Data from Alterations in Gemin5 Expression Contribute to Alternative mRNA Splicing Patterns and Tumor Cell Motility
Jong‐Heun LeeChristine E. HorakChand KhannaZhaojing MengLi‐Rong YuTimothy D. VeenstraPatricia S. Steeg
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<div>Abstract<p>The role of Gemin5 in alternative mRNA splicing, tumor cell motility, and proteomic instability was investigated. Isotope Capture Affinity Tag proteomic analysis was conducted on MDA-MB-435 tumor cells transfected with either a control vector (C-100) or the Nm23-H1 metastasis suppressor (H1-177). Ingenuity pathway analysis revealed that RNA posttranscriptional processing was the most prominent class of differentially expressed proteins. Within this category, overexpression of Acinus1, Poly(a) binding protein, HNRPA2B1, Bop1, and Gemin5 was confirmed in less metastatic H1-177 cells. Overexpression of the latter four proteins was also observed in the lower metastatic antisense Ezrin transfectant of a murine osteosarcoma model system, confirming the general relevance of the trends. Gemin5, a component of the spliceosomal complex, was chosen for further study. Analysis of global mRNA splicing by SpliceArray chips revealed that 16 genes were differentially spliced in C-100 compared with H1-177 cells; transient transfection of <i>gemin5</i> into C-100 cells restored the splice pattern to that of H1-177 cells. Alternative splicing patterns for the engulfment and cell motility 1 and thrombospondin 4 genes were confirmed by semiquantitative reverse transcription-PCR. Gemin5 overexpression coordinately reduced C-100 cell motility by 50%, and siRNA-mediated reduction of Gemin5 expression increased the motility of H1-177 cells by 2-fold (<i>P</i> < 0.004). The data provide the first demonstration that alterations in the expression of a spliceosome protein can effect both specific splicing events and tumor cell motility. The data also show that changes in mRNA splicing patterns accompany metastatic progression, which may contribute to proteome instability. [Cancer Res 2008;68(3):639–44]</p></div>Keywords:
Expression (computer science)
Abstract Most human genes are alternatively spliced, allowing for a large expansion of the proteome.The multitude of regulatory inputs to splicing limits the potential to infer general principles from investigating native sequences. Here, we created a rationally designed library of >32,000 splicing events to dissect the complexity of splicing regulation through systematicsequence alterations. Measuring RNA and protein splice isoforms allowed us to investigate bothcause and effect of splicing decisions, quantify diverse regulatory inputs and accurately predict (R2=0.75–0.85) isoform ratios from sequence and secondary structure. By profiling individual cells, we measure the cell-to-cell variability of splicing decisions and show that it can be encoded in the DNA and influenced by regulatory inputs, opening the door for a novel,single-cell perspective on splicing regulation.
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Alternative splicing of pre-mRNA is a key mechanism for increasing the complexity of proteins in humans, causing a diversity of expression of transcriptomes and proteomes in a tissue-specific manner. Alternative splicing is regulated by a variety of splicing factors. However, the changes and errors of splicing regulation caused by splicing factors are strongly related to many diseases, something which represents one of this study’s main interests. Further understanding of alternative splicing regulation mediated by cellular factors is also a prospective choice to develop specific drugs for targeting the dynamic RNA splicing process. In this review, we firstly concluded the basic principle of alternative splicing. Afterwards, we showed how splicing isoforms affect physiological activities through specific disease examples. Finally, the available treatment methods relative to adjusting splicing activities have been summarized.
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Alternative splicing (AS) is an important mechanism used to generate greater transcriptomic and proteomic diversity from a finite genome. Nearly all human gene transcripts are alternatively spliced and can produce protein isoforms with divergent and even antagonistic properties that impact cell functions. Many AS events are tightly regulated in a cell-type or tissue-specific manner, and at different developmental stages. AS is regulated by RNA-binding proteins, including cell- or tissue-specific splicing factors. In the past few years, technological advances have defined genome-wide programs of AS regulated by increasing numbers of splicing factors. These splicing regulatory networks (SRNs) consist of transcripts that encode proteins that function in coordinated and related processes that impact the development and phenotypes of different cell types. As such, it is increasingly recognized that disruption of normal programs of splicing regulated by different splicing factors can lead to human diseases. We will summarize examples of diseases in which altered expression or function of splicing regulatory proteins has been implicated in human disease pathophysiology. As the role of AS continues to be unveiled in human disease and disease risk, it is hoped that further investigations into the functions of numerous splicing factors and their regulated targets will enable the development of novel therapies that are directed at specific AS events as well as the biological pathways they impact.
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The importance of alternative splicing in regulating apoptosis has been suggested by findings of functionally antagonistic proteins generated by alternative splicing of several genes involved in apoptosis. Among these, Ich-1 (also named as caspase-2) encodes a member of the caspase family of proteases. Two forms of Ich-1 are produced as a result of alternative splicing: Ich-1L, which causes apoptosis, and Ich-1S, which prevents apoptosis. The precise nature of Ich-1 alternative splicing and its regulation remain unknown. Here, we show that the production of Ich-1L and Ich-1S transcripts results from alternative exclusion or inclusion of a 61-bp exon. Several splicing factors can regulate Ich-1 splicing. Serine-arginine-rich proteins SC35 and ASF/SF2 promote exon skipping, decreasing the ratio of Ich-1S to Ich-1L transcripts; whereas heterogeneous nuclear ribonucleoprotein A1 facilitates exon inclusion, increasing this ratio. Furthermore, in cultured cells, SC35 overexpression increases apoptosis; whereas heterogeneous nuclear ribonucleoprotein A1 overexpression decreases apoptosis. These results provide the first direct evidence that splicing factors can regulate Ich-1 alternative splicing and suggest that alternative splicing may be an important regulatory mechanism for apoptosis.
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Through alternative splicing, most human genes produce multiple isoforms in a cell-, tissue-, and disease-specific manner. Numerous studies show that alternative splicing is essential for development, diseases, and their treatments. Despite these important examples, the extent and biological relevance of splicing are currently unknown.To solve this problem, we developed pairedGSEA and used it to profile transcriptional changes in 100 representative RNA-seq datasets. Our systematic analysis demonstrates that changes in splicing, on average, contribute to 48.1% of the biological signal in expression analyses. Gene-set enrichment analysis furthermore indicates that expression and splicing both convey shared and distinct biological signals.These findings establish alternative splicing as a major regulator of the human condition and suggest that most contemporary RNA-seq studies likely miss out on critical biological insights. We anticipate our results will contribute to the transition from a gene-centric to an isoform-centric research paradigm.
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During pre-mRNA splicing events, introns are removed from the pre-mRNA, and the remaining exons are connected together to form a single continuous molecule. Alternative splicing is a common mechanism for the regulation of gene expression in eukaryotes. More than 90% of human genes are known to undergo alternative splicing. The most common type of alternative splicing is exon skipping, which is also known as cassette exon. Other known alternative splicing events include alternative 5' splice sites, alternative 3' splice sites, intron retention, and mutually exclusive exons. Alternative splicing events are controlled by regulatory proteins responsible for both positive and negative regulation. In this review, we focus on neuronal splicing regulators and discuss several notable regulators in depth. In addition, we have also included an example of splicing regulation mediated by the RBFox protein family. Lastly, as previous studies have shown that a number of splicing factors are associated with neuronal diseases such as Alzheime's disease (AD) and Autism spectrum disorder (ASD), here we consider their importance in neuronal diseases wherein the underlying mechanisms have yet to be elucidated.
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