Journal: Human Molecular Genetics

Article Title: RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

doi: 10.1093/hmg/dds240

Figure Lengend Snippet: Gene ontologies of RBFOX1 -dependent differentially spliced genes

Article Snippet: Primary antibodies were against HA-tag (1:2000, Covance, Princeton, NJ, USA), RBFOX1 (rabbit polyclonal, 1:500, Aviva Systems Biology, San Diego, CA, USA) or β-actin (mouse monoclonal, 1:250 000, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Gene Expression

Journal: Human Molecular Genetics

Article Title: RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

doi: 10.1093/hmg/dds240

Figure Lengend Snippet: WGCNA in the RBFOX1 knockdown cell line reflects pathways important to neurodevelopment and to autism. For clarity, only the most highly connected module members are shown. Genes with the highest connectivity (i.e. hubs) are indicated in red. (A) Blue module. (B). Yellow module. Differentially expressed ASD genes are in purple.

Article Snippet: Primary antibodies were against HA-tag (1:2000, Covance, Princeton, NJ, USA), RBFOX1 (rabbit polyclonal, 1:500, Aviva Systems Biology, San Diego, CA, USA) or β-actin (mouse monoclonal, 1:250 000, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Knockdown

Journal: Human Molecular Genetics

Article Title: RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

doi: 10.1093/hmg/dds240

Figure Lengend Snippet: RNA sequencing detects altered alternative splicing patterns in PHNP cells with RBFOX1 knockdown. (A) Heat map showing clustering of gene expression among five biological replicates representing three experimental conditions; wild-type (black), RBFOX1 knockdown (shRBFOX1, red) and non-targeting RNA interference (shGFP, green). Analysis is of the top 250 most significant genes, using the Bayes method with a Spearman correction. (B) Analysis of the intronic regions 400 nucleotides upstream and downstream of the alternative exons whose splicing was most significantly affected by RBFOX1 knockdown for the presence of the binding sites for RBFOX1, NOVA1 or PTBP1. Observed sites are shown as well as the number predicted by iterative analysis of an equivalent number of random introns culled from all human genes. Enrichment of the various sites is indicated by gray boxes and arrows. Significance is based on the normal distribution. ns, not significant. A schematic illustration of the predicted effects on alternative splicing based on the location of the RBFOX1-binding site is shown, with downstream sites enhancing and upstream sites repressing exon inclusion. The correlation between RBFOX1-binding site location and splicing changes identified by RNA sequencing in this study is shown. (C) Validation of splicing changes detected by RNA sequencing. Exons are labeled using a sequential annotation based on location within the gene. Genomic coordinates can be found in Supplementary Material, File S1. qRT-PCR or semi-qRT-PCR was used to calculate the ratio of exon inclusion in the RBFOX1 knockdown cells lines when compared with the shGFP control line. A selection of 25 genes is shown with the differential fold change (log base 2) in exon inclusion detected by RNA sequencing shown in red and the observed fold change by RT-PCR shown in blue. Standard error of the mean is indicated by black bars. (D) Comparison of the RBFOX1 gene set with published gene lists. The number of overlapping genes is indicated along with the percentage they represent from each list. Lists were derived from the references indicated and are also shown in Supplementary Material, File S3. Online sources for gene lists include the Genes to Cognition (G2C) database (http://www.genes2cognition.org/), the Organelle DB (http://organelledb.lsi.umich.edu/), the Online Mendelian Inheritance in Man database (http://www.omim.org/), the GeneTests database (http://www.ncbi.nlm.nih.gov/sites/GeneTests/) and the Simons Foundation Autism Research Initiative database (https://sfari.org/). Lists referenced as supplemental are composites of multiple lists derived from the above sources. P-values were determined based on hypergeometric probability. ER, endoplasmic reticulum.

Article Snippet: Primary antibodies were against HA-tag (1:2000, Covance, Princeton, NJ, USA), RBFOX1 (rabbit polyclonal, 1:500, Aviva Systems Biology, San Diego, CA, USA) or β-actin (mouse monoclonal, 1:250 000, Sigma-Aldrich, St Louis, MO, USA).

Techniques: RNA Sequencing, Alternative Splicing, Knockdown, Gene Expression, Binding Assay, Biomarker Discovery, Labeling, Quantitative RT-PCR, Control, Selection, Reverse Transcription Polymerase Chain Reaction, Comparison, Derivative Assay

Journal: Human Molecular Genetics

Article Title: RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

doi: 10.1093/hmg/dds240

Figure Lengend Snippet: Characterization of differential gene expression in RBFOX1 knockdown cells. (A) A selection of 44 genes is shown with the differential fold change (log base 2) in gene expression detected by RNA sequencing shown in red and the observed fold change by qRT-PCR shown in blue. Standard error of the mean is indicated by black bars. (B) Comparison of the RBFOX1 differentially expressed gene set with published gene lists. The number of overlapping genes is indicated along with the percentage they represent from each list. Lists were derived from the references indicated and are also shown in Supplementary Material, File S6. Online sources for gene lists are as described for Figure 2. Lists referenced as supplemental are composites of multiple lists derived from the above sources. P-values were determined based on hypergeometric probability. ER, endoplasmic reticulum.

