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Diversification of stem cell molecular repertoire by alternative splicing.

ABSTRACT: 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.

SUBMITTER: Pritsker M

PROVIDER: S-EPMC1242282 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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Diversification of stem cell molecular repertoire by alternative splicing.

Pritsker Moshe M Doniger Tirza T TT Kramer Laurie C LC Westcot Stephanie E SE Lemischka Ihor R IR

Proceedings of the National Academy of Sciences of the United States of America 20050923 40

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 ...[more]

PMID: 16183747

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Project description:BACKGROUND:The CEA gene family is one of the most rapidly evolving gene families in the human genome. The founder gene of the family is thought to be an ancestor of the inhibitory immune checkpoint molecule CEACAM1. Comprehensive analyses of mammalian genomes showed that the CEA gene family is subject to tremendous gene family expansion and contraction events in different mammalian species. While in some species (e.g. rabbits) less than three CEACAM1 related genes exist, were in others (certain microbat species) up to 100 CEACAM1 paralogs identified. We have recently reported that the horse has also an extended CEA gene family. Since mechanisms of gene family expansion and diversification are not well understood we aimed to analyze the equine CEA gene family in detail. RESULTS:We found that the equine CEA gene family contains 17 functional CEACAM1-related genes. Nine of them were secreted molecules and eight CEACAMs contain transmembrane and cytoplasmic domain exons, the latter being in the focus of the present report. Only one (CEACAM41) gene has exons coding for activating signaling motifs all other CEACAM1 paralogs contain cytoplasmic exons similar to that of the inhibitory receptor CEACAM1. However, cloning of cDNAs showed that only one CEACAM1 paralog contain functional immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic tail. Three receptors have acquired a stop codon in the transmembrane domain and two have lost their inhibitory motifs due to alternative splicing events. In addition, alternative splicing eliminated the transmembrane exon sequence of the putative activating receptor, rendering it to a secreted molecule. Transfection of eukaryotic cells with FLAG-tagged alternatively spliced CEACAMs indicates that they can be expressed in vivo. Thus detection of CEACAM41 mRNA in activated PBMC suggests that CEACAM41 is secreted by lymphoid cells upon activation. CONCLUSIONS:The results of our study demonstrate that alternative splicing after gene duplication is a potent mechanism to accelerate functional diversification of the equine CEA gene family members. This potent mechanism has created novel CEACAM receptors with unique signaling capacities and secreted CEACAMs which potentially enables equine lymphoid cells to control distantly located immune cells.

Project description:The epigenetic modulatory SAGA complex is involved in various developmental and stress responsive pathways in plants. Alternative transcripts of the SAGA complex's enzymatic subunit GCN5 have been identified in Brachypodium distachyon. These splice variants differ based on the presence and integrity of their conserved domain sequences: the histone acetyltransferase domain, responsible for catalytic activity, and the bromodomain, involved in acetyl-lysine binding and genomic loci targeting. GCN5 is the wild-type transcript, while alternative splice sites result in the following transcriptional variants: L-GCN5, which is missing the bromodomain and S-GCN5, which lacks the bromodomain as well as certain motifs of the histone acetyltransferase domain. Absolute mRNA quantification revealed that, across eight B. distachyon accessions, GCN5 was the dominant transcript isoform, accounting for up to 90% of the entire transcript pool, followed by L-GCN5 and S-GCN5. A cycloheximide treatment further revealed that the S-GCN5 splice variant was degraded through the nonsense-mediated decay pathway. All alternative BdGCN5 transcripts displayed similar transcript profiles, being induced during early exposure to heat and displaying higher levels of accumulation in the crown, compared to aerial tissues. All predicted protein isoforms localize to the nucleus, which lends weight to their purported epigenetic functions. S-GCN5 was incapable of forming an in vivo protein interaction with ADA2, the transcriptional adaptor that links the histone acetyltransferase subunit to the SAGA complex, while both GCN5 and L-GCN5 interacted with ADA2, which suggests that a complete histone acetyltransferase domain is required for BdGCN5-BdADA2 interaction in vivo. Thus, there has been a diversification in BdGCN5 through alternative splicing that has resulted in differences in conserved domain composition, transcript fate and in vivo protein interaction partners. Furthermore, our results suggest that B. distachyon may harbor compositionally distinct SAGA-like complexes that differ based on their histone acetyltransferase subunit.

Dataset Information

Diversification of stem cell molecular repertoire by alternative splicing.

Publications

Diversification of stem cell molecular repertoire by alternative splicing.

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