The Rbfox family of splicing factors regulate alternative splicing during animal development and in disease, impacting thousands of exons in the maturing brain, heart and muscle. Rbfox proteins have long been known to bind to the RNA sequence GCAUG with high affinity and specificity, but just half of Rbfox binding sites contain a GCAUG motif in vivo. We incubated recombinant RBFOX2 with over 60,000 mouse and human transcriptomic sequences to reveal substantial binding to several moderate-affinity, non-GCAYG sites at a physiologically relevant range of RBFOX2 concentrations. We find that these ‘secondary motifs’ bind Rbfox robustly in cells and that several together can exert regulation comparable to GCAUG in a trichromatic splicing reporter assay. Furthermore, secondary motifs regulate RNA splicing in neuronal development and in neuronal subtypes where cellular Rbfox concentrations are highest, enabling a second wave of splicing changes as Rbfox levels increase.
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nsRBNS raw data are available under accession code GSE152510, and processed data are available in Supplementary Table 2. Owing to their large volume, FACS data are available from the corresponding author upon reasonable request. Data used in other analyses can be found at PDB 2ERR (Fig. 2a), GEO GSE54794 (Fig. 3a,b), SRA SRP128054 and SRP035321 (Fig. 3c–e), SRA PRJNA185305 (Fig. 5) and SRA SRP055008 (Fig. 6a,b). Source data are provided with this paper.
Custom code generated during the current study is available from the corresponding author upon reasonable request.
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We thank past and present members of the Burge laboratory, J. Conboy and I. Jarmoskaite for helpful comments on the manuscript. We gratefully acknowledge the courtesy of the laboratory of C. Zhang (Columbia University), who shared intermediate results from Weyn-Vanhentenryck et al.4 used for Fig. 6 (gene expression, PSI values and exon coordinates). M.J. received EMBO Long-Term Fellowship ALTF-1130-2015. All other authors were supported by NIH grant 5-R01-GM085319.
The authors declare no competing interests.
Peer review information Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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a, Correlations among seven natural sequence nsRBNS experiments. Pearson correlations are reported for any sequence with an enrichment (R) value greater than 1. Darker color indicates a higher correlation (R 1.1.463 cor.test function). n = 38467. b, Correlation of nsRBNS R with eCLIP enrichment at oligo-derived regions for all oligonucleotides or sequence regions containing a single GCAUG Rbfox primary motif (n = 2946). c, R value distribution of nsRBNS sequences containing 0 (n = 21596) or 1-3 (n = 2397) NGCAU motifs. d, R value distribution of nsRBNS sequences containing 0 (n = 11077) or 1-3 (n = 12916) AU motifs. e, RBFOX2 eCLIP in HepG2 at library positions in the transcriptome for 0 (n = 7041) or 1-3 (n = 711) NGCAU motifs. RBFOX2 peaks were compared to a no-protein control to determine enrichments. f, RBFOX2 eCLIP in HepG2 at library positions in the transcriptome for 0 (n = 4610) or 1-3 (n = 3142) AU motifs. RBFOX2 peaks were compared to a no-protein control to determine enrichments.
Extended Data Fig. 2 Different nsRBNS libraries emphasize different 5mer binding preferences for RBFOX2.
a, R value distribution of nsRBNS sequences containing 1-2 copies of different 6mer classes UGNNUG (n = 7725), CGNNUG (n = 1751), AGNNUG (n = 6260), GGNNUG (n = 4935). b-c, Comparison of random (b) and intronic natural sequence (c) RBNS with 3′ UTR nsRBNS 5mer enrichments. Primary and secondary motifs are labelled in red and blue, respectively. Dotted lines show 2.5 standard deviations above the mean. d, Filter binding with radiolabeled oligonucleotides containing three copies of the indicated sequence brought to equilibrium with six concentrations of RBFOX2. Primary motifs in gold, secondary motifs in teal, controls in grey. Error bars indicate +/− SD for three replicates.
a, Some secondary motifs show sharp peaks near 0 in a metaplot centered at the motif in introns (black) and 3′ UTRs (grey) in RBFOX2 iCLIP data27. 5′ ends of iCLIP reads containing the motif of interest 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. See Methods for read counts. b, AU-rich nsRBNS motifs do not show characteristic read peaks near 0 in a metaplot centered at the motif in introns (black) and 3′ UTRs (grey) in RBFOX2 iCLIP data27. iCLIP reads containing the motif of interest 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. See methods for read counts. c, Schematic showing the generation of a clip enrichment (CE) score from iCLIP data. After generation of a metaplot, the read count at the peak apex was divided by the read count at its lowest point to generate a CE score analogous to an enrichment. d, Correlation of iCLIP- and nsRBNS-enriched 5mers in 3′ UTRs (n = 1024). CLIP enrichment (CE) scores were computed for iCLIP peaks. Secondary motifs indicated in teal, primary motifs indicated in gold, outlined circles indicate 5-mers that overlap primary motifs.
