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Transgenic alternative-splicing reporters reveal tissue-specific expression profiles and regulation mechanisms in vivo

Nature Methods volume 3, pages 909–915 (2006)Cite this article

Abstract

Alternative splicing of pre-mRNAs allows multicellular organisms to create a huge diversity of proteomes from a finite number of genes. But extensive studies in vitro or in cultured cells have not fully explained the regulation mechanisms of tissue-specific or developmentally regulated alternative splicing in living organisms. Here we report a transgenic reporter system that allows visualization of expression profiles of mutually exclusive exons in Caenorhabditis elegans. Reporters for egl-15 exons 5A and 5B showed tissue-specific profiles, and we isolated mutants defective in the tissue specificity. We identified alternative-splicing defective-1 (asd-1), encoding a new RNA-binding protein of the evolutionarily conserved Fox-1 family, as a regulator of the egl-15 reporter. Furthermore, an asd-1; fox-1 double mutant was defective in the expression of endogenous egl-15 (5A) and phenocopied egl-15 (5A) mutant. This transgenic reporter system can be a powerful experimental tool for the comprehensive study of expression profiles and regulation mechanisms of alternative splicing in metazoans.

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Figure 1: Alternative splicing reporter worms.
Figure 2: Screening for mutants defective in tissue-specific alternative splicing of egl-15 reporter.
Figure 3: Identification of asd-1 gene.
Figure 4: Regulation of egl-15 reporter in body-wall muscles.
Figure 5: Regulation of the endogenous egl-15 exon 5s by asd-1 and fox-1.

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Acknowledgements

We thank T. Hirose and T. Fukuhara at Tokyo Medical and Dental University for their discussion. We thank A. Fire at Stanford University School of Medicine, R.Y. Tsien at the University of California, San Diego, H. Nishitoh at TMDU and Caenorhabditis Genetics Center for materials. We thank K. Kawamata, Y. Yamamoto, K. Yamada, G. Ohno and N. Egawa for their technical assistance. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H.K. and M.H.) and Grants-in-aid from National Institute of Biomedical Innovation, Japan (to M.H.).

Author information

Authors and Affiliations

  1. School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan

    Hidehito Kuroyanagi & Masatoshi Hagiwara

  2. Medical Research Institute, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan

    Hidehito Kuroyanagi & Masatoshi Hagiwara

  3. Department of Physiology, Tokyo Women's Medical University School of Medicine, Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan

    Tetsuo Kobayashi & Shohei Mitani

  4. CREST, JST, Hon-cho, Kawaguchi, Saitama, 332-0012, Japan

    Tetsuo Kobayashi & Shohei Mitani

Authors
  1. Hidehito Kuroyanagi
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  2. Tetsuo Kobayashi
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  3. Shohei Mitani
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  4. Masatoshi Hagiwara
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Contributions

H.K. contributed to the overall experiments. T.K. and S.M. contributed to mutant screening and chromosome mapping. H.K. and M.H. organized this work. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Hidehito Kuroyanagi or Masatoshi Hagiwara.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Mutation in UGCAUG stretch does not affect E5B-GFP selection in hypodermis. (PDF 87 kb)

Supplementary Fig. 2

Inactivation of E5B-GFP leads to selection of E5A-RFP even though UGCAUG stretch is mutagenized. (PDF 62 kb)

Supplementary Fig. 3

Inactivation of E5B-GFP leads to selection of E5A-RFP in hypodermis. (PDF 83 kb)

Supplementary Fig. 4

Model of regulation of endogenous egl-15 exon 5s in sex myoblasts. (PDF 23 kb)

Supplementary Methods (PDF 47 kb)

Supplementary Note 1

Alternative splicing reporter worms with a pair of separate mini-genes. (PDF 94 kb)

Supplementary Note 2

Exon 5B sequence is not required for inclusion or suppresesion of E5B-GFP. (PDF 105 kb)

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Kuroyanagi, H., Kobayashi, T., Mitani, S. et al. Transgenic alternative-splicing reporters reveal tissue-specific expression profiles and regulation mechanisms in vivo. Nat Methods 3, 909–915 (2006). https://doi.org/10.1038/nmeth944

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