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RBFOX and SUP-12 sandwich a G base to cooperatively regulate tissue-specific splicing

Nature Structural & Molecular Biology volume 21, pages 778–786 (2014)Cite this article

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Abstract

Tissue-specific alternative pre-mRNA splicing is often cooperatively regulated by multiple splicing factors, but the structural basis of cooperative RNA recognition is poorly understood. In Caenorhabditis elegans, ligand binding specificity of fibroblast growth factor receptors (FGFRs) is determined by mutually exclusive alternative splicing of the sole FGFR gene, egl-15. Here we determined the solution structure of a ternary complex of the RNA-recognition motif (RRM) domains from the RBFOX protein ASD-1, SUP-12 and their target RNA from egl-15. The two RRM domains cooperatively interact with the RNA by sandwiching a G base to form the stable complex. Multichromatic fluorescence splicing reporters confirmed the requirement of the G and the juxtaposition of the respective cis elements for effective splicing regulation in vivo. Moreover, we identified a new target for the heterologous complex through an element search, confirming the functional significance of the intermolecular coordination.

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Figure 1: The RBFOX family and SUP-12 cooperatively regulate alternative splicing of egl-15 pre-mRNA.
Figure 2: Solution structures of SUP-12–RNA6 and ASD-1–SUP-12–RNA12 complexes.
Figure 3: Close-up views of the ASD-1–SUP-12–RNA12 and SUP-12–RNA6 complexes.
Figure 4: Recognition of the U8 and G9 bases is weakened in the G7A-mutant complex.
Figure 5: ASD-1 RRM guides SUP-12 RRM to form the stable ASD-1–SUP-12–RNA12 ternary complex.
Figure 6: The sandwiched base G7 and not U8 is crucial for regulation of muscle-specific alternative splicing in vivo.
Figure 7: A highly conserved UGCAUGGUGUG stretch in cle-1 intron 16 successfully predicts the activation of exon 16 by the RBFOX family and SUP-12.

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Acknowledgements

We thank H. Kurokawa for technical assistance and T. Imada, K. Ake and T. Nakayama for help with manuscript preparation. This work was supported by the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the National Project on Protein Structural and Functional Analyses (to Sh.Y.) of the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), Grants-in-Aid for Scientific Research on Innovative Areas “RNA Regulation” (no. 20112004 to H.K.; nos. 21112522 and 23112723 to Y.M.) and “Transcription Cycle” (no. 25118506 to H.K.) from MEXT, Grants-in-Aid for Scientific Research (B) (no. 23370080 to Y.M.; no. 26291003 to H.K.) from the Japan Society for the Promotion of Science (JSPS) and Precursory Research for Embryonic Science and Technology (PRESTO) from Japan Science and Technology Agency (JST) to H.K.

Author information

Author notes

  1. Kanako Kuwasako and Mari Takahashi: These authors contributed equally to this work.

Authors and Affiliations

  1. RIKEN Systems and Structural Biology Center, Yokohama, Japan

    Kanako Kuwasako, Mari Takahashi, Kengo Tsuda, Seiko Yoshikawa, Fahu He, Naohiro Kobayashi, Mikako Shirouzu, Takuhiro Ito, Akiko Tanaka, Shigeyuki Yokoyama & Yutaka Muto

  2. RIKEN Center for Life Science Technologies, Yokohama, Japan

    Kanako Kuwasako, Mari Takahashi, Kengo Tsuda, Mikako Shirouzu, Takuhiro Ito, Akiko Tanaka & Yutaka Muto

  3. Faculty of Pharmacy and Research Institute of Pharmaceutical Sciences, Musashino University, Nishitokyo, Japan

    Kanako Kuwasako & Yutaka Muto

  4. Graduate School of Nanobioscience, Yokohama City University, Yokohama, Japan

    Satoru Unzai

  5. Institute for Protein Research, Osaka University, Suita, Japan

    Naohiro Kobayashi

  6. Tatsuo Miyazawa Memorial Program, RIKEN Genomic Sciences Center, Yokohama, Japan

    Peter Güntert

  7. Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Frankfurt am Main, Germany

    Peter Güntert

  8. Frankfurt Institute of Advanced Studies, Goethe University Frankfurt, Frankfurt am Main, Germany

    Peter Güntert

  9. Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Hachioji, Japan

    Peter Güntert

  10. RIKEN Structural Biology Laboratory, Yokohama, Japan

    Shigeyuki Yokoyama

  11. Graduate School of Biomedical Science, Tokyo Medical and Dental University, Tokyo, Japan

    Masatoshi Hagiwara & Hidehito Kuroyanagi

  12. Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Masatoshi Hagiwara

  13. Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

    Hidehito Kuroyanagi

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  1. Kanako Kuwasako
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Contributions

K.K. and M.T. performed the structure determinations of the SUP-12–RNA6 complex and the ASD-1–SUP-12–RNA12 complex by NMR. S.U. performed the analytical ultracentrifugation experiments. Se.Y., M.S., T.I. and A.T. assisted with sample preparation. K.T., F.H., N.K., P.G. and Sh.Y. assisted with the structural determination. H.K. performed the in vivo splicing assays. The project was directed by M.H. and Y.M. All authors contributed to the preparation of the manuscript.

