Journal: Human Reproduction (Oxford, England)

Article Title: Subphenotype meta-analysis of testicular cancer genome-wide association study data suggests a role for RBFOX family genes in cryptorchidism susceptibility

doi: 10.1093/humrep/dey066

Figure Lengend Snippet: Genes containing suggestive intragenic markers common to undescended testis (UDT) and testicular cancer consortium (TECAC) case–case and case–control GWAS analyses.

Article Snippet: We quantified transcript expression by quantitative reverse transcriptase-PCR (qRT-PCR) using a protocol reported previously ( Johnson et al. , 2010 ; Barthold et al. , 2008 , 2013 ) and TaqMan gene expression assays (Thermo Fisher Scientific, USA) specific for Rbfox1 (Rn01761235_m1), Rbfox2 (Rn01197021_m1), Rbfox3 (Rn01464214_m1) and Gapdh (Rn99999916_s1).

Techniques: Expressing, Protein Binding, Migration, Activation Assay, Sterility, Modification, Transduction

Journal: Human Reproduction (Oxford, England)

Article Title: Subphenotype meta-analysis of testicular cancer genome-wide association study data suggests a role for RBFOX family genes in cryptorchidism susceptibility

doi: 10.1093/humrep/dey066

Figure Lengend Snippet: Custom gene lists overrepresented among suggestive undescended testis (UDT) and testicular cancer consortium (TECAC) intragenic GWAS signals.

Article Snippet: We quantified transcript expression by quantitative reverse transcriptase-PCR (qRT-PCR) using a protocol reported previously ( Johnson et al. , 2010 ; Barthold et al. , 2008 , 2013 ) and TaqMan gene expression assays (Thermo Fisher Scientific, USA) specific for Rbfox1 (Rn01761235_m1), Rbfox2 (Rn01197021_m1), Rbfox3 (Rn01464214_m1) and Gapdh (Rn99999916_s1).

Techniques: Expressing

Journal: Human Reproduction (Oxford, England)

Article Title: Subphenotype meta-analysis of testicular cancer genome-wide association study data suggests a role for RBFOX family genes in cryptorchidism susceptibility

doi: 10.1093/humrep/dey066

Figure Lengend Snippet: Relative expression of Rbfox1 and Rbfox2 transcripts in E17 and E19 gubernacula comparing Long Evans wild type (LE/wt) and an inbred strain of UDT-susceptible (LE/orl) rats (n = 5 samples per group; bars indicate mean ± SE). Rbfox2 expression was significantly increased at E19 in the LE/orl strain (*P < 0.05).

Article Snippet: We quantified transcript expression by quantitative reverse transcriptase-PCR (qRT-PCR) using a protocol reported previously ( Johnson et al. , 2010 ; Barthold et al. , 2008 , 2013 ) and TaqMan gene expression assays (Thermo Fisher Scientific, USA) specific for Rbfox1 (Rn01761235_m1), Rbfox2 (Rn01197021_m1), Rbfox3 (Rn01464214_m1) and Gapdh (Rn99999916_s1).

Techniques: Expressing

Journal: Human Reproduction (Oxford, England)

Article Title: Subphenotype meta-analysis of testicular cancer genome-wide association study data suggests a role for RBFOX family genes in cryptorchidism susceptibility

doi: 10.1093/humrep/dey066

Figure Lengend Snippet: Single confocal Z-stack images of representative E19 LE/wt gubernacula obtained from whole mount samples after benzyl alcohol:benzyl benzoate (BABB) tissue clearing; three replicate samples imaged showed a similar protein distribution. Proteins visualized include myosin heavy chain (MHC; specific for differentiated muscle), Draq5 (nuclear staining), RBFOX1 and RBFOX2. (A) Expression of RBFOX1 is enriched in the developing cremaster muscle, particularly the inner layer, but is also present in the mesenchymal cells of the gubernacular core and cord, and is both nuclear and cytoplasmic. (B) Expression of RBFOX2 is primarily nuclear and enhanced in the ‘myogenic zone’ around developing muscle, within a growth zone (the position is outlined by a white dotted circle) observable in the distal gubernacular tip, and throughout the peritoneal layer and cord of the gubernaculum (scale bars: 100 μm).

Article Snippet: We quantified transcript expression by quantitative reverse transcriptase-PCR (qRT-PCR) using a protocol reported previously ( Johnson et al. , 2010 ; Barthold et al. , 2008 , 2013 ) and TaqMan gene expression assays (Thermo Fisher Scientific, USA) specific for Rbfox1 (Rn01761235_m1), Rbfox2 (Rn01197021_m1), Rbfox3 (Rn01464214_m1) and Gapdh (Rn99999916_s1).

Techniques: Staining, Expressing

Journal: Nature structural & molecular biology

Article Title: Concentration-dependent splicing is enabled by Rbfox motifs of intermediate affinity

doi: 10.1038/s41594-020-0475-8

Figure Lengend Snippet: a. Ribbon structure of RBFOX1 (teal) bound to UGCAUG (gold). Generated with data from Auweter et al. (PDB 2ERR). Protein–RNA hydrogen bonds are indicated in black. b. Relative per-base conservation, as represented by PhyloP score, for each position of GCAUG at all instances of the motif in 3' UTRs (dark teal, n = 26707) and introns (light teal, n = 91791). c. Primary (gold), secondary (teal), polyA and polyC (light grey), and GCAGG, GUAAG, and GUCCG (dark grey) R values are shown across four concentrations of RBFOX2 nsRBNS experiments. d. Analysis of the fraction of oligonucleotides bound in nsRBNS at three concentrations of RBFOX2 for primary (gold (GCAUG and GCACG), n GCAUG-1 = 5435, n GCAUG-2 = 384, n GCACG-1 = 1246, n GCACG-2 = 30) and secondary (teal, n Secondary-1 = 15676, n Secondary-2 = 6722, n Secondary-6 = 64) motifs. An oligonucleotide was considered bound if it had an R value of at least 1.1. e. nsRBNS R values at 1.1 μM RBFOX2 concentration for all pentamers diverging from GCAUG or GCACG by 1 or 2 bases. Secondary motifs identified here are outlined in black.

