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

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.The results of the whole-genome sequencing projects have thus far identified far fewer genes than previously expected, and it is particularly puzzling that highly diverse organisms such as fruit fly, mouse and human seem to have rather similar numbers of genes. Therefore, the number of genes alone does not account for the increased complexity of higher organisms, and other regulatory principles must contribute to the use of genetic information during development and homeostasis 1,2 . This phenomenon is attributed in part to the mechanisms of alternative splicing, which allow for combinatorial assembly of different exons from the same primary transcript. It was estimated that B38-59% of the genes in mouse and human are subject to alternative splicing 2-6 and that, furthermore, B15% of the genetic disorders in humans are caused by mutations related to defects in pre-mRNA splicing [7][8][9] .Although the identification of all splice variants is important for understanding the transcriptome, only a few large-scale analyses have been made by computational means for exon prediction within genomes or using expressed-sequence tags (ESTs) and cDNA sequences. Predicted exons were used to design exon-and intron 10 -specific oligonucleotide arrays to monitor pre-mRNA splicing on a genome-wide scale 11 , including experiments using exon-junction arrays 12 or predicted exons on chromosome 22 (ref. 1). But these approaches are limited to predicted or known exons and do not allow new exon discovery.To identify genes regulated at the level of alternative splicing by experimental means, we developed an approach to selectively clone differentially spliced exons from distinct biological samples into alternative splicing libraries (ASLs) and to sequence alternative splicing sequence enriched tags (ASSETs). As ASSETs encompass exons along with their related splice junctions, they not only allow exon and gene identification but also enable the analysis of different splicing types, which are beyond the reach of present computational predictions.As cancer-r...

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