Corresponding authors

Correspondence to Masatoshi Hagiwara, Hidehito Kuroyanagi or Yutaka Muto.

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Integrated supplementary information

Supplementary Figure 1 Multiple sequence alignment of the single RRM domains of the RBFOX family proteins and the SUP-12–RBM24–RBM38 family proteins in C. elegans and humans.

The RRM domains of ASD-1 (G5EEW7) and FOX-1 (Q10572) from C. elegans, RBFOX1, 2, and 3 from Homo sapiens (Q9NWB1, O43251, and A6NFN3, respectively), RBM24 (Q9BX46) and RBM38 (Q9H0Z9) from H. sapiens, and C. elegans SUP-12 (O45189) were aligned by using Clustal X. C.e, C. elegans; H.s, Homo sapiens. Star and round marks indicate residues of the RBFOX family and SUP-12, respectively, involved in the RNA-recognition in the binary and/or the ternary complexes and the colors of the marks represents the pattern of involvement as indicated. The information about the RBFOX RRM/RNA binary complex was from Auweter et al 11. The amino acids are colored as follows: green, aromatic amino acid; brown, aliphatic; blue, positively charged; pink, negatively charged; purple, hydroxyl or sulfur-containing; orange, G and P. Secondary structure elements for ASD-1 RRM and SUP-12 RRM are depicted above and below the sequence alignment, respectively. Blue boxes, β-sheets; pink ovals, α-helices. The conserved motifs of the RRM domains, RNP1 and RNP2, are indicated with red lines.

Supplementary Figure 2 SUP-12 RRM efficiently binds to 5′-GUGUGC-3′.

(a) The 1H-15N HSQC spectrum of SUP-12 RRM, showing the amide chemical shift changes in the absence (black) and presence (ratio of SUP-12 RRM: RNA=1:2, red) of RNA6. Assignments are shown in the 3-letter amino acid code with the position numbers. (b) Quantification of the chemical-shift perturbation values of SUP-12 RRM upon binding to RNA6 (ratio of protein:RNA=1:2). The perturbation values greater than the baseline plus three times the standard deviation of the baseline (3 x 0.08 ppm) were considered as significant perturbations (i.e., the significant level is 0.34 ppm, indicated by a dashed red line). Black letters indicate amino acid residues with significant chemical shift changes. (c,d) Solution structures of the SUP-12–RNA6 complex. (c) A stereo view of the backbone traces of the 20 conformers of the complex. The backbone of SUP-12 RRM is colored magenta. The RNA molecule is green.

(d) Ribbon and stick representations of the complex. Upper panel, a stereo view; lower panel, another view of the complex rotated by 45° as indicated. The side chains of SUP-12 RRM involved in the RNA-recognition are represented as follows: green, carbons; red, oxygen; blue, nitrogen. The RNA is represented by a ball-and-stick model, where carbon, oxygen, nitrogen and phosphorus atoms are colored dark gray, red, blue and yellow, respectively.

Supplementary Figure 3 SUP-12 RRM alone can bind to RNA oligomers containing UG elements.

(a) Schematic representation of the 3′-end portion of egl-15 intron 4 and the sequences of RNA oligomers used for ITC measurements. (b–e) ITC measurements of SUP-12 RRM binding to four kinds of RNA oligomers 5′-UGCAUGG-3′ (b), 5′-GUGUGC-3′ (c), 5′- CUUUGUUUUCAG-3′ (d) and 5′-CUUUGUU-3′ (e). (f–h), ITC measurements of ASD-1 RRM binding to three kinds of RNA oligomers 5′-UGCAUGG-3′ (f), 5′-GUGUGC-3′ (g) and 5′-CUUUGUUUUCAG-3′ (h). Raw data as a function of time are shown in the top panels, and plots of the total heat released as a function of the molar ratio of RNA are shown in the bottom panels. The experimental data were fitted to a theoretical titration curve. The continuous lines represent the non-linear least-squares best fit to the experimental data, using a one-site model. The results shown are one of two technical replicates.

Supplementary Figure 4 ASD-1 RRM and SUP-12 RRM form a stable ternary complex with 5′-UGCAUGGUGUGC-3′.

