Abstract
The U2AF heterodimer has been well studied for its role in defining functional 3′ splice sites in pre-mRNA splicing, but many fundamental questions still remain unaddressed regarding the function of U2AF in mammalian genomes. Through genome-wide analysis of U2AF-RNA interactions, we report that U2AF has the capacity to directly define ~88% of functional 3′ splice sites in the human genome, but numerous U2AF binding events also occur in intronic locations. Mechanistic dissection reveals that upstream intronic binding events interfere with the immediate downstream 3′ splice site associated either with the alternative exon, to cause exon skipping, or with the competing constitutive exon, to induce exon inclusion. We further demonstrate partial functional impairment with leukemia-associated mutations in U2AF35, but not U2AF65, in regulated splicing. These findings reveal the genomic function and regulatory mechanism of U2AF in both normal and disease states.
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Acknowledgements
The authors are grateful to members of the Fu laboratory for numerous stimulating discussions during the course of this investigation. This work was supported by China 973 program grants (2011CB811300 and 2012CB910800), a Chinese 111 program grant (B06018) and US National Institutes of Health grants (HG004659 and GM049369) to X.-D.F. and a US National Institutes of Health grant (DK098808) to D.-E.Z. and X.-D.F.
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T.W., Y. Zhang, D.-E.Z. and X.-D.F. designed the experiments; C.S. and T.W. performed the biochemical experiments; B.Y., J.H., Y. Zhou, A.D. and J.Z. were responsible for bioinformatics analysis of genomics data; J.Q. and H.L. performed RASL-seq and RNA-seq; P.T., L.J., G.C., H.S. and D.-E.Z. contributed to additional data analysis; and T.W., C.S., B.Y. and X.-D.F. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Mapping of U2AF65 CLIP tags to the human genome.
(a) Western analysis of the IP efficiency of anti-U2AF65 antibody. (b) Comparison between U2AF65 CLIP-seq data generated in the current study with the published U2AF65 iCLIP-seq data17. (c,d,e) Profiles of base deletion (c), insertion (d) and substitution (e) detected by CIMS analysis of U2AF65-RNA interactions37. (f) Preferential deletion mutation on uridine residues in CIMS. (g) Footprint of U2AF65 binding on RNA based on CIMS.
Supplementary Figure 2 Enriched motifs in U2AF65-binding sites.
(a) List of top 20 enriched hexamers. (b) Percentage of U2AF65 binding sites that contain one or more top 50 motifs (red), compared with randomly selected 50 hexamers (blue). (c) S65 scores of U2AF65 binding sites in 3’splice sites and non-3’splice sites18.
Supplementary Figure 3 RT-PCR validation of U2AF65 RNAi–induced alternative splicing.
(a) Induced exon skipping events in response to U2AF65 RNAi. (b) Induced exon inclusion events in response to U2AF65 RNAi.
Supplementary Figure 4 U2AF65 binding profile on representative genes and insertion mutational analysis of U2AF65-binding site–induced changes in alternative splicing.
(a) U2AF65 binding on the alternative exon in GANAB. (b) U2AF65 binding on the alternative exon in ANKRD10. PCR-validated splicing changes are shown in the right. (c) The minigene constructs illustrating the insertion of the U2AF65 binding site from an intronic region in DROSHA (see Fig. 4d) into an U2AF65 insensitive gene C1orf43 either in an upstream or a downstream intronic location. Bottom panel: RT-PCR analysis of wt and mutant minigenes in response to U2AF65 RNAi.
Supplementary Figure 5 Similar effects of U2AF65 RNAi and U2AF35 RNAi on alternative splicing.
(a) Examples of induced exon skipping events in response to U2AF65 or U2AF35 RNAi. (b) Examples of induced exon inclusion events in response to U2AF65 or U2AF35 RNAi. (c) Concordant splicing response to U2AF65 and U2AF35 RNAi determined by RT-PCR. ∆PSI: Difference of Percentage of Splicing In (PSI) between HeLa cells treated with U2AF35 or U2AF65 siRNA and negative control (NC) siRNA. (d) Heatmap showing similar U2AF65 RNAi-induced splicing events with or without exogenously expressed U2AF35. (e) RT-PCR validation of a set of U2AF65 RNAi-induced splicing events in the presence or absence of overexpressed U2AF35.
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Supplementary Data Set 1
Original uncropped gels and blots presented in main figures (PDF 3834 kb)
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Shao, C., Yang, B., Wu, T. et al. Mechanisms for U2AF to define 3′ splice sites and regulate alternative splicing in the human genome. Nat Struct Mol Biol 21, 997–1005 (2014). https://doi.org/10.1038/nsmb.2906
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DOI: https://doi.org/10.1038/nsmb.2906
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