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Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges

Nature Structural & Molecular Biology volume 20, pages 1434–1442 (2013)Cite this article

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Abstract

Alternative splicing (AS) enables programmed diversity of gene expression across tissues and development. We show here that binding in distal intronic regions (>500 nucleotides (nt) from any exon) by Rbfox splicing factors important in development is extensive and is an active mode of splicing regulation. Similarly to exon-proximal sites, distal sites contain evolutionarily conserved GCATG sequences and are associated with AS activation and repression upon modulation of Rbfox abundance in human and mouse experimental systems. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns and showed that a conserved long-range RNA-RNA base-pairing interaction (an RNA bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene. Thus we demonstrate a previously unknown RNA-mediated mechanism for AS control by distally bound RNA-binding proteins.

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Figure 1: Characteristics of Rbfox binding in distal intronic regions.
Figure 2: The Rbfox binding motif TGCATG is the most enriched hexamer in conserved regions in distal intronic space around alternatively spliced exons.
Figure 3: Both proximal and distal Rbfox motifs regulate splicing.
Figure 4: Distal conserved regions containing Rbfox sites control splicing of upstream alternative exons.
Figure 5: An RNA bridge between ENAH E11a and a conserved distal RBFOX site is necessary for exon inclusion.

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Acknowledgements

The authors would like to thank A. Pasquinelli, N. Chi, K. Willert and L. Goldstein and members of the Yeo, Conboy and Goldstein labs for critical reading of the manuscript. M.T.L. is supported as a National Science Foundation GK12 Fellow. This work was supported by grants from the National Institute of Health to G.W.Y. (U54 HG007005, R01 HG004659, R01 GM084317 and R01 NS075449) and to J.G.C. (HL045182 and DK094699) and partially supported by grants to J.W.G. (CA112970 and CA126551). J.G.C. also acknowledges support from DK032094. This work was also supported by the Director, Office of Science, and Office of Biological & Environmental Research of the US Department of Energy under Contract No. DE-AC02-05CH1123. D.L.B. and L.T.G. were supported by US National Institutes of Health grant RO1 GM49662 to D.L.B. D.L.B. is an Investigator of the Howard Hughes Medical Institute. M.T.L. and G.W.Y. are grateful for a gift from P. Yang at Genentech that supported M.T.L. G.W.Y. is supported as an Alfred P. Sloan Research Fellow.

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Author notes

  1. Katlin B Massirer

    Present address: Present address: State University of Campinas, Sao Paulo, Brazil.,

Authors and Affiliations

  1. Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USA

    Michael T Lovci, Justin Arnold, Tiffany Y Liang, Thomas J Stark, Katlin B Massirer, Gabriel A Pratt & Gene W Yeo

  2. Stem Cell Program, University of California, San Diego, La Jolla, California, USA

    Michael T Lovci, Justin Arnold, Tiffany Y Liang, Thomas J Stark, Katlin B Massirer, Gabriel A Pratt & Gene W Yeo

  3. Institute for Genomic Medicine, University of California, San Diego, La Jolla, California, USA

    Michael T Lovci, Justin Arnold, Tiffany Y Liang, Thomas J Stark, Katlin B Massirer, Gabriel A Pratt & Gene W Yeo

  4. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Dana Ghanem, Henry Marr, Sherry Gee, Marilyn Parra & John G Conboy

  5. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA

    Lauren T Gehman & Douglas L Black

  6. Howard Hughes Medical Institute, University of California, Los Angeles, California, USA

    Lauren T Gehman & Douglas L Black

  7. Molecular Engineering Laboratory, Biomedical Sciences Institutes, Agency for Science, Technology & Research, Singapore

    Shawn Hoon & Gene W Yeo

  8. School of Biological Sciences, Nanyang Technological University, Singapore

    Shawn Hoon

  9. Department of Biomedical Engineering, Oregon Health & Science University, Portland, Oregon, USA

    Joe W Gray

  10. Yong Loo Lin School of Medicine, National University of Singapore, Singapore

    Gene W Yeo

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Contributions

M.T.L. and G.A.P. conducted the bioinformatics analyses. D.G., H.M., J.A., S.G., M.P., T.Y.L., T.J.S., S.H. and K.B.M. conducted biological experiments. L.T.G. and D.L.B. generated the Rbfox mutant mice and isolated brain RNA. J.W.G., J.G.C. and G.W.Y. designed the study. M.T.L., D.G., J.G.C. and G.W.Y. wrote the manuscript with input from all authors.