Article Snippet: The GFP of a pEGFP rbFOX1 plasmid (Addgene #63085) was replaced with Cerulean (Cerulean-N1 Addgene #54742) to produce a Cerulean:Rbfox1 vector.

Techniques: Generated, Concentration Assay

Journal: Nature structural & molecular biology

Article Title: Concentration-dependent splicing is enabled by Rbfox motifs of intermediate affinity

doi: 10.1038/s41594-020-0475-8

Figure Lengend Snippet: a. Primary and secondary motif reads peak near 0 in a metaplot centered at the motif in introns (black) and 3' UTRs (grey) in RBFOX2 iCLIP data (GEO GSE54794). iCLIP reads containing the motif of interest (see for read counts) were aligned with position one of the pentamer at 0 and normalized to the minimum read count in an 80-nt window (50-nt window shown). Y-axis range was reduced for secondary motifs. b. Correlation of intronic iCLIP- and nsRBNS-enriched 5 mers ( n = 1024). Secondary motifs indicated in teal, primary motifs indicated in gold. Grey dots indicate “hitchhiking” motifs that overlap the primary motif GCAUG by at least three bases but do not have intrinsic Rbfox affinity. c. RBFOX1 HiTS-CLIP data , (SRA SRP128054, SRP035321) enrichments for primary motifs and secondary six motifs in both 3' UTRs (left, n = 2963 and 989) and introns (right, n = 847 and 1431) relative to transcriptomic frequencies in mouse whole brain and ventral spinal neurons, respectively. Enrichment was calculated based on the 5 mer composition of 100-base CLIP peak regions centered around the apex of the CLIP peak relative to 5 mer composition of the transcriptomic region. d. Four secondary motifs (GCUUG, GAAUG, GUUUG, GUAUG) are indicated among the top 200 highly enriched 5 mers derived from intronic HiTS-CLIP peaks from mouse ventral spinal neurons. Primary motifs in gold, secondary motifs in teal. Peaks calculated as above. e. High-confidence CLIP peaks in two different Rbfox1 HiTS-CLIP datasets (SRA SRP128054, SRP035321) in ventral spinal neurons and mouse whole brain cells attributable to primary (light gold), or four secondary (teal; GCUUG, GAAUG, GUUUG, and GUAUG), or both (dark gold) motifs in both 3' UTRs ( n = 15487 and n = 24972, respectively) and introns ( n = 4800 and n = 1519, respectively). Fold enrichments above transcriptomic background are indicated. Peaks containing neither primary nor two or more secondary motifs are shown in grey.

Article Snippet: The GFP of a pEGFP rbFOX1 plasmid (Addgene #63085) was replaced with Cerulean (Cerulean-N1 Addgene #54742) to produce a Cerulean:Rbfox1 vector.

Techniques: Derivative Assay

Journal: Nature structural & molecular biology

Article Title: Concentration-dependent splicing is enabled by Rbfox motifs of intermediate affinity

doi: 10.1038/s41594-020-0475-8

Figure Lengend Snippet: a. Experimental design of Rbfox1 splicing reporter. One GCAUG primary motif or six copies of a secondary motif (GCUUG, GAAUG, or GUAUG) were cloned in a 250-base window downstream of an alternative exon in the RG6 dual fluorescent splicing reporter. Plasmids were co-transfected in HEK293T cells with a plasmid expressing fluorescently labelled RBFOX1 to monitor cellular protein levels. b. Semi-quantitative PCR with 5’ 6-FAM-labelled primer indicates exon inclusion in the presence of both primary and secondary motifs in an Rbfox1 -dependent manner. c. Mean percent spliced in (PSI) values of exons containing primary motifs (gold), secondary motifs (teal), or motif permutations (grey) in the downstream intron after expression of Rbfox1 . Error bars show SD of technical replicates in triplicate. For GAAUG, the median permutation value was used due to the introduction of a splicing silencer in its permuted form. d. Per-cell inclusion:exclusion (EGFP:dsRED, y-axis) ratio for the RG6 alternative exon as Rbfox expression (Cerulean, x-axis) increases as measured by flow cytometry. Primary motifs (gold), six copies of three indicated secondary motifs (teal), primary (intact and permuted) and secondary motifs without co-transfection of Rbfox (dark grey), and a permuted primary motif with co-expressed Rbfox (light grey) are shown. Representative data from one of two replicates (the other is shown in ). Bin numbers can be found in . The center line represents the median, lower and upper hinges the first and third quartiles, respectively, and whiskers extend to the smallest or largest value (at most 1.5*IQR (interquartile range) of the hinge. Outliers are not shown. Notches extend 1.58*IQR/sqrt(n), giving a roughly 95% confidence interval on the medians. Uncropped gel image is available as source data online.

