journal: bioRxiv

Article Title: Correction of RBFOX1 deficit rescues Huntington’s disease mis-splicing and pathology

doi: 10.1101/2024.11.06.622223

Figure Lengend Snippet: (A) Gel shows RT-PCR amplification of Tg-RBFOX1 or TgU2AF2 mRNA in wild-type, MildTgRBFOX1 or MildTgU2AF2 and StrongTgRBFOX1 or StrongTgU2AF2 mice. (B) Histogram shows brain weight of WT (n=5/n=8), MildTgRBFOX1 (n=7) or MildTgU2AF2 (n=5) and Strong TgRBFOX1 (n=7) or Strong TgU2AF2 (n=6) mice (ANOVA, followed by Tukey’s post hoc test; *P < 0.05;**P < 0.01; ***P < 0.001).

Article Snippet: Human RBFOX1 cDNA (from pENTR-A2BP1, Addgene, 16176) or human U2AF2 cDNA (from pCDNA3-U2AF65-DYK, GenScript, OHu17874) were independently cloned into a plasmid containing a bidirectional TetO sequence that also harbors the β-galactosidase (β-Gal) reporter with a NLS (pBI-G, Clontech, 631004).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: bioRxiv

Article Title: Correction of RBFOX1 deficit rescues Huntington’s disease mis-splicing and pathology

doi: 10.1101/2024.11.06.622223

Figure Lengend Snippet: ( A ) Venn diagram showing the 83 genes in the intersection between the 245 genes with exons mis-spliced in Huntington’s disease 15 and the 543 genes with RBFOX-direct target exons 25 ( B ) Venn diagram showing the 76 genes in the intersection between the 245 genes with exons mis-spliced in Huntington’s disease 15 and the 966 genes that are functional targets of Rbfox1 according to Supplementary Table 1. Representation factor (RF) was determined with Two-sided Fisher’s Exact test, using as background genes the human-mouse orthologous genes coincidentally detected in the human and mouse RNA-seq datasets used to define the Huntington’s disease mis-splicing signature 15 ( n = 12,882).

Article Snippet: Human RBFOX1 cDNA (from pENTR-A2BP1, Addgene, 16176) or human U2AF2 cDNA (from pCDNA3-U2AF65-DYK, GenScript, OHu17874) were independently cloned into a plasmid containing a bidirectional TetO sequence that also harbors the β-galactosidase (β-Gal) reporter with a NLS (pBI-G, Clontech, 631004).

Techniques: Functional Assay, RNA Sequencing

Journal: bioRxiv

Article Title: Cell-type-specific splicing of transcription regulators and Ptbp1 by Rbfox1/2/3 in the developing neocortex

doi: 10.1101/2024.09.09.612108

Figure Lengend Snippet: a. Rbfox3/2/1 and Ptbp1 mRNA levels in bulk RNA-Seq data of E14.5 Eomes:EGFP (-), Eomes:EGFP (+) and Tubb3:EGFP (+) cells. Red-blue represents high-low gene expression. b. Transcription levels of Ptbp1 and Rbfox3/2/1 in E14.5 RGCs, IPCs, immature and mature neurons. Re-analysis of our published scRNA-Seq data. c. Feature plots of E10.5-E18.5 scRNA-Seq data showing Rbfox3 expression in immature neurons and a fraction of Eomes -positive cells. d. RNA ISH results of E14.5 sagittal brain sections showing the expression of Rbfox3 (this study, with a probe against the coding sequence) in comparison to Rbfox1 and Rbfox2 (adapted from Genepaint). Scale bar, 400μm. e. Eomes/Tbr2 immunostaining and Rbfox3 RNA ISH co-labeling showed overlapping signals in the SVZ/IZ. Rbfox3 immunostaining was performed on an adjacent section to the RNA ISH one. Scale bar, 50μm. f. Co-Immunostaining of Rbfox1 and Rbfox2 in the E14.5 dorsal forebrain. CP: cortical plate; VZ: ventricular zone. Scale bar, 100μm. g. Co-immunostaining results of Rbfox3 with Eomes and Ptbp1 in the E14.5 mouse dorsal forebrains. Rbfox3 was expressed in 39% of Eomes-positive cells while overlapped with less than 2.5% of Ptbp1-positive cells. Scale bar, 100μm. h. Co-immunostaining results of Rbfox1 (Sigma-Aldrich HPA040809, rabbit) and Rbfox3 (Millipore MAB377, mouse), or Rbfox1 (Millipore MABE159, mouse) and Rbfox2 (Bethyl A300-864A, rabbit) in the dorsal forebrains of P0 mice. Scale bar, 100μm. See also Figure S3.

