A genomic view of alternative splicing Lee, Christopher; Modrek, Barmak
Nature genetics,
200201, 2002, 2002-Jan, 2002-1-00, 20020101, Letnik:
30, Številka:
1
Journal Article
Recenzirano
Recent genome-wide analyses of alternative splicing indicate that 40-60% of human genes have alternative splice forms, suggesting that alternative splicing is one of the most significant components ...of the functional complexity of the human genome. Here we review these recent results from bioinformatics studies, assess their reliability and consider the impact of alternative splicing on biological functions. Although the 'big picture' of alternative splicing that is emerging from genomics is exciting, there are many challenges. High-throughput experimental verification of alternative splice forms, functional characterization, and regulation of alternative splicing are key directions for research. We recommend a community-based effort to discover and characterize alternative splice forms comprehensively throughout the human genome.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
One of the most interesting opportunities in comparative genomics is to compare not only genome sequences but additional phenomena, such as alternative splicing, using orthologous genes in different ...genomes to find similarities and differences between organisms. Recently, genomics studies have suggested that 40-60% of human genes are alternatively spliced and have catalogued up to 30,000 alternative splice relationships in human genes. Here we report an analysis of 9,434 orthologous genes in human and mouse, which indicates that alternative splicing is associated with a large increase in frequency of recent exon creation and/or loss. Whereas most exons in the mouse and human genomes are strongly conserved in both genomes, exons that are only included in alternative splice forms (as opposed to the constitutive or major transcript form) are mostly not conserved and thus are the product of recent exon creation or loss events. A similar comparison of orthologous exons in rat and human validates this pattern. Although this says nothing about the complex question of adaptive benefit, it does indicate that alternative splicing in these genomes has been associated with increased evolutionary change.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
We have developed an automated method for discovering tissue‐specific regulation of alternative splicing through a genome‐wide analysis of expressed sequence tags (ESTs). Using this approach, we have ...identified 667 tissue‐specific alternative splice forms of human genes. We validated our muscle‐specific and brain‐specific splice forms for known genes. A high fraction (8/10) were reported to have a matching tissue specificity by independent studies in the published literature. The number of tissue‐specific alternative splice forms is highest in brain, while eye_retina, muscle, skin, testis and lymph have the greatest enrichment of tissue‐specific splicing. Overall, 10–30% of human alternatively spliced genes in our data show evidence of tissue‐specific splice forms. Seventy‐eight percent of our tissue‐specific alternative splices appear to be novel discoveries. We present bioinformatics analysis of several tissue‐specific splice forms, including automated protein isoform sequence and domain prediction, showing how our data can provide valuable insights into gene function in different tissues. For example, we have discovered a novel kidney‐specific alternative splice form of the WNK1 gene, which appears to specifically disrupt its N‐terminal kinase domain and may play a role in PHAII hypertension. Our database greatly expands knowledge of tissue‐specific alternative splicing and provides a comprehensive dataset for investigating its functional roles and regulation in different human tissues.
We have identified 6201 alternative splice relationships in human genes, through a genome-wide analysis of expressed sequence tags (ESTs). Starting with approximately 2.1 million human mRNA and EST ...sequences, we mapped expressed sequences onto the draft human genome sequence and only accepted splices that obeyed the standard splice site consensus. A large fraction (47%) of these were observed multiple times, indicating that they comprise a substantial fraction of the mRNA species. The vast majority of the detected alternative forms appear to be novel, and produce highly specific, biologically meaningful control of function in both known and novel human genes, e.g. specific removal of the lysosomal targeting signal from HLA-DM beta chain, replacement of the C-terminal transmembrane domain and cytoplasmic tail in an FC receptor beta chain homolog with a different transmembrane domain and cytoplasmic tail, likely modulating its signal transduction activity. Our data indicate that a large proportion of human genes, probably 42% or more, are alternatively spliced, but that this appears to be observed mainly in certain types of molecules (e.g. cell surface receptors) and systemic functions, particularly the immune system and nervous system. These results provide a comprehensive dataset for understanding the role of alternative splicing in the human genome, accessible at http://www.bioinformatics.ucla.edu/HASDB.
