Diversification of stem cell molecular repertoire by alternative splicing.

Complete information regarding transcriptional and posttranscriptional gene regulation in stem cells is necessary to understand the regulation of self-renewal and differentiation. Alternative splicing is a prevalent mode of posttranscriptional regulation, and occurs in approximately one half of all mammalian genes. The frequency and functional impact of alternative splicing in stem cells are yet to be determined. In this study we combine computational and experimental methods to identify splice variants in embryonic and hematopoietic stem cells on a genome-wide scale. Using EST collections derived from stem cells, we detect alternative splicing in >1,000 genes. Systematic RT-PCR and sequencing studies show confirmation of computational predictions at a level of 80%. We find that alternative splicing can modify multiple components of signaling pathways important for stem cell function. We also analyze the distribution of splice variants across different classes of genes. We find that tissue-specific genes have a higher tendency to undergo alternative splicing than ubiquitously expressed genes. Furthermore, the patterns of alternative splicing are only weakly conserved between orthologous genes in human and mouse. Our studies reveal extensive modification of the stem cell molecular repertoire by alternative splicing and provide insights into its overall role as a mechanism of generating genomic diversity.

[1]  Christopher J. Lee,et al.  Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss , 2003, Nature Genetics.

[2]  Phillip A. Sharp,et al.  Split genes and RNA splicing , 1994, Cell.

[3]  P. Bork,et al.  Alternative splicing and genome complexity , 2002, Nature Genetics.

[4]  Philip Lijnzaad,et al.  The Ensembl genome database project , 2002, Nucleic Acids Res..

[5]  M. Surani,et al.  The Polycomb-Group GeneEzh2 Is Required for Early Mouse Development , 2001, Molecular and Cellular Biology.

[6]  E. Birney,et al.  Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs , 2002, Nature.

[7]  D. Melton,et al.  "Stemness": Transcriptional Profiling of Embryonic and Adult Stem Cells , 2002, Science.

[8]  D. Linch,et al.  Transmigration of CD34+ cells across specialized and nonspecialized endothelium requires prior activation by growth factors and is mediated by PECAM-1 (CD31). , 1998, Blood.

[9]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[10]  Christopher J. Lee,et al.  Genome-wide detection of alternative splicing in expressed sequences of human genes , 2001, Nucleic Acids Res..

[11]  K. Guegler,et al.  Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation , 2004, Nature Biotechnology.

[12]  Bosiljka Tasic,et al.  Alternative pre-mRNA splicing and proteome expansion in metazoans , 2002, Nature.

[13]  Tomaso Poggio,et al.  Identification and analysis of alternative splicing events conserved in human and mouse. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[14]  I. Weissman,et al.  Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin in immature lymphocytes. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[15]  J. V. Moran,et al.  Initial sequencing and analysis of the human genome. , 2001, Nature.

[16]  Terry Gaasterland,et al.  Splice variation in mouse full-length cDNAs identified by mapping to the mouse genome. , 2002, Genome research.

[17]  John T. Dimos,et al.  A Stem Cell Molecular Signature , 2002, Science.

[18]  M. Boguski,et al.  dbEST — database for “expressed sequence tags” , 1993, Nature Genetics.

[19]  K. Georgopoulos,et al.  The Ikaros gene encodes a family of functionally diverse zinc finger DNA-binding proteins , 1994, Molecular and cellular biology.

[20]  Christopher J. Lee,et al.  A genomic view of alternative splicing , 2002, Nature Genetics.

[21]  Bert W O'Malley,et al.  Coordinate Regulation of Transcription and Splicing by Steroid Receptor Coregulators , 2002, Science.

[22]  J. Valcárcel,et al.  Alternative pre-mRNA splicing: the logic of combinatorial control. , 2000, Trends in biochemical sciences.

[23]  G C Overton,et al.  The genetic program of hematopoietic stem cells. , 2000, Science.

[24]  S. Brenner,et al.  Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[25]  B. Graveley.  Alternative splicing: increasing diversity in the proteomic world. , 2001, Trends in genetics : TIG.

[26]  S. Salesse,et al.  p210BCR/ABL-induced alteration of pre-mRNA splicing in primary human CD34+ hematopoietic progenitor cells , 2004, Leukemia.

[27]  A. J. Lopez,et al.  Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation. , 1998, Annual review of genetics.

[28]  Hans-Christian Pedersen,et al.  Characterization of Two Novel Nuclear BTB/POZ Domain Zinc Finger Isoforms , 2002, Journal of Biological Chemistry.

[29]  Christopher J. Lee,et al.  Genome-wide detection of tissue-specific alternative splicing in the human transcriptome. , 2002, Nucleic acids research.

[30]  D. Bentley.  The mRNA assembly line: transcription and processing machines in the same factory. , 2002, Current opinion in cell biology.

[31]  Gene W. Yeo,et al.  Variation in alternative splicing across human tissues , 2004, Genome Biology.

[32]  Holm Zaehres,et al.  LIF/STAT3 Signaling Fails to Maintain Self‐Renewal of Human Embryonic Stem Cells , 2004, Stem cells.

[33]  B. Berger,et al.  Human and Mouse Gene Structure: Comparative Analysis and Application to Exon Prediction , 2000 .

[34]  F. Campagne,et al.  TissueInfo: high-throughput identification of tissue expression profiles and specificity. , 2001, Nucleic acids research.

[35]  J. Castle,et al.  Genome-Wide Survey of Human Alternative Pre-mRNA Splicing with Exon Junction Microarrays , 2003, Science.