Article Snippet: The following primary antibodies were used: anti-Rbfox1 (Millipore MABE159, mouse, 1:800; Sigma-Aldrich HPA040809, rabbit, 1:500), anti-Rbfox2 (Bethyl A300-864A, rabbit, 1:500), anti-Rbfox3/NeuN (Cell Signaling Technology 24307T, rabbit, 1:500; Millipore MAB377, mouse, 1:500), anti-Cux1(Santa Cruz sc-13024, rabbit, 1:1000), anti-Ctip2 (Abcam ab18465, rat, 1:1000), anti-Tuj1 (BioLegend 801201, mouse, 1:1000), anti-GFP (Abcam ab13970, chicken, 1:3000), anti-Flag (Sigma-Aldrich F1804, mouse, 1:500), anti-Ptbp1 (Abcam ab5642, goat, 1:200), anti-Tbr2 (Thermo Fisher Scientific 14-4875-82, rat, 1:200), anti-BrdU (AbD Serotec MCA2060GA, rat, 1:500), anti-Ki67 (Abcam, ab15580, 1:200).

Techniques: RNA Sequencing Assay, Expressing, Sequencing, Comparison, Immunostaining, Labeling

Journal: bioRxiv

Article Title: Cell-type-specific splicing of transcription regulators and Ptbp1 by Rbfox1/2/3 in the developing neocortex

doi: 10.1101/2024.09.09.612108

Figure Lengend Snippet: a. Simultaneous in-utero delivery of CRISPR/Cas9 guide RNAs at E13.5 significantly decreased Rbfox1/2/3 expression at P0 (antibodies to all three Rbfox1/2/3 proteins, red) and led to neuronal migration defects. EGFP (green) shows 2A-EGFP in the construct, and Flag (purple) shows anti-Flag signals where the Flag tag was fused with SpCas9. Scale bar, 100μm. b. Quantification of Rbfox1/2/3 positive cells (mixture of three Rbfox1/2/3 antibodies) in control and Rbfox1/2/3 triple knockout brain slices (unpaired two-tailed Student’s t-test, n=4 sections from four P0 brains for each group). c. Quantification of Rbfox1/2/3 triple knockout (tKO) cells showing abnormal accumulation of cells in the deep layers and depletion of cells in the superficial layers (unpaired two-tailed Student’s t-test, n=4 P0 brains for each group). d. RT-PCR results showing that Rbfox1/2/3 triple knockout in the brain regulated alternative splicing of transcription regulators such as Meis2 and Tead1 . e. IUE of Rbfox1/2/3 tKO at E13.5 and examination of brain tissues at E15.5. Antibodies against Pax6 (red), Eomes (red), and Rbfox3 (grey). EGFP (green) shows 2A-EGFP in the CRISPR/Cas9 construct. Scale bar, 100μm. f. Quantification of the distribution of Rbfox1/2/3 tKO cells (EGFP+) in the embryonic neocortex (unpaired two-tailed Student’s t-test, n=4 sections from four E15.5 brains for each group). g. Portions of Pax6-, Eomes-, and Rbfox3-positive cells in EGFP-positive cells in control and Rbfox1/2/3 triple knockout brain slices. Unpaired two-tailed Student’s t-test, n=4 sections from four E15.5 brains for each group. See also Figure S5.