Asthma is canonically thought of as a disorder of excessive Th2-driven inflammation in the airway, although recent studies have described heterogeneity with respect to asthma pathophysiology. We have ...previously described distinct phenotypes of asthma based on the presence or absence of a three-gene "Th2 signature" in bronchial epithelium, which differ in terms of eosinophilic inflammation, mucin composition, subepithelial fibrosis, and corticosteroid responsiveness. In the present analysis, we sought to describe Th2 inflammation in human asthmatic airways quantitatively with respect to known mediators of inflammation and intercellular communication. Using whole-genome microarray and quantitative real-time PCR analysis of endobronchial biopsies from 27 mild-to-moderate asthmatics and 13 healthy controls with associated clinical and demographic data, we found that asthmatic Th2 inflammation is expressed over a variable continuum, correlating significantly with local and systemic measures of allergy and eosinophilia. We evaluated a composite metric describing 79 coexpressed genes associated with Th2 inflammation against the biological space comprising cytokines, chemokines, and growth factors, identifying distinctive patterns of inflammatory mediators as well as Wnt, TGF-β, and platelet-derived growth factor family members. This integrated description of the factors regulating inflammation, cell migration, and tissue remodeling in asthmatic airways has important consequences for the pathophysiological and clinical impacts of emerging asthma therapeutics targeting Th2 inflammation.
We have constructed a database of alternatively spliced protein forms (ASP), consisting of 13 384 protein isoform sequences of 4422 human genes (www.bioinformatics.ucla.edu/ASP). We identified fifty ...protein domain types that were selectively removed by alternative splicing at much higher frequencies than average (p-value < 0.01). These include many well-known protein-interaction domains (e.g., KRAB; ankyrin repeats; Kelch) including some that have been previously shown to be regulated functionally by alternative splicing (e.g., collagen domain). We present a number of novel examples (Kruppel transcription factors; Pbx2; Enc1) from the ASP database, illustrating how this pattern of alternative splicing changes the structure of a biological pathway, by redirecting protein interaction networks at key switch points. Our bioinformatics analysis indicates that a major impact of alternative splicing is removal of protein−protein interaction domains that mediate key linkages in protein interaction networks. ASP expands the available dataset of human alternatively spliced protein forms from 1989 human genes (SwissProt release 42) to 5413 (nonredundant set, ASP + SwissProt), a nearly 3-fold increase. ASP will enhance the existing pool of protein sequences that are searched by mass spectroscopy software during the identification of peptide fragments. Keywords: alternative splicing • human proteome • protein interaction domains • bioinformatics
Recently there has been much interest in assessing the role of alternative splicing in evolution. We have sought to measure functional selection pressure on alternatively spliced single‐exon skips, ...by calculating the fraction that are an exact multiple of 3 nt in length and therefore preserve protein reading‐frame in both the exon‐inclusion and exon‐skip splice forms. The frame‐preservation ratio (defined as the number of exons that are an exact multiple of three in length, divided by the number of exons that are not) was slightly above random for both constitutive exons and alternatively spliced exons as a whole in human and mouse. However, orthologous exons that were observed to be alternatively spliced in the expressed sequence tag data from two or more organisms showed a substantially increased bias to be frame‐preserving. This effect held true only for exons within the protein coding region, and not the untranslated region. In five animal genomes (human, mouse, rat, zebrafish, Drosophila), we observed an association between these conserved alternative splicing events and increased selection pressure for frame‐preservation. Surprisingly, this effect became stronger as a function of decreasing exon inclusion level: for alternatively spliced exons that were included in a majority of the gene’s transcripts, the frame‐preservation bias was no higher than that of constitutive exons, whereas for alternatively spliced exons that were included in only a minority of the gene’s transcripts, the frame‐preservation bias increased nearly 20‐fold. These data indicate that a subpopulation of modern alternative splicing events was present in the common ancestors of these genomes, and was under functional selection pressure to preserve the protein reading frame.
