(A) Diagram of Rbfox1 domains and deletion mutants.
(B) Co-immunoprecipitation of LASR with Rbfox1 and mutants. Soluble and high molecular weight (HMW) nuclear fractions were prepared from Flp-In T-REx 293 cells stably expressing either full-length HA-FLAG-SV40NLS-Rbfox1 (FL) or a deletion mutant as diagrammed in (A). Anti-FLAG immunoprecipitates were eluted with FLAG peptide, separated on gels, and stained with SYPRO Ruby. Arrowheads indicate HA-FLAG-SV40NLS-Rbfox1 protein and its mutants. LASR subunits are indicated on the right.
(C) Sedimentation of Rbfox1 complexes in glycerol density gradients. HMW fractions of cells expressing Rbfox1 or an Rbfox1 fragment were loaded onto 10–50% glycerol density gradients. Gradient fractions from top to bottom run from left to right. Proteins from odd fractions were immunoblotted with anti-FLAG antibody. 40S and 60S markers are indicated below.
(D) Amino acid sequences encoded by exons B40 and M43 in Rbfox1 and Rbfox2 (top). Sedimentation of Rbfox1 and Rbfox2 splice variants in glycerol density gradients as in (C) (bottom).
(E) CTD sequence alignments for the nuclear B40 isoforms of Rbfox1, Rbfox2 and Rbfox3. Tyrosines examined by mutagenesis are shown in red. Sequences of C1, C2 and C3 fragments of CTD are indicated by arrows.
(F) Sedimentation of Rbfox1 tyrosine mutants in glycerol density gradients. Rbfox1 proteins containing increasing numbers of mutated tyrosines were separated as in (C). Three to ten tyrosine residues were mutated to serine, alanine or phenylalanine as indicated on the left.
(G) Immunoprecipitation of LASR with HA-FLAG-Rbfox1 containing tyrosine-to-serine mutations. Rbfox proteins and LASR subunits are indicated on the right.
See also .

(A) Top: Diagram of Rbfox1 mutants containing different fragments of the CTD. Bottom: Immunoprecipitation of LASR with the Rbfox proteins. Arrowheads indicate the Rbfox protein and its mutants. LASR subunits are indicated on the right. The asterisk marks a non-specific band seen in the control IP.
(B) Sedimentation of Rbfox1 CTD mutants in glycerol density gradients.
See also .

(A) Top: Diagram of the SNAP-tagged Rbfox1 CTD with a C-terminal 6xHis tag and its ten-tyrosine-to-serine mutant (CTD-YS). Bottom: Epifluorescence microscopic images of aggregates formed by SNAP-CTD and SNAP-CTD-YS in 37.5 mM and 150 mM NaCl in vitro, at protein concentrations indicated on the top. Images were taken 21 hours and 48 hours after lowering the salt concentration. Scale bar: 5 µm.
(B) Top: Diagram of the SNAP-tagged Rbfox1 C2 fragment and its ten-tyrosine-to-serine mutant. Bottom: Confocal fluorescence microscopy images of structures formed by SNAP-C2 and SNAP-C2-YS in 37.5 mM and 150 mM NaCl. Images were taken 24 hours after lowering the salt concentration. Scale bar: 5 µm.
(C) Top: Diagram of the SNAP and mEGFP-tagged Rbfox1 C2 fragment and its ten-tyrosine-to-serine mutant. Bottom: Confocal fluorescence microscopy images of the C2 fragment and C2-YS mutant. Solutions contained 0.5 µM of each protein and images were taken 24 hours after the addition of 10% PEG8000. Scale bar: 5 µm.
(D) Top: Diagram of the mEGFP-FLAG-tagged Rbfox1 protein and its mutants. Bottom: Confocal fluorescence microscopy images of cells stably expressing mEGFP-tagged Rbfox1 and its mutants. All images were taken with the same gain settings and other parameters, except the mEGFP signal of SV40NLS-ΔCTD where the gain was increased for better visualization. Additional images are provided in . Scale bar: 10 µm.
(E) Analysis of EGFP fluorescence in cells stably expressing mEGFP-FLAG-Rbfox1 (WT), and mEGFP-FLAG-Rbfox1 (10Y). The pixel intensity was measured across multiple nuclei, excluding the nucleoli, to obtain the mean and standard deviation of the fluorescent signal. Standard deviation from the mean was plotted against the mean fluorescence for 57 WT and 64 10Y mutant cells and fitted by linear regression. A P-value of 0.00263 was determined using a two-sided analysis of covariance test for comparison of WT versus 10Y slopes.
See also , and .

