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

Nature Methods volume 3, pages 909915 (2006) | Download Citation

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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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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

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

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Hidehito Kuroyanagi or Masatoshi Hagiwara.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Mutation in UGCAUG stretch does not affect E5B-GFP selection in hypodermis.

  2. 2.

    Supplementary Fig. 2

    Inactivation of E5B-GFP leads to selection of E5A-RFP even though UGCAUG stretch is mutagenized.

  3. 3.

    Supplementary Fig. 3

    Inactivation of E5B-GFP leads to selection of E5A-RFP in hypodermis.

  4. 4.

    Supplementary Fig. 4

    Model of regulation of endogenous egl-15 exon 5s in sex myoblasts.

  5. 5.

    Supplementary Methods

  6. 6.

    Supplementary Note 1

    Alternative splicing reporter worms with a pair of separate mini-genes.

  7. 7.

    Supplementary Note 2

    Exon 5B sequence is not required for inclusion or suppresesion of E5B-GFP.

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DOI

https://doi.org/10.1038/nmeth944

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