(a) 1H-15N HSQC spectrum of the ASD-1–SUP-12–RNA12 complex. Assignments of the ASD-1 and SUP-12 amino acid residues are shown in the 3-letter amino acid code with the position numbers in orange and red, respectively. (b) Correlation between the calculated and the experimental RDC values of the ASD-1–SUP-12–RNA12 complex. (c) Solution structures of the ASD-1–SUP-12–RNA12 complex. A stereo view of the backbone traces of the 20 conformers of the ASD-1–SUP-12–RNA12 complex. The backbones of ASD-1 RRM and SUP-12 RRM are colored orange and magenta, respectively. The RNA molecule is blue for UGCAUG and green for GUGUGC. Note that average root-mean-square deviation to mean structure for the ternary complex was larger than that for the binary complex due to the quality loss of the NMR spectra caused by the increase in the molecular weight. (d) A stereo view of the complex in ribbon and stick representations. The side chains of the RRM domains and the RNA molecule are represented as in Supplementary Figure 2d except the carbon atoms in the ASD-1 side-chains in green. (e) A superposition of the 20 conformers of the ASD-1-SUP-12- RNA12 ternary complex. The interface region between ASD-1 RRM and SUP-12 RRM is demonstrated. The backbones of ASD-1 RRM and SUP-12 RRM are colored gray and light gray, respectively. The side-chains of ASD-1 (Asp128, Glu130, lle132 and Arg177) and SUP-12 (Tyr44 and Arg103) are colored orange and green, respectively. The G7 base is colored blue.

Supplementary Figure 5 The G7-recognition modes of SUP-12 RRM are different between the binary complex and the ternary complex.

(a, b) The hydrogen bond between the imino proton of the G7 base and the main-chain in the binary complex is missing in the ternary complex. 2D spectra of the imino-proton region for the bound RNA molecules in the SUP-12–RNA6 (a) and the ASD-1–SUP-12–RNA12 (b) complexes. The assignments of the H1 and H3 atoms in the RNA molecules are shown with blue and green lines.

(c–f) ITC measurements between SUP-12 RRM and four kinds of RNA oligomers, 5′-GUGUGC-3′ (RNA6) (c) (reproduced from Supplementary Fig. 3c), 5′-AUGUGC-3′ (d), 5′-CUGUGC-3′ (e) and 5′-UUGUGC-3′ (f). Raw data were analyzed according to the method described in the legend of Supplementary Figure 3 by using a one-site model. The results shown are one of two technical replicates.

Supplementary Figure 6 The G7 base fixed the spatial relationship between ASD-1 and SUP-12.

(a) The pocket for the U8 and G9 accommodation in the ASD-1–SUP-12–RNA12 complex. Guanine (light blue), adenine (pink) and cytosine (yellow) nucleotides are superposed on the U8 nucleotide (light green). The G9 nucleotide is colored light yellow. ASD-1 RRM and SUP-12 RRM are colored gray and white, respectively. (b, c) Dynamics of the wild-type and the mutant complexes. Measurement of T1, T2, proton-nitrogen heteronuclear NOEs and T1/T2 values for the ASD-1–SUP-12–RNA12 (b) and the ASD-1–SUP-12– RNA12(G7A) (c) complexes. The relaxation values for the ASD-1 RRM C-term and SUP-12 RRM N-term in the ternary complexes with the wild-type RNA is missing (b) because enough relaxation data were not acquired for these regions due to chemical shift broadening. On the other hand, we acquired enough data for these regions in the ternary complex with the mutant RNA (c) probably because of independent mobility of the two RRM domains on the RNA.

Supplementary Figure 7 The UGCAUGGUGUG stretch is conserved in the introns of the common target pre-mRNAs for the RBFOX family and SUP-12.

(a) Nucleotide sequence alignment of egl-15 intron 4 from C. elegans, C. briggsae, C. japonica, C. brenneri and C. remanei. Residues conserved in three or more species are colored orange. Binding sites for U1 snRNP (U1), U2AF, the RBFOX family (ASD-1 and FOX-1) and SUP-12 are indicated. An asterisk indicates the position of U8 that are not fully conserved in nematodes. (b) Nucleotide sequence alignment of cle-1 intron 16 from C. elegans, C. remanei, C. briggsae and C. brenneri. Residues conserved in three or more species are colored orange. The conserved UGCAUGGUGUG stretch is denoted with asterisks. Binding sites for U1 snRNP (U1) and U2AF are indicated.

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Kuwasako, K., Takahashi, M., Unzai, S. et al. RBFOX and SUP-12 sandwich a G base to cooperatively regulate tissue-specific splicing. Nat Struct Mol Biol 21, 778–786 (2014). https://doi.org/10.1038/nsmb.2870

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