Article Snippet: The following primary antibodies were used: anti-Rbfox1 (Millipore MABE159, mouse, 1:800; Sigma-Aldrich HPA040809, rabbit, 1:500), anti-Rbfox2 (Bethyl A300-864A, rabbit, 1:500), anti-Rbfox3/NeuN (Cell Signaling Technology 24307T, rabbit, 1:500; Millipore MAB377, mouse, 1:500), anti-Cux1(Santa Cruz sc-13024, rabbit, 1:1000), anti-Ctip2 (Abcam ab18465, rat, 1:1000), anti-Tuj1 (BioLegend 801201, mouse, 1:1000), anti-GFP (Abcam ab13970, chicken, 1:3000), anti-Flag (Sigma-Aldrich F1804, mouse, 1:500), anti-Ptbp1 (Abcam ab5642, goat, 1:200), anti-Tbr2 (Thermo Fisher Scientific 14-4875-82, rat, 1:200), anti-BrdU (AbD Serotec MCA2060GA, rat, 1:500), anti-Ki67 (Abcam, ab15580, 1:200).

Techniques: In Utero, CRISPR, Expressing, Migration, Construct, FLAG-tag, Control, Triple Knockout, Two Tailed Test, Reverse Transcription Polymerase Chain Reaction, Alternative Splicing

Journal: bioRxiv

Article Title: Cell-type-specific splicing of transcription regulators and Ptbp1 by Rbfox1/2/3 in the developing neocortex

doi: 10.1101/2024.09.09.612108

Figure Lengend Snippet: a. Sashimi plots showing that Ptbp1 mammal-specific alternative exon8 (B) displayed higher inclusion in cells enriched for neurons ( Tubb3:EGFP (+)) than RGCs ( Eomes:EGFP (-)), and transient expression of Rbfox3 (IUE) increased its inclusion levels in vivo . In addition, Ptbp1 poison exon9N ( below, red arrow) was upregulated by Rbfox3 expression. Conservation scores at the bottom show that the two alternative exons and the GCAUG motifs are conserved in mammals. b. Zoom in view of the downstream intron of Ptbp1 exon8 showing the conserved GCAUG Rbfox binding motif_1. c. RT-PCR results show that transient Rbfox3 expression in RGCs promoted Ptbp1 exon8 inclusion. d. Ptbp1 poison exon9N introduces premature stop codons that were predicted to induce NMD. e. Heat map of RNA-Seq results showing that transient Rbfox3 expression downregulated Ptbp1 , Rbfox1 , and Rbfox2 mRNA levels. Colors indicate scaled expression. f. The downstream intron of Ptbp1 poison exon9N contains two conserved GCAUG Rbfox binding motif_2 and motif_3. g. Mini gene-splicing assays in Neuro2a cells showing that Rbfox3 expression promoted Ptbp1 exon8 and exon9N inclusion. Deleting Motif_1 attenuated the Rbfox3-mediated effect on exon8, while Motifs_2/3 were required for Rbfox3-mediated exon 9N inclusion. Numbers indicate PSI values. h. Transient Rbfox3 expression by IUE (E13.5 > E14.5) leads to a decreased proportion of Ptbp1-positive cells (yellow/green). Green, anti-EGFP; red, anti-Ptbp1. Scale bar, 50μm. i. Quantifications of Ptbp1-positive cells in the VZ of control and Rbfox3 IUE brain slices (unpaired two-tailed Student’s t-test, n=3 sections from two E14.5 brains for each group). See also Figure S6.

Article Snippet: The following primary antibodies were used: anti-Rbfox1 (Millipore MABE159, mouse, 1:800; Sigma-Aldrich HPA040809, rabbit, 1:500), anti-Rbfox2 (Bethyl A300-864A, rabbit, 1:500), anti-Rbfox3/NeuN (Cell Signaling Technology 24307T, rabbit, 1:500; Millipore MAB377, mouse, 1:500), anti-Cux1(Santa Cruz sc-13024, rabbit, 1:1000), anti-Ctip2 (Abcam ab18465, rat, 1:1000), anti-Tuj1 (BioLegend 801201, mouse, 1:1000), anti-GFP (Abcam ab13970, chicken, 1:3000), anti-Flag (Sigma-Aldrich F1804, mouse, 1:500), anti-Ptbp1 (Abcam ab5642, goat, 1:200), anti-Tbr2 (Thermo Fisher Scientific 14-4875-82, rat, 1:200), anti-BrdU (AbD Serotec MCA2060GA, rat, 1:500), anti-Ki67 (Abcam, ab15580, 1:200).

