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. Author manuscript; available in PMC: 2021 Feb 17.
Published in final edited form as: Nat Struct Mol Biol. 2020 Aug 17;27(10):901–912. doi: 10.1038/s41594-020-0475-8

Figure 5. Secondary motifs enable splicing regulation at distinct concentration Rbfox concentration ranges in neuronal differentiation.

Figure 5.

a. Total expression of Rbfox1, Rbfox2, and Rbfox3 in transcripts per million (TPM) based on RNA-seq during a neuronal differentiation time course39 (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.