Article Snippet: The GFP of a pEGFP rbFOX1 plasmid (Addgene #63085) was replaced with Cerulean (Cerulean-N1 Addgene #54742) to produce a Cerulean:Rbfox1 vector.

Techniques: Clone Assay, Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cotransfection

Journal: Nature structural & molecular biology

Article Title: Concentration-dependent splicing is enabled by Rbfox motifs of intermediate affinity

doi: 10.1038/s41594-020-0475-8

Figure Lengend Snippet: a. Six secondary motifs approximate the exon inclusion of one primary motif in an Rbfox1-dependent manner at the protein level, replicate 2. RG6 plasmids containing one primary motif or six secondary motifs were co-transfected in HEK293T cells with fluorescently labelled Rbfox1 and monitored by flow cytometry for the inclusion isoform (GFP), exclusion isoform (dsRED), and Rbfox1 (Cerulean) expression at the single-cell level. Controls including a scrambled motif co-transfected with Rbfox1 (light grey) and scrambled and intact motifs without Rbfox1 (grey) are also shown. Bins detailed in . b. The slope of linear fit of two flow cytometry replicates were null-subtracted and normalized to their permuted controls. Error bars represent standard error of the mean (SEM).

Article Snippet: The GFP of a pEGFP rbFOX1 plasmid (Addgene #63085) was replaced with Cerulean (Cerulean-N1 Addgene #54742) to produce a Cerulean:Rbfox1 vector.

Techniques: Transfection, Flow Cytometry, Expressing

Journal: Nature structural & molecular biology

Article Title: Concentration-dependent splicing is enabled by Rbfox motifs of intermediate affinity

doi: 10.1038/s41594-020-0475-8

Figure Lengend Snippet: a. Total expression of Rbfox1 , Rbfox2 , and Rbfox3 in transcripts per million (TPM) based on RNA-seq during a neuronal differentiation time course (SRA PRJNA185305). b. Correlation of Rbfox primary (gold) and secondary (teal) motifs in the downstream intron with delta PSI at both low-moderate (RG-DS1) and moderate-high (DS1-DS3) transitions of Rbfox expression during in vitro neuronal differentiation. Increased color intensity represents 0, 1, or 2+ motifs in the downstream intron. Primary motifs, RG–DS1: 0 ( n = 1521), 1 ( n = 269), 2+ ( n = 90), DS1–DS3: 0, ( n = 3611), 1 ( n = 596), 2+ ( n = 171). Secondary motifs, RG–DS1: 0 ( n = 2763), 1 ( n = 838), 2+ ( n = 159), DS1–DS3: 0, ( n = 6461), 1 ( n = 1882), 2+ ( n = 413). The center line of the boxplot represents the median, lower and upper hinges the first and third quartiles, respectively, and whiskers extend to the smallest or largest value (at most 1.5*IQR (interquartile range) of the hinge. Outliers are not shown. Notches extend 1.58*IQR/sqrt(n), giving a roughly 95% confidence interval on the medians. *** P < 0.001 (two-sided Wilcoxon Rank-Sum). c. Gene Ontology categories of splicing events driven by primary motifs ( n = 388) (top) and secondary motifs ( n = 561) (bottom) during neuronal differentiation. Events were compared to all expressed genes at DS3 and terms were filtered for FDR < 0.1, B > 99, and b > 9. d. Correlation of Rbfox primary (gold) and secondary (teal) motifs throughout stages of neuronal differentiation subsequent to RG stage. Pearson correlation of secondary motif presence with delta PSI is shown at intervals of neuronal differentiation from radial glia stage to mature 28-day glutamatergic neurons. Events: RG–DS1 ( n = 940), RG–DS3 ( n = 3436), RG–MAT16 ( n = 3860) RG–MAT21 ( n = 3942) RG–MAT28 ( n = 4119). Size of point indicates correlation coefficient, intensity indicates uncorrected p-value < 0.05. e. Reporter design of CD47 intron 10-containing RG6. All secondary motifs were ablated from the 617-nt intron and sequentially reintroduced into the reporter to examine the effects of individual secondary motifs. f. Mean percent spliced in (PSI) values of exons containing secondary motifs in the downstream intron after expression of Rbfox1 . Error bars show SD of triplicate technical replicates.

Article Snippet: The GFP of a pEGFP rbFOX1 plasmid (Addgene #63085) was replaced with Cerulean (Cerulean-N1 Addgene #54742) to produce a Cerulean:Rbfox1 vector.

Techniques: Expressing, RNA Sequencing, In Vitro

Journal: Nature structural & molecular biology

Article Title: Concentration-dependent splicing is enabled by Rbfox motifs of intermediate affinity