(A) Diagram of the DUP-E33 and DUP-E9* minigene reporters. The downstream and upstream UGCAUG sites are indicated. The sequence of the mutant element is shown below.
(B) In vivo splicing assays of E33WT, E33mt, E9*WT, and E9*mt minigene reporters transiently expressed in cells carrying various Rbfox proteins. Gel electrophoresis of RT-PCR splicing assays is shown with spliced products indicated on the right. Bar graphs of PSI (percentage spliced in) calculated from three independent experiments are shown above each gel. Error bars indicate the SEM. **** indicates p < 0.0001 by unpaired, one-tailed Student's t test between the Rbfox1 CTD deletion (ΔCTD) and full length Rbfox1 (FL).
(C) Immunoblot of Rbfox protein expression for the splicing assays in (B). Whole cell lysates were prepared from Flp-In T-REx 293 Rbfox2^−/− cells transiently expressing full-length HA-FLAG-SV40NLS-Rbfox1 and its CTD deletion mutant and probed with antibodies to FLAG and SNRNP70.
(D) Assay of E33WT and E33mt exon splicing regulated by Rbfox tyrosine mutants. Gel electrophoresis of RT-PCR products from the DUP-E33WT and DUP-E33mt minigenes upon co-expression with the HA-FLAG-Rbfox1 tyrosine-to-serine mutants indicated on top. Spliced products are indicated on the right. Bar graphs of PSI calculated from three independent experiments are shown above each gel. Error bars indicate SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001 by unpaired, one-tailed Student's t test between each Rbfox1 tyrosine-to-serine mutant and wild type Rbfox1.
(E) Assay of DUP-E9*WT and DUP-E9*mt minigenes coexpressed with Rbfox tyrosine mutants. Products were separated and analyzed as in (D). Bar graphs showing PSI calculated from three independent experiments are shown above each gel. Error bars indicate the SEM. n.s., not significant by unpaired, one-tailed Student's t test between each Rbfox1 tyrosine-to-serine mutants and wild type Rbfox1.
(F) Immunoblot of Rbfox protein expression in the cells used for splicing assays in (D, E). Whole cell lysates were prepared from Flp-In T-REx 293 Rbfox2^−/− cells transiently expressing HA-FLAG-Rbfox1 tyrosine mutants and probed with antibodies to FLAG and GAPDH.
(G) Diagram of the DUP-E33MS2 and DUP-E9*MS2 minigene reporters. The downstream and upstream MS2 stem-loop sites are indicated.
(H) Diagram of Rbfox1 mutants fused to MS2 coat protein (MCP). All proteins were FLAG-SV40NLS tagged.
(I) Splicing assays of the DUP-E33MS2 and DUP-E9*MS2 minigenes coexpressed with Rbfox1 mutants. Products were separated and analyzed as in (D). Bar graphs showing PSI calculated from three independent experiments are shown below each gel. Error bars indicate the SEM. Significance differences are indicated by * p < 0.05, ** p < 0.01, or n.s. (not significant) by unpaired, two-tailed Student's t test between each MCP-Rbfox1 fusion and MCP protein alone, or the pair indicated.
(J) Immunoblot of MCP-Rbfox1 fusion protein expression in cells used for splicing assays in (I). Whole cell lysates were prepared as in (F) and probed with antibodies to FLAG and SNRNP70.
See also and .

(A) Heat map of exon In/Ex values for 206 cassette exons altered by Rbfox1 in Flp-In T-REx 293 Rbfox2^−/− cells and measured by RASL-seq. Values were subjected to hierarchical clustering with rows corresponding to exons and columns to conditions of an expressed Rbfox protein and replicates. These conditions included the F126A mutant (FA), no protein (Ctrl), wild type Rbfox1 (WT), and the 10 tyrosine to serine mutant (10Y), each with three replicates. The clustering defined three exon groups exhibiting different responses to Rbfox1 and the 10Y mutant.
(B) Boxplots of the Log2 fold change between the WT In/Ex ratio and the 10Y In/Ex ratio plotted separately for Groups 1, 2 and 3. Each dot represents a cassette exon splicing event. The middle line indicates the median fold change in In/Ex ratio, with the box enclosing the quartiles of fold change values above and below the median.
See also and –.