Bioinformatics of alternative splicing and its regulation

The sequencing of the human genome and ensuing wave of data generation have brought new light upon the extent and importance of alternative splicing as an RNA regulatory mechanism. Alternative splicing could potentially explain the complexity of protein repertoire during evolution, and defects in the splicing mechanism are responsible for diseases as complex as cancer. Among the challenges that rise in light of these discoveries are cataloguing splice variation in the human and other eukaryotic genomes, and identifying and characterizing the splicing regulatory elements that control their expression. Bioinformatics efforts tackling these two questions are just at the beginning. This article is a survey of these methods.     

癌症与可变剪接

可变剪接在发育、分化和癌症等过程中发挥着非常重要的作用。近年来,越来越多的研究表明可变剪接与癌症有着密切的关系,许多癌症相关基因受可变剪接调控。由于癌症特异性的剪接变体具有明显的诊断价值,使得对癌症与可变剪接的研究成为热点。简要概述了癌症相关基因的可变剪接、可变剪接变体的鉴定方法、可变剪接与癌症治疗等研究进展。     

The landscape of human mutually exclusive splicing

Mutually exclusive splicing of exons is a mechanism of functional gene and protein diversification with pivotal roles in organismal development and diseases such as Timothy syndrome, cardiomyopathy and cancer in humans. In order to obtain a first genomewide estimate of the extent and biological role of mutually exclusive splicing in humans, we predicted and subsequently validated mutually exclusive exons (MXEs) using 515 publically available RNA‐Seq datasets. Here, we provide evidence for the expression of over 855 MXEs, 42% of which represent novel exons, increasing the annotated human mutually exclusive exome more than fivefold. The data provide strong evidence for the existence of large and multi‐cluster MXEs in higher vertebrates and offer new insights into MXE evolution. More than 82% of the MXE clusters are conserved in mammals, and five clusters have homologous clusters in Drosophila. Finally, MXEs are significantly enriched in pathogenic mutations and their spatio‐temporal expression might predict human disease pathology.     

Abnormal alternative splicing promotes tumor resistance in targeted therapy and immunotherapy

Abnormally alternative splicing events are common hallmark of diverse types of cancers. Splicing variants with aberrant functions play an important role in cancer development. Most importantly, a growing body of evidence has supported that alternative splicing might play a significant role in the therapeutic resistance of tumors. Targeted therapy and immunotherapy are the future directions of tumor therapy; however, the loss of antigen targets on the tumor cells surface and alterations in drug efficacy have resulted in the failure of targeted therapy and immunotherapy. Interestingly, abnormal alternative splicing, as a strategy to regulate gene expression, is reportedly involved in the reprogramming of cell signaling pathways and epitopes on the tumor cell surface by changing splicing patterns of genes, thus rendering tumors resisted to targeted therapy and immunotherapy. Accordingly, increased knowledge regarding abnormal alternative splicing in tumors may help predict therapeutic resistance during targeted therapy and immunotherapy and lead to novel therapeutic approaches in cancer. Herein, we provide a brief synopsis of abnormal alternative splicing events in cancer progression and therapeutic resistance.     

Detection and measurement of alternative splicing using splicing-sensitive microarrays

Splicing and alternative splicing are major processes in the interpretation and expression of genetic information for metazoan organisms. The study of splicing is moving from focused attention on the regulatory mechanisms of a selected set of paradigmatic alternative splicing events to questions of global integration of splicing regulation with genome and cell function. For this reason, parallel methods for detecting and measuring alternative splicing are necessary. We have adapted the splicing-sensitive oligonucleotide microarrays used to estimate splicing efficiency in yeast to the study of alternative splicing in vertebrate cells and tissues. We use gene models incorporating knowledge about splicing to design oligonucleotides specific for discriminating alternatively spliced mRNAs from each other. Here we present the main strategies for design, application, and analysis of spotted oligonucleotide arrays for detection and measurement of alternative splicing. We demonstrate these strategies using a two-intron yeast gene that has been altered to produce different amounts of alternatively spliced RNAs, as well as by profiling alternative splicing in NCI 60 cancer cell lines.     