doi: 10.1038/s41594-020-0475-8

Figure Lengend Snippet: a. Differentiated neuronal cell types from Weyn-Vanhentenryck et al . (SRA SRP055008), arranged by combined Rbfox1, Rbfox2, and Rbfox3 expression (log 10 of sum of RPKM values). Cell types were grouped into Lowest, Low, Medium, High, and Highest Rbfox expression categories. Cells analyzed: olfactory sensory neurons (OMP+) (OSN), enterochromaffin cells (EC), taste receptor cells (TRC), rod or cone photoreceptors, jugular or nodose visceral sensory ganglia, dopaminergic neurons (DN), motor neurons (MN), dorsal root ganglia sensory neurons (Nav1.8+ or Avil+) (DRG), trigeminal ganglia, Purkinje neurons (PN), and cerebellar granule neurons (CGN). b. Linear regression of 1,909 alternatively spliced exons comparing the Rbfox expression groups indicated in a. Horizontal bars represent the Pearson r value (left) and uncorrected significance (right) of the correlation between the number of occurrences of primary or secondary (GCUUG, GAAUG, GUUUG, and GUAUG) motifs and ΔPSI values between Medium and Low (1.), between High and Low (2.), or between Highest and Low (3.) Rbfox expression regimes. Grey dotted and dashed lines indicate 0.05 and 0.01 significance thresholds, respectively. c. An equilibrium model for Rbfox binding to intronic sequences in the nucleus at various expression levels of Rbfox (low, grey area; high, teal area). GCAUG indicated by gold, GCACG indicated by yellow, secondary motifs indicated in teal, non-specific 5 mers in grey. d. Graphical summary of how Rbfox proteins (golden ellipses) regulate distinct splicing events at different expression levels (teal shading). Secondary motifs are functionally relevant only at higher Rbfox levels, occurring at later stages of neuronal differentiation or in cell types with high Rbfox expression, while primary motifs are functionally relevant at earlier stages and in cell types with medium as well as high Rbfox levels.

Article Snippet: The GFP of a pEGFP rbFOX1 plasmid (Addgene #63085) was replaced with Cerulean (Cerulean-N1 Addgene #54742) to produce a Cerulean:Rbfox1 vector.

Techniques: Expressing, Binding Assay

Journal: Nature structural & molecular biology

Article Title: Concentration-dependent splicing is enabled by Rbfox motifs of intermediate affinity

doi: 10.1038/s41594-020-0475-8

Figure Lengend Snippet: RBNS 7-mer enrichments (R-value) for 1.1 μΜ RBFOX2 (a) and 1.3 μΜ RBFOX3 (b) binding were first corrected for non-specific contributions (R’ see ) and then linearly correlated with known dissociation constants (K d ) for RBFOX1 binding 1,2 . Correlation coefficients between log(R’) and log(K d ) were r=-0.955, P-value=8.379 × 10 −9 (a) and r=−0.915, P-value=6.7 × 10 −7 (b). Scatter plots show estimated K d as a function of the original, uncorrected R-value. Resulting 7-mer K d estimates were highly correlated between RBFOX2 and RBFOX3 (c) with r=0.763, P-value ≈ 0. Data for all 7-mers are shown on a logarithmic scale. Primary motif containing 7-mers are highlighted in gold (GCAUG), yellow (GCACG), and teal (secondary motifs GCUUG, GAAUG, GUUUG, GUGUG, GUAUG, GCCUG). Grouping 7-mers by their 5-mer content allows to estimate average K d s for each 5-mer (see ). A histogram of these 5-mer dissociation constants is shown in (d), with primary and secondary motifs highlighted as in (c). Motifs GCUUG, GAAUG and GUUUG were considered strong motifs. 136 non-primary or secondary 5-mers with partial overlap to primary motifs GCAUG, GCACG were excluded.

Article Snippet: The GFP of a pEGFP rbFOX1 plasmid (Addgene #63085) was replaced with Cerulean (Cerulean-N1 Addgene #54742) to produce a Cerulean:Rbfox1 vector.

Techniques: Binding Assay

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: T1 receptor levels in vitro and in vivo. ( A ) Overexpression of Rbfox1 in mouse primary hippocampal neurons leads to a specific up-regulation of TrkB.T1. Western blot analysis of wild-type mouse primary hippocampal neurons transfected with a lentiviral vector expressing Rbfox1 (Rbfox1) or eGFP (Ctrl) as a control. Neurons were transfected after 4 days in vitro and analyzed 72 hr after transfection. Ntrk2 (TrkB) protein levels were tested with an antibody against the TrkB extracellular domain to detect all TrkB isoforms (left panel) or an antibody specifically recognizing TrkB.T1 (right panel). Antibodies against Rbfox1 were used to verify Rbfox1 overexpression; Gapdh and β-III-Tubulin were used as control of loading and as a neuronal marker respectively. ( B ) Quantification of TrkB full-length (TrkB.FL) and TrkB.T1 protein from experiments as in A; n = 3 ± SEM. * = p ≤ 0.05, ** = p ≤ 0.01 (Student’s t-test). ( C ) Quantitative PCR analysis of TrkB.FL and TrkB.T1 expression from mouse primary hippocampal neurons as in ( A ); n = 3 ± SEM; ** = p ≤ 0.01 (Student’s t-test). ( D ) Immunoblot analysis of littermates wild-type R26-Rbfox1 +/+ (WT) and transgenic R26-Rbfox1 +/flox ( R26-Fox1 ) primary hippocampal neurons transfected with a Cre-expressing adenovirus (Ad-Cre) and analyzed as in ( A ). ( E ) Immunoblot of hippocampi derived from control Nes-Cre animals (Ctrl) and transgenic Nes-Cre;R26-Rbfox1 +/flox animals (N-cre;Fox1) analyzed with antibodies as in ( A ). ( F ) Immunoblot quantification analysis of TrkB.FL, TrkB.T1 and Rbfox1 protein levels of N-cre;Fox1 relative to Ctrl as in ( E ); n = 6 ± SEM; * = p ≤ 0.05; ** = p ≤ 0.01 (Student’s t-test).

Article Snippet: The Rbfox1 transgenic mouse model (R26-Fox1) was generated by targeting the Gt(ROSA)26Sor locus (Chromosome 6) with a CTV vector (Addgene #15912) to conditionally express murine Rbfox1.

