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Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas

Nature Methods volume 1, pages 233–239 (2004)Cite this article

Abstract

It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line–specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.

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Figure 1: Preparation of ASLs.
Figure 2: Library distribution of genes identified by EST and ASSETs sequencing (Table 1).
Figure 3: Efficiency of selection step.
Figure 4: Computational analysis of ASSETs.
Figure 5: Alternative splicing patterns.
Figure 6: Real-time PCR on selected ASSETs.

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Acknowledgements

We thank E.V. Sviderskaya for providing cells from the Wellcome Trust Functional Genomics Cell Bank, W.J. Pavan for supplying RNA and encouragement, T. Hayashi for support and encouragement, H. Nishibe for secretarial assistance and N. Harigai for technical assistance. A Research Grant for the RIKEN Genome Exploration Research Project from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government to Y.H. and a RIKEN Presidential Research Grant for Innovation Tool Development to P.C. supported this study.

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Authors and Affiliations

  1. Genome Science Laboratory, RIKEN, Wako main campus, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan

    Akira Watahiki, Kazunori Waki, Norihito Hayatsu, Toshiyuki Shiraki, Mari Nakamura, Jun Kawai, Yoshihide Hayashizaki & Piero Carninci

  2. Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennodai Tsukuba, Ibaraki, 305-8575, Japan

    Akira Watahiki & Yoshihide Hayashizaki

  3. Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC), Yokohama Institute 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Kanagawa, Japan

    Kazunori Waki, Norihito Hayatsu, Toshiyuki Shiraki, Shinji Kondo, Mari Nakamura, Daisuke Sasaki, Takahiro Arakawa, Jun Kawai, Yoshihide Hayashizaki & Piero Carninci

  4. K.K. Dnaform, Tsukuba Branch, 3-1 Chuo 8-chome, Ami Machi, Inashiki Gun, Ibaraki, 300-0332, Japan

    Matthias Harbers

  5. Science of Biological Supramolecular Systems, Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehiro-Cho, Tsurumi-Ku, Yokohama, 230-0045, Japan

    Yoshihide Hayashizaki

Authors
  1. Akira Watahiki
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Correspondence to Matthias Harbers or Piero Carninci.

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Supplementary information

Supplementary Fig. 1

New splicing events identified by sequencing of ASLs. (PDF 22 kb)

Supplementary Fig. 2

Computational Analysis and Annotation of ESTs and ASSETS (PDF 41 kb)

Supplementary Fig. 3

RT-PCR Analysis of randomly Selected ASSETS (PDF 271 kb)

Supplementary Fig. 4

ASSETS and full-length sequences for Aph2 (PDF 28 kb)

Supplementary Fig. 5

Curves for Real-time RT-PCR Experiments (PDF 267 kb)

Supplementary Table 1

Computational Analysis of ASSETS – 1 (PDF 27 kb)

Supplementary Table 2

Computational Analysis of ASSETS – 2 (PDF 13 kb)

Supplementary Table 3

Annotation of EST and ASSETS (PDF 12 kb)

Supplementary Table 4

High-quality targets Selected to Characterized Splice Types (PDF 101 kb)

Supplementary Table 5

Statistical analysis of splice types found in high- quality targets, and their location within the transcripts. (PDF 842 kb)

Supplementary Table 6

Important Genes Identified by ASSETS (PDF 14 kb)

Supplementary Table 7

Primers and Probes Used for Colony Hybridization (PDF 39 kb)

Supplementary Table 8

ASSETS specific Primer Sets for RT-PCR (PDF 15 kb)

Supplementary Table 9

ASSETS specific Primer Sets for Real-time PCR (PDF 27 kb)

Supplementary Methods

Preparation of ASLs, Colony Hybridization, RT-PCR, real-time PCR, and Sequencing (PDF 44 kb)

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Watahiki, A., Waki, K., Hayatsu, N. et al. Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas. Nat Methods 1, 233–239 (2004). https://doi.org/10.1038/nmeth719

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