可变剪接与疾病的生物信息学研究概况

可变剪接是真核基因转录后期的重要调控机制,它使得同一条蛋白质编码基因能够产生多种转录体,极大的扩展了遗传信息的应用.研究发现,可变剪接与人类疾病有着密切的联系.错误的剪接会导致疾病,增加疾病的易感性与病变程度,甚至直接导致癌变.现对可变剪接调控机制与疾病的生物信息学研究进展进行综述.     

SOX2 modulates alternative splicing in transitional cell carcinoma

Aberrant alternative splicing of key cellular regulators may play a pivotal role in cancer development. To investigate the potential influence of altered alternative splicing on the development of transitional cell carcinoma (TCC), splicing activity in the TCC cell lines TSGH8301 and BFTC905 was examined using the SV40-immortalized uroepithelial cell line SV-HUC-1 as a reference. Our results indicate a significant alteration in splice site selection in the TCC cell lines. By gene expression profiling and subsequent validation, we discovered that sex-determining region Y-box protein 2 (SOX2) is specifically upregulated in BFTC905. Furthermore, ectopic expression of SOX2 modulates alternative splicing of the splicing reporter in vivo. More significantly, using an in vitro pull-down assay, it was found that SOX2 exhibits RNA-binding capability. Our observations suggest that SOX2 modulates alternative splicing by functioning as a splicing factor.     

Tumor microenvironment–associated modifications of alternative splicing

Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ∼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.     

Regulation of mammalian pre-mRNA splicing

In eukaryotes, most protein-coding genes contain introns which are removed by precursor messenger RNA (pre-mRNA) splicing. Alternative splicing is a process by which multiple messenger RNAs (mRNAs) are generated from a single pre-mRNA, resulting in functionally distinct proteins. Recent genome-wide analyses of alternative splicing indicated that in higher eukaryotes alternative splicing is an important mechanism that generates proteomic complexity and regulates gene expression. Mis-regulation of splicing causes a wide range of human diseases. This review describes the current understanding of pre-mRNA splicing and the mechanisms that regulate mammalian pre-mRNA splicing. It also discusses emerging directions in the field of alternative splicing. Supported by the Program of “one Hundred Talented people” of the Chinese Academy of Sciences.     

Splicing factor poly(rC)-binding protein 1 is a novel and distinctive tumor suppressor

A lot of evidence has been found on the link between tumorigenesis and the aberrant expression of splicing factors. A number of splicing factors have been reported to be either oncogenic or overexpressed in cancer cells. However, splicing factors can also play negative roles in tumorigenesis. In the current review, we focus on splicing factor poly(rC)-binding protein 1 (PCBP1), a novel tumor suppressor that is characterized by downregulation in many cancer types and shows inhibition of tumor formation and metastasis. Notably, the messenger RNA levels of PCBP1 are not significantly decreased in most cancer types. In fact, PCBP1 protein is often degraded or shows a loss-of-function through phosphorylation in cancer cells. PCBP1 is highly homologous to its family member, PCBP2. Interestingly, PCBP2 appears to be an oncogenic splicing factor. A growing body of evidence has shown that PCBP1 regulates alternative splicing, translation, and RNA stability of many cancer-related genes. Taking together, PCBP1 has distinctive tumor suppressive functions, and increasing PCBP1 expression may represent a new approach for cancer treatment.     

哺乳动物细胞mRNA剪接调控的分子机制

mRNA的可变剪接是指一个单一的mRNA前体(pre-mRNA)经过不同的剪接加工方式生成多种mRNA变异体(variants)的过程,这些变异体最终可以编码合成具有不同结构和功能的蛋白质。在过去的10多年中,大量数据表明,可变剪接是增加转录组和蛋白质组多样性的重要资源,也是调控哺乳动物细胞基因表达的重要步骤。可变剪接具有高度的组织与发育阶段特异性,并受到外界信号的控制。剪接调控的紊乱与疾病的发生发展密切相关。该文将对哺乳动物细胞mRNA剪接调控的分子机制进行阐述。     

miR‐200/375 control epithelial plasticity‐associated alternative splicing by repressing the RNA‐binding protein Quaking

Members of the miR‐200 family are critical gatekeepers of the epithelial state, restraining expression of pro‐mesenchymal genes that drive epithelial–mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR‐200c and another epithelial‐enriched miRNA, miR‐375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA‐binding protein Quaking (QKI). During EMT, QKI‐5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI‐5 is both necessary and sufficient to direct EMT‐associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial‐derived cancer types. Importantly, several actin cytoskeleton‐associated genes are directly targeted by both QKI and miR‐200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT. These findings demonstrate the existence of a miR‐200/miR‐375/QKI axis that impacts cancer‐associated epithelial cell plasticity through widespread control of alternative splicing.     