Techniques: In Vitro, In Vivo, Over Expression, Western Blot, Transfection, Plasmid Preparation, Expressing, Control, Marker, Real-time Polymerase Chain Reaction, Transgenic Assay, Derivative Assay

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: ( A ) Schematic representation of the targeting vector used to generate the Rbfox1 conditional transgenic mouse model. The Gt(ROSA)26Sor locus (Rosa26) was targeted using a vector including the Rbfox1 cDNA that can be activated by the CAG promoter after cre-mediated removal of the loxP-flanked STOP-cassette. ( B ) Cre expression under the nestin promoter is specific to neurons in the hippocampus. β-Gal staining of the hippocampal area of control (R26-LacZ) and Nes-Cre;R26-LacZ (N-cre;R26-LacZ) mice showing robust Nes-cre activity in the granule cells of dentate gyrus (DG) and CA1 and CA3 pyramidal neurons. ( C ) Rbfox1 expression induced by nestin-cre is restricted to the neuronal compartment of the hippocampus. Immunofluorescence staining of N-cre ( Nes -Cre) and N-cre;Fox1 ( Nes-Cre;R26-Rbfox1 +/flox ) hippocampi showing similar pattern of Rbfox1 expression. Staining for Rbfox1 (green staining) and glial fibrillary acidic protein (GFAP; red staining) also shows no difference between the control and the N-cre;Fox1 sections. DAPI staining was used for nuclei. ( D ) Higher magnification immunofluorescence staining of DG area from panels in ( A ).

Article Snippet: The Rbfox1 transgenic mouse model (R26-Fox1) was generated by targeting the Gt(ROSA)26Sor locus (Chromosome 6) with a CTV vector (Addgene #15912) to conditionally express murine Rbfox1.

Techniques: Plasmid Preparation, Transgenic Assay, Expressing, Staining, Control, Activity Assay, Immunofluorescence

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: ( A ) Western blot analysis of TrkC.FL, TrkC.T1 and Rbfox2 expression in the same hippocampal samples used in , including control Nes -Cre animals (Ctrl) and N-cre;Fox1 ( Nes-Cre;R26-Rbfox1 +/flox ) animals. ( B ) Quantifications of the immunoblots bands as in ( A ); n = 6 ± SEM.

Article Snippet: The Rbfox1 transgenic mouse model (R26-Fox1) was generated by targeting the Gt(ROSA)26Sor locus (Chromosome 6) with a CTV vector (Addgene #15912) to conditionally express murine Rbfox1.

Techniques: Western Blot, Expressing, Control

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: T1 receptor levels. ( A ) Western blot analysis of control (Ctrl) and Rbfox1 +/- (Het) mouse hippocampus with antibodies against TrkB, Rbfox1, Rbfox2, Rbfox3 and Gapdh (used as loading control). ( B ) quantifications of immunoblots bands from ( A ) relative to Ctr (100%); n = 4 ± SEM; *p≤0.05; **p≤0.01 (Student’s t-test). ( C ) Overexpression of Rbfox2, but not Rbfox3 up-regulate TrkB.T1 expression in primary hippocampal neurons. Western blot analysis of wild-type mouse primary hippocampal neurons transfected with a lentiviral vector expressing eGFP as a control (Ctrl), Rbfox2 (Fox2) or Rbfox3 (Fox3). Neurons were transfected after 4 days in vitro and analyzed 72 hr after transfection by immunoblot with antibodies against TrkB extracellular domain to detect all TrkB isoforms, GFP, Rbfox2, Rbfox3 and β-III-Tubulin as loading control and neuronal marker. ( D ) Quantification of TrkB full-length (TrkB.FL) and TrkB.T1 protein from experiments as in ( C ); n = 3 ± SEM. *** = p ≤ 0.001 (Student’s t-test).

Article Snippet: The Rbfox1 transgenic mouse model (R26-Fox1) was generated by targeting the Gt(ROSA)26Sor locus (Chromosome 6) with a CTV vector (Addgene #15912) to conditionally express murine Rbfox1.

Techniques: Western Blot, Control, Over Expression, Expressing, Transfection, Plasmid Preparation, In Vitro, Marker

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: T1-isoform and its RNA binding function is necessary to increase TrkB.T1 receptor levels. ( A ) Rbfox1 iCLIP analysis of mouse brain chromatin associated high molecular weight nuclear fraction (HMW) or soluble nuclear fraction (Soluble) showing association of Rbfox1 to TrkB transcripts in both HMW (vertical red bars) and soluble (vertical orange bars) nuclear fractions. Extracellular domain (ECD), specific TrkB.T1 region ( T1 ) and tyrosine kinase domain (TK domain) are also indicated. ( B ) Schematic representation of the murine Ntrk2 gene. Exon lengths are indicated in base pairs (bp) while intron lengths are indicated as kilo-bases (kb). White and black stars indicate the start and stop codons respectively. ( C ) Agarose gels of RT-PCR amplification products from an RNA immunoprecipitation (RIP) analysis of wild type primary hippocampal neurons with an Rbfox1 antibody; input samples are from the PCR amplification of the RNA and antibody mixture before the immunoprecipition (IP) with mouse IgG or Rbfox1 antibodies. Indicated exonic (blue and green arrows) and intronic (red arrows) primers were used for the analysis. Note the presence of RT-PCR amplification bands following Rbfox1 IP for both TrkB.FL and TrkB.T1 and a proximal intronic region upstream of the specific TrkB.T1 exon, Camta1 (positive control; ; ), but not Sirt1 (negative control). ( D ) Pulse-chase mRNA stability assay in primary hippocampal neurons derived from littermate control R26-Rbfox1 +/flox (Ctrl) and Nes -Cre; R26-Rbfox1 +/flox (N-cre;Fox1) embryos. Nascent RNA of primary neurons was labeled with 5-EU for 5 hr (pulsing) followed by QPCR analysis at 0 and 9 hr using c-DNA specific primers (purple arrows in schematic) for TrkB.FL and TrkB.T1. Values at 9 hr are expressed as percentage relative to 0 hr. n = 6 ± SEM; ns = p > 0.05; * = p ≤ 0.05 (Student’s t-test). ( E ); Rbfox1 RNA binding activity is required to promote TrkB.T1 up-regulation. Western blot analysis of wild-type mouse primary hippocampal neurons transfected with an adenovirus expressing WT Rbfox1 (Rbfox1) or an Rbfox1 with a mutation in the RNA binding domain (Rbfox1-F158A). Neurons were transfected after 4 days in vitro and analyzed 48 hr after transfection. ( F ) Quantification of TrkB.FL and TrkB.T1 protein from experiments as in E; n = 3 ± SEM. * = p ≤ 0.05, (Student’s t-test).