5mer enrichment of top 200 5mers in two HiTS-CLIP datasets in both introns and 3′ UTRs. 5mer enrichment was calculated by determining the frequencies of all 1,024 5mers in CLIP peaks in each region and dataset and subsequently normalizing to control peaks from that region. Peaks were analyzed from (a) Mouse ventral spinal neuron (VSN) 3′ UTR HiTS-CLIP, (b,c) Mouse whole brain intronic HiTS-CLIP, and (c) Mouse whole brain 3′ UTR HiTS-CLIP. Gold indicates primary motifs, teal indicates secondary motifs, outlines indicate 5-mers that overlap with primary motifs.
Graphs were drawn with pseudocolor in FlowJo. a, Gating strategy to select for single, live, intact cells. Events were gated through three serial gates to obtain approximately 25000 events for downstream analysis. Total number of events in each graph, and the percentage of events within the gate in each graph are shown. (FSC: forward scatter; SSC: side scatter; A: area; H: height; W: width.) b,c, Compensated values of the three fluorophores used (dsRED, EGFP, Cerulean), in positive and control samples with (b) primary and (c) secondary motifs.
Extended Data Fig. 6 Secondary motifs promote inclusion in a splicing reporter in an RBFOX1-dependent manner at the protein level.
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 Supplementary Table 5. 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).
Extended Data Fig. 7 Secondary motifs become engaged at specific intervals of neuronal differentiation.
Pearson correlation of secondary motif presence with exon inclusion at intervals of neuronal differentiation beginning with embryonic stem cells and progressing to mature 28-day glutamatergic neurons (ESC–NESC (n = 448), NESC–RG (n = 1478), RG–DS1 (n = 940), DS1–DS3 (n = 2189), DS3–MAT16 (n = 1600), MAT16–MAT21 (n = 378), MAT21–MAT28 (n = 373)). Size of point indicates correlation coefficient, intensity indicates p-value < 0.05.
Extended Data Fig. 8 Estimation of secondary motif-dependent Rbfox events across neuronal cell types.
In a comparison of neuronal cell types with medium to highest Rbfox mRNA expression, exons likely to be regulated by Rbfox are significantly enriched in secondary motifs (P < .0084 Fisher’s exact test, ndown = 13; nup = 28). Of 864 alternative exons with increased splicing, 11% are primary, 26.4% primary and secondary, and 3.2% are 4+ secondary motif-associated. Exons with one to three secondary motif instances are also significantly enriched (P < 0.0012, Fisher’s exact test, ndown = 263; nup = 354). Stars indicate significant groupings.
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 Methods) and then linearly correlated with known dissociation constants (Kd) for RBFOX1 binding1,2. Correlation coefficients between log(R’) and log(Kd) were r = −0.95, P-value=8.3 ×10-9 (a) and r = -0.91, P-value=6.7 ×10-7 (b). Scatter plots show estimated Kd as a function of the original, uncorrected R-value. Resulting 7-mer Kd estimates were highly correlated between RBFOX2 and RBFOX3 (c) with r = 0.76, 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 Kds for each 5-mer (see Methods). 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.
a, A high nuclear mRNA expression weighted histogram of potential intronic Rbfox binding sites (1,000,000 mRNAs/cell with average half-life time of 3 hours). Motif 5mers in gold (GCAUG), yellow (GCACG), and teal (GCUUG, GAAUG, GUUUG, GUAUG). b, A low nuclear mRNA expression weighted histogram of potential intronic Rbfox binding sites (10x lower mRNA copies/cell and a half-life time of 4 hours). c, d, Predicted average Rbfox occupancies on 5mer motifs as a function of the nuclear Rbfox concentration in low (c) and high (d) mRNA scenarios. The low mRNA scenario predicts that the fraction of Rbfox bound to secondary motifs surpasses primary motifs at Rbfox levels > 1 μΜ. This is lower than estimates from the high mRNA scenario in main Fig. 6 (~14 μΜ). Non-specific binding depicted in grey. e, Filter binding with radiolabeled oligonucleotide containing three copies of a primary (GCAUG) or secondary (GCUUG, GAAUG, GUUUG) were incubated to equilibrium in the presence of unlabeled, single copy GCAUG oligonucleotide at six concentrations of RBFOX2. As protein concentration increased, so did the fraction bound of labeled RNA for both primary and secondary motifs. Error bars indicate + /- SD of three replicates.
Oligonucleotides in the nsRBNS 3′-UTR library.
Enrichment (R) values of individual oligonucleotides at all concentrations.
Oligonucleotide sequences used in filter binding experiments.
Sequences cloned into RG6 for secondary motif reporter assays.
Cell count per sample for experiments in Fig. 4 and Extended Data Figs. 5 and 6.
Assignment of approximate dissociation constants to all Rbfox 5-mer motif variants by calibrating random RBNS data for human RBFOX2 and RBFOX3 to SPR data.
Summary of statistical tests.
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Begg, B.E., Jens, M., Wang, P.Y. et al. Concentration-dependent splicing is enabled by Rbfox motifs of intermediate affinity. Nat Struct Mol Biol 27, 901–912 (2020). https://doi.org/10.1038/s41594-020-0475-8