Although activating mutations and gains in copy number are key mechanisms for oncogene activation, the relationship between
the two is not well understood. In this study, we focused on KRAS copy ...gains and mutations in non–small cell lung cancer. We found that KRAS copy gains occur more frequently in tumors with KRAS activating mutations and are associated with large increases in KRAS expression. These copy gains tend to be more focal in tumors with activating mutations than in those with wild-type KRAS . Fluorescence in situ hybridization analysis revealed that some tumors have homogeneous low-level gains of the KRAS locus, whereas others have high-level amplification of KRAS , often in only a fraction of tumor cells. Associations between activating mutation and copy gains were also observed for
other oncogenes ( EGFR in non–small cell lung cancer, BRAF and NRAS in melanoma). Activating mutations were associated with copy gains only at the mutated oncogene locus but not other oncogene
loci. However, KRAS activating mutations in colorectal cancer were not associated with copy gains. Future work is warranted to clarify the relationship
among the different mechanisms of oncogene activation. (Mol Cancer Res 2009;7(8):1244–52)
T-helper type 2 (Th2) inflammation, mediated by IL-4, IL-5, and IL-13, is considered the central molecular mechanism underlying asthma, and Th2 cytokines are emerging therapeutic targets. However, ...clinical studies increasingly suggest that asthma is heterogeneous.
To determine whether this clinical heterogeneity reflects heterogeneity in underlying molecular mechanisms related to Th2 inflammation.
Using microarray and polymerase chain reaction analyses of airway epithelial brushings from 42 patients with mild-to-moderate asthma and 28 healthy control subjects, we classified subjects with asthma based on high or low expression of IL-13-inducible genes. We then validated this classification and investigated its clinical implications through analyses of cytokine expression in bronchial biopsies, markers of inflammation and remodeling, responsiveness to inhaled corticosteroids, and reproducibility on repeat examination.
Gene expression analyses identified two evenly sized and distinct subgroups, "Th2-high" and "Th2-low" asthma (the latter indistinguishable from control subjects). These subgroups differed significantly in expression of IL-5 and IL-13 in bronchial biopsies and in airway hyperresponsiveness, serum IgE, blood and airway eosinophilia, subepithelial fibrosis, and airway mucin gene expression (all P < 0.03). The lung function improvements expected with inhaled corticosteroids were restricted to Th2-high asthma, and Th2 markers were reproducible on repeat evaluation.
Asthma can be divided into at least two distinct molecular phenotypes defined by degree of Th2 inflammation. Th2 cytokines are likely to be a relevant therapeutic target in only a subset of patients with asthma. Furthermore, current models do not adequately explain non-Th2-driven asthma, which represents a significant proportion of patients and responds poorly to current therapies.
Studies have shown that genetic and sex differences strongly influence gene expression in mice. Given the diversity and complexity of transcripts produced by alternative splicing, we sought to use ...microarrays to establish the extent of variation found in mouse strains and genders. Here, we surveyed the effect of strain and sex on liver gene and exon expression using male and female mice from three different inbred strains.
71 liver RNA samples from three mouse strains - DBA/2J, C57BL/6J and C3H/HeJ - were profiled using a custom-designed microarray monitoring exon and exon-junction expression of 1,020 genes representing 9,406 exons. Gene expression was calculated via two different methods, using the 3'-most exon probe ("3' gene expression profiling") and using all probes associated with the gene ("whole-transcript gene expression profiling"), while exon expression was determined using exon probes and flanking junction probes that spanned across the neighboring exons ("exon expression profiling"). Widespread strain and sex influences were detected using a two-way Analysis of Variance (ANOVA) regardless of the profiling method used. However, over 90% of the genes identified in 3' gene expression profiling or whole transcript profiling were identified in exon profiling, along with 75% and 38% more genes, respectively, showing evidence of differential isoform expression. Overall, 55% and 32% of genes, respectively, exhibited strain- and sex-bias differential gene or exon expression.
Exon expression profiling identifies significantly more variation than both 3' gene expression profiling and whole-transcript gene expression profiling. A large percentage of genes that are not differentially expressed at the gene level demonstrate exon expression variation suggesting an influence of strain and sex on alternative splicing and a need to profile expression changes at sub-gene resolution.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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