Article Snippet: The Rbfox1 transgenic mouse model (R26-Fox1) was generated by targeting the Gt(ROSA)26Sor locus (Chromosome 6) with a CTV vector (Addgene #15912) to conditionally express murine Rbfox1.

Techniques: RNA Binding Assay, High Molecular Weight, Reverse Transcription Polymerase Chain Reaction, Amplification, RNA Immunoprecipitation, Positive Control, Negative Control, Pulse Chase, Stability Assay, Derivative Assay, Control, Labeling, Activity Assay, Western Blot, Transfection, Expressing, Mutagenesis, In Vitro

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: T1 3'UTR does not contribute to TrkB.T1 mRNA expression changes caused by Rbfox1 overexpression. ( A ) Western blot analysis of lysates from a Doxycycline (Dox) inducible HEK293- Rbfox1 cell line transfected with a TrkB.T1 cDNA containing the 3'UTR sequence. Rbfox1 expression was induced by 0.5 µg/ml Dox and samples were analyzed 48 hr after the transfection of the TrkB.T1-3'UTR cDNA. ( B ) Quantification of the TrkB.T1 specific bands from ( A ); n = 3 ± SEM. ( C ) Western blot analysis of lysates from the neuroblastoma cell line Neuro-2a (N2A) stably expressing TrkB.T1 with the 3’UTR sequence (N2A-TrkB.T1-3’UTR). Rbfox1 cDNA was transiently transfected into N2A-TrkB.T1-3’UTR cells and TrkB.T1 levels were analyzed 48 hr after the transfection and quantified in ( D ); n = 3 ± SEM.

Article Snippet: The Rbfox1 transgenic mouse model (R26-Fox1) was generated by targeting the Gt(ROSA)26Sor locus (Chromosome 6) with a CTV vector (Addgene #15912) to conditionally express murine Rbfox1.

Techniques: Expressing, Over Expression, Western Blot, Transfection, Sequencing, Stable Transfection

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: T1 isoform levels in the hippocampus of Rbfox1 overexpressing animals. ( A ) Schematic representation of the murine Ntrk2 (TrkB) gene transcripts annotated from ENSEMBL (highlighted in green). In each transcript exons and introns are indicated with vertical and horizontal lines respectively. TrkB.FL (Ntrk2-201) and TrkB.T1 (Ntrk2-202) are indicated (Picture modified from Ensembl genome browser; ENSMUSG00000055254). ( B ) RNA-Seq specific analysis of the Ntrk2 transcripts in control (Ctrl; Nes-Cre ) and N-cre;Fox1 hippocampus ( Nes-Cre;R26-Rbfox1 +/flox ). Isoform IDs are indicated on the Y axis while the average of RNA-sequencing reads for every transcript is indicated on the X axis. TrkB.T1 (Ntrk2-202; red box) is the only TrkB isoform significantly modulated in the N-cre;Fox1 hippocampus. ( C ) Immunoblot analysis of wild type (WT) and TrkB.T1 knockout (T1-/-) hippocampus blotted with an antibody against the TrkB extracellular domain.

Article Snippet: The Rbfox1 transgenic mouse model (R26-Fox1) was generated by targeting the Gt(ROSA)26Sor locus (Chromosome 6) with a CTV vector (Addgene #15912) to conditionally express murine Rbfox1.

Techniques: Modification, RNA Sequencing, Control, Western Blot, Knock-Out

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: ( A ) Dot plot showing the top enriched biological process Gene Ontology (GO) terms. Note that significant over-represented GO terms (q-value <0.05) are neuronal related processes. GO terms are displayed on the left Y axis. ( B ) Venn diagram showing the number of overlapping gene-isoforms differentially expressed in N-cre;Fox1 and Rbfox1 knockout ( Rbfox1 -KO) hippocampus .

Article Snippet: The Rbfox1 transgenic mouse model (R26-Fox1) was generated by targeting the Gt(ROSA)26Sor locus (Chromosome 6) with a CTV vector (Addgene #15912) to conditionally express murine Rbfox1.