Article Snippet: Primary antibodies were against HA-tag (1:2000, Covance, Princeton, NJ, USA), RBFOX1 (rabbit polyclonal, 1:500, Aviva Systems Biology, San Diego, CA, USA) or β-actin (mouse monoclonal, 1:250 000, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Gene Expression, Knockdown, Selection, RNA Sequencing, Quantitative RT-PCR, Comparison, Derivative Assay

Journal: Human Molecular Genetics

Article Title: RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

doi: 10.1093/hmg/dds240

Figure Lengend Snippet: Characterization of RBFOX1 expression in human brain and fetal-derived neural progenitor cells. (A) A schematic illustration of the RBFOX1 genomic organization is shown. Figure adapted from Underwood et al. (9). Untranslated exons are shown in light gray, translated exons are in white. The brain-specific exon 16 is shown in dark gray, whereas the muscle-specific exon 17 is shown in black. The location of the RNA-binding domain (RRM) is indicated. (B) The expression level of RBFOX1 was assessed by qRT-PCR, with mRNA from PHNP cells differentiated for the indicated times. Primers were directed against exons 8–9 to detect all RBFOX1 isoforms (light gray). Autoregulatory alternative splicing of exon 11 eliminates RNA binding, so primers against exons 9–11 were utilized to detect isoforms with an active RNA-binding domain (dark gray). (C) In situ hybridization was performed with a human fetal brain, age of 19 weeks, using an S35-labeled antisense riboprobe directed against exons 8–13 of RBFOX1. Two representative coronal and sagittal sections are shown. The sense probe is used as a control (lower panels). (D) To quantitate the pattern of RBFOX1 isoforms expressed in the indicated tissues and cell lines, RT-PCR was performed using primers to amplify exons 15–20, which represent the largest region of alternative splicing diversity in the gene. Amplified products were subcloned and sequenced. Total clones are indicated with the representative counts and percentages of the various alternative spliced isoforms. The most highly expressed patterns are highlighted in gray. c, caudate; cp, cortical plate; gz, germinal zone; p, putamen; t, thalamus.

Article Snippet: Primary antibodies were against HA-tag (1:2000, Covance, Princeton, NJ, USA), RBFOX1 (rabbit polyclonal, 1:500, Aviva Systems Biology, San Diego, CA, USA) or β-actin (mouse monoclonal, 1:250 000, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Expressing, Derivative Assay, RNA Binding Assay, Quantitative RT-PCR, Alternative Splicing, In Situ Hybridization, Labeling, Control, Reverse Transcription Polymerase Chain Reaction, Amplification, Clone Assay

Journal: Human Molecular Genetics

Article Title: RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

doi: 10.1093/hmg/dds240

Figure Lengend Snippet: Gene ontologies of RBFOX1 -related differentially expressed genes

Article Snippet: Primary antibodies were against HA-tag (1:2000, Covance, Princeton, NJ, USA), RBFOX1 (rabbit polyclonal, 1:500, Aviva Systems Biology, San Diego, CA, USA) or β-actin (mouse monoclonal, 1:250 000, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Expressing, Migration, Transmission Assay

Journal: Human Molecular Genetics

Article Title: RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

doi: 10.1093/hmg/dds240

Figure Lengend Snippet: A model of RBFOX1 function in PHNPs. During neuronal differentiation, RBFOX1 is induced and directly modulates (solid arrows) an RNA splicing network (black box) which in turns coordinately regulates a transcriptional network of additional genes (black box). Downstream genes present within these networks can further modulate either RNA splicing or transcription to generate additional layers of control (dashed arrows). RBFOX1 can further alter its own splicing and downregulate the activity of these networks as indicated. Additional regulatory factors and/or programs can also contribute to the coordinate regulation of these networks (dotted arrows). The number of factors identified in this study at each of the steps is indicated with selected proteins of interest as mediators noted with their predicted regulatory contribution indicated, see text for complete details. The genes within these networks affect a number of key cellular developmental processes that promote neural development and synaptogenesis, leading to the formation of mature neurons. Disruption of these pathways, particularly genes in the transcriptional network, can lead to neurodevelopmental and/or neuropsychiatric phenotypes in humans. The asterisk indicates factors involved in splicing and/or other aspects of RNA-processing.

Article Snippet: Primary antibodies were against HA-tag (1:2000, Covance, Princeton, NJ, USA), RBFOX1 (rabbit polyclonal, 1:500, Aviva Systems Biology, San Diego, CA, USA) or β-actin (mouse monoclonal, 1:250 000, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Control, Activity Assay, Disruption