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Correspondence to John G Conboy or Gene W Yeo.

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Integrated supplementary information

Supplementary Figure 1 CLIP-seq to identify Rbfox binding sites, motifs and gene ontology analyses.

(a) Autoradiograph of Rbfox protein-RNA complexes from mouse brain immunoprecipitated with Rbfox-specific antibodies and trimmed with optimized unit (U). concentrations of micrococcal nuclease (MNase). (b) Venn diagram showing the number of genes bound by Rbfox1 and Rbfox2 in mouse brain. (c) List of the top three motifs significantly enriched in the Rbfox1 CLIP compared to appropriate background controls. Similar results were found for Rbfox2 (not shown). (d) Box-plots of hexamer Z-scores, comparing Rbfox1 CLIP clusters to randomly located clusters in each genic region. GCAUG and UGCAUG, the known Rbfox motifs, and a GU-rich 6-mer were enriched in all genic regions except 5′ UTRs. (e) Venn diagrams showing the number of Rbfox1 (red) and Rbfox2 (blue) CLIP-seq clusters that overlap (yellow) each other by ≥ 1nt (“All clusters”). Clusters are then restricted to those within 50 nt of a GCAUG motif in mouse (“GCAUG in mouse”), or where the GCAUG motif is conserved (“Conserved GCAUG”) at increasing branch-length (BL) scores (the higher the score, the more conserved the GCAUG sequence across multiple genomes). Analyses of proximal (“PI”) and distal intronic (“DI”) clusters are displayed on the left and right columns. (f) Bar plots represent the fraction of clusters that overlap relative to the Rbfox1 (red) or Rbfox2 (blue) clusters corresponding to the different restrictions in (e). (g) Dendrogram showing hierarchical clustering with Euclidean distance of statistically significant gene ontology terms (in at least one experimental condition) using negative log10 P values that measure the enrichment of genes within the ontology terms for various experimental conditions.

Supplementary Figure 2 Genome browser views of selected alternatively spliced genes containing distal RBFOX binding sites.

(a-e) Exons and introns are indicated in blue (UCSC genes) or red (Ensembl Genes) with thick lines representing exons and thin lines overlaid with arrows representing introns. The direction of the arrows denotes the direction of transcription. Read density from Rbfox1 and Rbfox2 CLIP-seq (orange and green tracks respectively) on several genes is shown with positive and negative values corresponding to the direction of transcription of aligned reads. Shank1 (a), Snap25 (b), Kcnma1 (c), Rbfox1 (d) and Rbfox2 (e) possess alternatively spliced exons (red arrows). PhastCons evolutionary conservation scores for placental mammals are displayed at the bottom of each panel in dark green. Genomic GCATG sites are indicated above the conservation track in black. Distal and proximal highly conserved regions are designated along the top with purple and green filled rectangles, respectively. Scale bars at the top define the size of region displayed.

Supplementary Figure 3 Discovery and characterization of conserved regions.

Contiguous regions within the human transcriptome were divided into three categories based on their degree of evolutionary conservation as determined by phastCons scores, S. Conserved regions that overlap known repetitive elements and transcription factor binding sites were removed. Pie charts illustrate the distribution of the three categories of lowly (L), moderately (M) and highly (H) conserved regions of approximately similar lengths (box-plots on the right) within different genic regions (5′UTR, exon, proximal intron, distal intron and 3′UTR) in protein-coding genes. Bar charts show the fraction of total nucleotides in each genic region covered by a highly-conserved region. (b) Results from a de novo motif search using HOMER1 for enriched hexamer motifs is shown for distal and proximal highly-conserved regions from (a). Up to 10 motifs are shown with their associated P values indicated to the right.

Supplementary Figure 4 Identification of Rbfox-dependent expression and splicing changes by RNA-seq.