Techniques: Knock-Out

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: RNA-seq analysis comparing gene-isoforms modulated by hippocampal Rbfox1 upregulation (N-cre;Fox1) and hippocampal neuron Rbfox1 and Rbfox3 knockout , or whole brain Rbfox1 -knockout . Venn diagram showing the limited overlap of genes modulated in N-cre;Fox1 hippocampus relative to hippocampal neurons with Rbfox1 and Rbfox3 double knockout ( A ), nuclear Rbfox1 and Rbfox3 knockout ( B ) or cytoplasmic Rbfox1 and Rbfox3 knockout ( C ). Note that the specific knockout of the nuclear or cytoplasmic Rbfox1 isoforms in was achieved by rescuing the complete Rbfox1 KO with the cytoplasmic ( Rbfox1 _C) or nuclear isoform ( Rbfox1 _N) respectively. ( D ) Venn diagram showing the overlap of genes modulated in N-cre;Fox1 hippocampus and whole brain Rbfox1 -KO.

Article Snippet: The Rbfox1 transgenic mouse model (R26-Fox1) was generated by targeting the Gt(ROSA)26Sor locus (Chromosome 6) with a CTV vector (Addgene #15912) to conditionally express murine Rbfox1.

Techniques: RNA Sequencing, Knock-Out, Double Knockout

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: T1 in synaptosomes. ( A ) Western blot analysis of TrkB isoform, PSD-95 and Rb-Fox1 expression in total homogenates (H), cytosolic (C) and synaptosome fraction (Syn) from hippocampus of control (Ctrl; Nes -Cre) and N-cre;Fox1 mouse brains. PSD-95 was used as control for enrichment of the synaptosomal fraction. ( B ) Immunoblot quantification of TrkB.FL, TrkB.T1 and Rbfox1 specific bands in the synaptosomal fraction as in ( A ); n = 3 ± SEM, *** = p ≤ 0.001, * = p ≤ 0.05 (Student’s t-test).

Article Snippet: The Rbfox1 transgenic mouse model (R26-Fox1) was generated by targeting the Gt(ROSA)26Sor locus (Chromosome 6) with a CTV vector (Addgene #15912) to conditionally express murine Rbfox1.

Techniques: Western Blot, Expressing, Control

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: T1 receptor allele. ( A ) Averaged time-course of the field excitatory postsynaptic potential (fEPSP) slope in hippocampal slices from control (Ctrl = Nes Cre, n = 10), N-cre;Fox1 ( Nes-Cre;R26-Rbfox1 +/flox , n = 10) and N-cre;Fox1;T1-Flx ( Nes -Cre; R26-Rbfox1 +/flox ; TrkB.T1 +/flox , n = 8) mice after extracellular application of BDNF (20 ng/ml). Values are expressed as fEPSP percentage of the baseline values (average of 5 min before BDNF application, mean ± SEM). BDNF application (20 min) is indicated by the black horizontal bar. ( B ) Histogram showing mean ± SEM of the fEPSP slope average at 50 min after BDNF infusion; ** = p ≤ 0.01; * = p ≤ 0.05; ns = p > 0.05 (One-way ANOVA followed by Tukey’s test). ( C ) Representative recording traces of slices before (baseline = black line) and 50 min after BDNF application (red line). ( D ) Western blot analysis of hippocampi from Ctrl, N-cre;Fox1 and N-cre;Fox1;T1-Flx mice probed with anti TrkB, Rbfox1 and Gapdh antibodies. ( E ) Input/Output relationship of Shaffer collateral projections on CA1 pyramidal neurons of Ctrl and N-cre;Fox1 hippocampal slices. Slope of fEPSP reported as percentage of the maximal recorded values plotted against the stimulation current. ( F ) Ratio between the second and first fEPSP evoked by the stimulation of Shaffer collateral and recorded in the CA1-radiatum at increasing time latency from 20 to 400 ms of Ctrl and N-cre;Fox1 hippocampal slices.

Article Snippet: The Rbfox1 transgenic mouse model (R26-Fox1) was generated by targeting the Gt(ROSA)26Sor locus (Chromosome 6) with a CTV vector (Addgene #15912) to conditionally express murine Rbfox1.

Techniques: Control, Western Blot

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: T1 receptor allele. ( A ) Averaged time-course of the field excitatory postsynaptic potential (fEPSP) slope in hippocampal slices from control (Ctrl = Nes Cre, n = 10), N-cre;Fox1( Nes-Cre;R26-Rbfox1 +/flox ; n = 10) and N-cre;Fox1;T1+/- ( Nes-Cre;R26-Rbfox1 +/flox ;TrkB.T1 +/- ; n = 13) mice after extracellular application of BDNF (20 ng/ml). Values are expressed as fEPSP percentage of the baseline values (average of 5 min before BDNF application, mean ± SEM). BDNF application (20 min) is indicated by the black horizontal bar. ( B ) Histogram showing mean ± SEM of the fEPSP slope average at 50 min after BDNF infusion; *** = p ≤ 0.001; ** = p ≤ 0.01; * = p ≤ 0.05 (Student’s t-test). ( C ) Representative recording traces of slices before (baseline = black line) and 50 min after BDNF application (red line). ( D ) Western blot analysis of hippocampus expression of TrkB, Rbfox1 and Gapdh of animals analyzed in ( A–C ).

Article Snippet: The Rbfox1 transgenic mouse model (R26-Fox1) was generated by targeting the Gt(ROSA)26Sor locus (Chromosome 6) with a CTV vector (Addgene #15912) to conditionally express murine Rbfox1.