Journal: Science Advances

Article Title: Trans-splicing facilitated by RNA pairing greatly expands sDscam isoform diversity but not homophilic binding specificity

doi: 10.1126/sciadv.abn9458

Figure Lengend Snippet: See also figs. S1 and S2. ( A ) Phylogenetic distribution of sDscam and isoform members in chelicerates. The variables Ig1s and Ig2s are indicated by green and red circles, respectively. Data from other species are referenced from our previous study . ( B ) Schematic of an sDscam locus. The 5′ untranslated region of sDscam β 4 is represented by a gray rectangle. The arrow indicates transcriptional direction. Cis- and trans-spliced isoforms are represented by blue lines (above) and other colored lines (below), respectively. The color connections are supported by RNA-seq and RT-PCR data. Var, variable; Con (C), constant. ( C ) Quantification of the cis- and trans-spliced isoforms. RPM, reads per million. ( D ) Validation of alternative combinations of 5′ and 3′ alternative exons. Because of the low expression of variable exons, nested PCR was required to amplify the products; only the primers used in the second PCR are depicted (table S2). ( E to J ) Evidence of trans-splicing between different genes. These combinations included sDscam β 2 and sDscam β 1 (E), sDscam β 3 and sDscam β 1 (F), sDscam β 2 –β3 and sDscam β 4 (G), sDscam β 1 / sDscam β 3 and sDscam β 2 (H), sDscam β 1 and sDscam β 3 (I), and sDscam β 2 and sDscam β 3 (J).

Article Snippet: The primary antibodies were used in isoform coimmunoprecipitation: anti-HA (hemagglutinin) tag rabbit polyclonal antibody (1:50; EarthOx, catalog no. E022180-01, RRID:AB_2811272).

Techniques: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Nested PCR

Journal: Science Advances

Article Title: Trans-splicing facilitated by RNA pairing greatly expands sDscam isoform diversity but not homophilic binding specificity

doi: 10.1126/sciadv.abn9458

Figure Lengend Snippet: See also fig. S12. ( A and B ) Domain-specific recognition of the N-variable mediated by the sDscamβ Ig domain shuffled isoform. Domain-shuffled chimeras of sDscamβ isoforms and their parental counterparts were assayed for their binding specificity. Chimeras in which the Ig1 domain was replaced by the corresponding domain swapped binding specificity, whereas the Ig2 replacement did not. ( C ) sDscamβ1 pairs with the same variable Ig1 domain do not display recognition specificity. Mean coaggregation indices are shown in the top right corner of each representative image (scale bars, 100 μm). ( D ) Schematic diagram of trans interactions of sDscamβ. Structural modeling shows that the Ig1 domain of sDscamβ interacts in an antiparallel manner.

Article Snippet: The primary antibodies were used in isoform coimmunoprecipitation: anti-HA (hemagglutinin) tag rabbit polyclonal antibody (1:50; EarthOx, catalog no. E022180-01, RRID:AB_2811272).

Techniques: Binding Assay

Journal: Science Advances

Article Title: Trans-splicing facilitated by RNA pairing greatly expands sDscam isoform diversity but not homophilic binding specificity

doi: 10.1126/sciadv.abn9458

Figure Lengend Snippet: See also fig. S13. ( A to D ) Cells coexpressing different combinations of differentially tagged sDscamβ isoform pairs were mixed and assayed for their coaggregation. β1 cis-spliced isoforms (A), β1 trans-spliced isoforms (B), β1/β2 cis-spliced isoforms (C), and β1/β2 trans-spliced isoforms (D) were measured. ( E ) The combination of cis- and trans-spliced sDscamβ pairs with the same variable Ig1-Ig2 domains did not exhibit the combinatorial homophilic specificity. ( F ) Analysis of the interaction of cells coexpressing three different GFP tags with cells expressing the same or different groups of mCherry tags. The underline marks the mismatched isoforms between the two cell groups. Mean coaggregation indices for (A) to (F) are shown in the top right corner of each representative image (scale bars, 100 μm). ( G ) Schematic diagram of the outcome of combinatorial homophilic specificity. The diagram shown here does not reflect cis multimers.

Article Snippet: The primary antibodies were used in isoform coimmunoprecipitation: anti-HA (hemagglutinin) tag rabbit polyclonal antibody (1:50; EarthOx, catalog no. E022180-01, RRID:AB_2811272).

Techniques: Expressing

Journal: Science Advances

Article Title: Trans-splicing facilitated by RNA pairing greatly expands sDscam isoform diversity but not homophilic binding specificity

doi: 10.1126/sciadv.abn9458

Figure Lengend Snippet: ( A ) Generation of extensive sDscam isoforms through a combination of alternative promoter choices and cis- and trans-alternative splicing. On the left is the schematic representation generating cis-spliced sDscamβ isoform diversity. This alternative cis-splicing process is mediated by a competing RNA secondary structure between the docking site and selector sequences. On the right is a schematic representation of the generation of trans-spliced sDscamβ isoforms. This trans-splicing process is facilitated by intronic intermolecular RNA secondary structures. ( B ) Schematic representation of sDscamβ diversity mediated by cotranscriptional RNA folding and alternative cis- and trans-splicing. These nascent transcripts generated from this single locus are geometrically close to each other before leaving their transcription sites, which facilitates trans-splicing between different sDscam β transcripts.

Article Snippet: The primary antibodies were used in isoform coimmunoprecipitation: anti-HA (hemagglutinin) tag rabbit polyclonal antibody (1:50; EarthOx, catalog no. E022180-01, RRID:AB_2811272).

Techniques: Generated