(a, b) Scatter plots of RPKM (reads per kilobase mapped in exons per million reads) values between Rbfox1 and Rbfox2 knockout (KO; y-axis) compared to wildtype (WT; x-axis) sibling pairs showed that 46 and 6 genes are significantly up-regulated (red points), and 11 and 9 genes are down-regulated (green points) upon loss of Rbfox1 and Rbfox2 in mouse brain, respectively (P < 0.001). (c–e) Scatter plots of RPKM values comparing ectopic expression (EE) of RBFOX1, RBFOX2 and RBFOX3 (y-axes) to a plasmid control (x-axis). (f) Scatter plots of percent-spliced-in (ψ) values of cassette exons comparing Rbfox1 and Rbfox2 KO (y-axes) to WT (x-axes); and ectopic expression (EE) of RBFOX1, RBFOX2 and RBFOX3 to plasmid control. Mouse exons that are alternatively spliced (|Δψ| ≥ 5%) upon loss of Rbfox1 (in red), Rbfox2 (in blue) and either (orange) are marked on top two plots. Human exons that are alternatively spliced upon EE of RBFOX1 (in red), RBFOX2 (in blue) and RBFOX3 (in green) are marked on the bottom three plots. A linear fit of the data (black line) and associated R2 value are shown. (g) Venn diagrams show the number of cassette exon splicing events that are in common upon loss of Rbfox1 and Rbfox2 in mouse (top) and upon ectopic expression of RBFOX proteins in human 293T cells (bottom), considering exons with |Δψ| ≥ 5% (any change, left), Δψ ≥ 5% (included, right top) and Δψ ≤ −5% (excluded, right bottom). RBFOX1-, RBFOX2- and RBFOX3-specific events are colored red, blue and green, with orange and dark green representing shared events. (h) A scatter plot shows Δψ values from RT-PCR2,3 (x-axis) and RNA-seq-derived RPKM (y-axis) measurements of exon inclusion for manually validated cassette splicing exon events in mouse brain for Rbfox1 (red) and Rbfox2 (blue). A linear fit to each set of points and associated R2 value are shown. (i) Bar-plots show a comparison between Δψ values using RT-PCR or RNA-seq. RT-PCR values were obtained from refs. 2,3.

Supplementary Figure 5 Distal association of Rbfox sites with Rbfox-dependent alternatively spliced exons.

(a, b) The fractions of alternatively spliced exons compared to unchanged that have GCATG motifs (at increasing levels of conservation) and CLIP-defined Rbfox1 and Rbfox2 sites either upstream or downstream, for both distal (“DI”) and proximal (“PI”) regions, are represented as vertical bars. Rbfox1 and Rbfox2 knockout (KO) mice compared to sibling WT pair are represented in (a). Ectopic expression (EE) of RBFOX1, RBFOX2 and RBFOX3 are represented in (B). The fractions are represented on the log-scale (y-axis). Blue bars represent exons with RNA-seq evidence for inclusion (Δψ > 5%); goldenrod bars represent exons with RNA-seq evidence for exclusion (Δψ < −5%); grey bars represent exons with no change by RNA-seq (−2% < Δψ < 2%). Ties with “*” symbols indicate statistically significant differences when categories of exons are compared (*P < 0.05, **P < 0.001; Fisher's exact test). The maximal change for each exon was used when combining evidence from all experiments, as represented by their unions (U; y-axis) (the final rows for (a) and (b)). (c) The cumulative distributions of Δψ values for exons with >1 GCAUG motif or CLIP-defined Rbfox binding site within either upstream or downstream, for both distal (“DI”) and proximal (“PI”) regions were compared to exons with no flanking conserved GCATG motifs. P values by two-sample Kolgomorov-Smirnov tests are indicated when statistically significant (P < 0.05).

Supplementary Figure 6 Un-cropped gel images.

Un-cropped gel images are shown for RT-PCR and biotin pull-down assays. These include slow-migrating bands, suspected heteroduplexes, which are excluded from main figures.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 12812 kb)

Supplementary Table 1

CLIP-seq library statistics (XLS 22 kb)

Supplementary Table 2

Gene ontology results (XLS 4488 kb)

Supplementary Table 3

Gene expression analysis (XLS 17451 kb)

Supplementary Table 4

Splicing analysis (XLS 15717 kb)

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Lovci, M., Ghanem, D., Marr, H. et al. Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges. Nat Struct Mol Biol 20, 1434–1442 (2013). https://doi.org/10.1038/nsmb.2699

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