Techniques: Control, Western Blot, Expressing

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: T1 heterozygous animals. ( A ) Averaged time-course of changes in field excitatory postsynaptic potential (fEPSP) slope after extracellular application of BDNF (20 ng/ml) in mouse hippocampal slices from control (Ctrl; Nes -cre, n = 10) and TrkB.T1 heterozygous animals (T1+/-; n = 8). Values are expressed as fEPSP percentage of baseline values (average of 5 min before BDNF application, mean ± SEM). BDNF application (20 min) is indicated by the black horizontal bar. ( B ) Histogram showing the average mean ± SEM of the fEPSP slope at 50 min after BDNF infusion (n.s.: Student’s t-test). ( C ) Western blot analysis of hippocampal expression of TrkB, Rbfox1 and Gapdh of animals as in ( A ).

Article Snippet: The Rbfox1 transgenic mouse model (R26-Fox1) was generated by targeting the Gt(ROSA)26Sor locus (Chromosome 6) with a CTV vector (Addgene #15912) to conditionally express murine Rbfox1.

Techniques: Control, Western Blot, Expressing

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet:

Article Snippet: The Rbfox1 transgenic mouse model (R26-Fox1) was generated by targeting the Gt(ROSA)26Sor locus (Chromosome 6) with a CTV vector (Addgene #15912) to conditionally express murine Rbfox1.

Techniques: Plasmid Preparation

Journal: Channels

Article Title: Characterization of Ca V 1.2 exon 33 heterozygous knockout mice and negative correlation between Rbfox1 and Ca V 1.2 exon 33 expressions in human heart failure

doi: 10.1080/19336950.2017.1381805

Figure Lengend Snippet: The mRNA expression of Rbfox1/2 and Ca V 1.2 α1 C in human hearts. (A) The mRNA levels of Rbfox1 were measured by real-time RT-PCR from the samples of human normal (NF, black, n = 8), dilated cardiomyopathy (DCM, red, n = 15) and ischemic cardiomyopathy (ICM, blue, n = 10) hearts. The relative expression was normalized by internal expression of human GAPDH and RPLPO mRNA ( P = 0.0103, one-way ANOVA; * P <0.05 vs. WT, Bonferroni post hoc test). (B) The mRNA levels of Rbfox2 were also measured by real-time RT-PCR from NF (n = 8), DCM (n = 15) and ICM (n = 10) hearts. (C) Ca V 1.2 α1 C mRNA expression was measured by real-time RT-PCR from NF (n = 8), DCM (n = 15) and ICM (n = 10) hearts (** P <0.01, one-way ANOVA; ** P <0.01 vs. WT, Bonferroni post hoc test). The correlations between alternative exon 33 expression and Rbfox1 (D) or Ca V 1.2 α1 C (E) mRNA level in human hearts were analyzed by linear regression, R 2 refers to the coefficient of determination.

Article Snippet: FAM labeled CACNA1C probe located at the junction of constitutive exon 3 and exon 4 (Assay ID Hs00167681_m1, Applied Biosystems), and RBFOX1/2 (Assay ID Hs01125659_m1 and Hs00204814_m1, Applied Biosystems) were used to measure the standard mRNA expressions.

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: RBFOX1 and RBFOX3 Mutations in Rolandic Epilepsy

doi: 10.1371/journal.pone.0073323

Figure Lengend Snippet: Hg19 genomic localization and overview of known transcript variants. Red bars represent microdeletion size and location for the patient. Red dashes indicate the genomic location of single nucleotide variants. (A) Overview RBFOX1 (B) RBFOX3 .

Article Snippet: Segregation in the families of the microdeletions was examined by real-time quantitative PCR using TaqMan CNV probes ( RBFOX1: Hs04461212_cn; RBFOX3: Hs03975574_cn) (Life Technologies, Darmstadt, Germany).

Techniques:

Journal: PLoS ONE

Article Title: RBFOX1 and RBFOX3 Mutations in Rolandic Epilepsy

doi: 10.1371/journal.pone.0073323

Figure Lengend Snippet: RBFOX1and RBFOX3 variants and phenotype of index-patients.

Article Snippet: Segregation in the families of the microdeletions was examined by real-time quantitative PCR using TaqMan CNV probes ( RBFOX1: Hs04461212_cn; RBFOX3: Hs03975574_cn) (Life Technologies, Darmstadt, Germany).

Techniques:

Journal: PLoS ONE

Article Title: RBFOX1 and RBFOX3 Mutations in Rolandic Epilepsy

doi: 10.1371/journal.pone.0073323

Figure Lengend Snippet: For three mutations for which DNA samples of family members were available, segregation analyses could be performed. The respective RBFOX1 and RBFOX3 truncating mutations co-segregated with a variable phenotype of either seizures or pathologic EEG patterns in most family members. Only a few individuals carried the respective familial mutation but did not present any clinical features, indicating incomplete penetrance of the mutations. However, subclinical phenotypes (e.g. EEG patterns) have not been investigated in these individuals (indicated by question mark). In family 3 the variant (deletion of two consecutive alanine residues at position 299–300 of RBFOX1) did not segregate with the epilepsy phenotype. Abbreviations: n.a = DNA was not available for testing; RE = rolandic epilepsy; CTS = centrotemporal spikes; ESES = encephalopathy with status epilepticus during sleep.

Article Snippet: Segregation in the families of the microdeletions was examined by real-time quantitative PCR using TaqMan CNV probes ( RBFOX1: Hs04461212_cn; RBFOX3: Hs03975574_cn) (Life Technologies, Darmstadt, Germany).

Techniques: Mutagenesis, Variant Assay