Techniques: Expressing, In Vivo, Binding Assay, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Cell-type-specific splicing of transcription regulators and Ptbp1 by Rbfox1/2/3 in the developing neocortex

doi: 10.1101/2024.09.09.612108

Figure Lengend Snippet: a. Sashimi plots showing the alternative splicing of Meis2 exon12 upon Rbfox3 expression (IUE E13.5 > E14.5) and Rbfox3/2/1 Clip-Seq tags (dashed rectangle for Rbfox3, Tag1 for Rbfox2, Tag2 and Tag3 for Rbfox1). The Clip-Seq results were derived from a previous study. b. Meis2 minigene constructs showing that the two Rbfox binding motifs ( GCAUG ) in Meis2 exon12 were mutated to G U AUG . c. Mini gene-splicing assay results show that mutating Rbfox binding motifs within Meis2 exon12 substantially inhibited its inclusion. Del_1, as well as Del_2 and Del_3 (Deletion of Tag2 and Tag3), showed synergistic effects with exon12 mutations. d. RNA-Seq results (variance stabilizing transformed read count, with standard deviation) showing that ectopic expression of Meis2 RGC-isoform (exon12 skipped), but not the neuron-isoform, promoted Tgfb3 mRNA expression. Differential gene expression was determined with the Wald test, corrected by the Benjamini and Hochberg method, and adjusted p-values are shown. e. RT-PCR validation of differential Tgfb3 expression regulated by Meis2 isoforms (bars showing SD, one-way ANOVA followed by Dunnett’s multiple comparisons test). f. Transient expression of empty vector or Meis2 isoforms in the radial glial progenitors by IUE at E13.5 and examining brain tissues at E15.5. Antibodies against EGFP (green), Pax6 (red), and Eomes (grey). Scale bar, 100μm. g. Quantification of the distribution of EGFP-positive cells in the embryonic neocortex. Unpaired two-tailed Student’s t-test, n=4 sections from four E15.5 brains for the control group, n=8 sections from five brains for the Meis2 neuron isoform group, and n=6 sections from three brains for the Meis2 RGC group. h. Quantification of Pax6- and Eomes-positive cells in EGFP-positive cells in control and Meis2 neuron- or RGC-isoform brain slices. Unpaired two-tailed Student’s t-test, n=4 sections from four E15.5 brains for the control group, n=8 sections from five brains for the Meis2 neuron isoform group, and n=6 sections from three brains for the Meis2 RGC group. See also Figure S7.

Article Snippet: The following primary antibodies were used: anti-Rbfox1 (Millipore MABE159, mouse, 1:800; Sigma-Aldrich HPA040809, rabbit, 1:500), anti-Rbfox2 (Bethyl A300-864A, rabbit, 1:500), anti-Rbfox3/NeuN (Cell Signaling Technology 24307T, rabbit, 1:500; Millipore MAB377, mouse, 1:500), anti-Cux1(Santa Cruz sc-13024, rabbit, 1:1000), anti-Ctip2 (Abcam ab18465, rat, 1:1000), anti-Tuj1 (BioLegend 801201, mouse, 1:1000), anti-GFP (Abcam ab13970, chicken, 1:3000), anti-Flag (Sigma-Aldrich F1804, mouse, 1:500), anti-Ptbp1 (Abcam ab5642, goat, 1:200), anti-Tbr2 (Thermo Fisher Scientific 14-4875-82, rat, 1:200), anti-BrdU (AbD Serotec MCA2060GA, rat, 1:500), anti-Ki67 (Abcam, ab15580, 1:200).

Techniques: Alternative Splicing, Expressing, Derivative Assay, Construct, Binding Assay, Splicing Assay, RNA Sequencing Assay, Transformation Assay, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Two Tailed Test, Control