Interpretation, Stratification and Evidence for Sequence Variants Affecting mRNA Splicing in Complete Human Genome Sequences

Information theory-based methods have been shown to be sensitive and specific for predicting and quantifying the effects of non-coding mutations in Mendelian diseases. We present the Shannon pipeline software for genome-scale mutation analysis and provide evidence that the software predicts variants affecting mRNA splicing. Individual information contents (in bits) of reference and variant splice sites are compared and significant differences are annotated and prioritized. The software has been implemented for CLC-Bio Genomics platform. Annotation indicates the context of novel mutations as well as common and rare SNPs with splicing effects. Potential natural and cryptic mRNA splicing variants are identified, and null mutations are distinguished from leaky mutations. Mutations and rare SNPs were predicted in genomes of three cancer cell lines (U2OS, U251 and A431), which were supported by expression analyses. After filtering, tractable numbers of potentially deleterious variants are predicted by the software, suitable for further laboratory investigation. In these cell lines, novel functional variants comprised 6-17 inactivating mutations, 1-5 leaky mutations and 6-13 cryptic splicing mutations. Predicted effects were validated by RNA-seq analysis of the three aforementioned cancer cell lines, and expression microarray analysis of SNPs in HapMap cell lines.     

Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation

Alternative splicing contributes to muscle development, but a complete set of muscle-splicing factors and their combinatorial interactions are unknown. Previous work identified ACUAA (“STAR” motif) as an enriched intron sequence near muscle-specific alternative exons such as Capzb exon 9. Mass spectrometry of myoblast proteins selected by the Capzb exon 9 intron via RNA affinity chromatography identifies Quaking (QK), a protein known to regulate mRNA function through ACUAA motifs in 3′ UTRs. We find that QK promotes inclusion of Capzb exon 9 in opposition to repression by polypyrimidine tract-binding protein (PTB). QK depletion alters inclusion of 406 cassette exons whose adjacent intron sequences are also enriched in ACUAA motifs. During differentiation of myoblasts to myotubes, QK levels increase two- to threefold, suggesting a mechanism for QK-responsive exon regulation. Combined analysis of the PTB- and QK-splicing regulatory networks during myogenesis suggests that 39% of regulated exons are under the control of one or both of these splicing factors. This work provides the first evidence that QK is a global regulator of splicing during muscle development in vertebrates and shows how overlapping splicing regulatory networks contribute to gene expression programs during differentiation.     

FoxM1与癌发生的相关剪接机制的探讨

FoxM1是一种原癌基因。它也是癌发生、发展密切相关的重要的转录因子。Fox M1是Forkhead Box转录因子家族重要成员,定位于染色体12p13.3,特异性表达于增殖期细胞中,在细胞终末分化时消失,是一个典型的与细胞增殖相关的转录因子,在细胞G/S及G/M期转换过程中发挥重要作用。它具有Fox M1A、B和C三种剪接异构体。Fox M1B和C在癌组织中高表达,发挥转录激活、促癌发生和发展的作用,而Fox M1A在癌组织中低表达,发挥转录抑制功能。癌组织中Fox M1B/C的优先选择对于Fox M1发挥促癌作用非常关键。因此对这一现象的成因即Fox M1癌相关选择性剪接机制的研究非常重要。     

Use of minigene systems to dissect alternative splicing elements

Pre-mRNA splicing is an essential step for gene expression in higher eukaryotes. The splicing efficiency of individual exons is determined by multiple features involving gene architecture, a variety of cis-acting elements within the exons and flanking introns, and interactions with components of the basal splicing machinery (called the spliceosome) and auxiliary regulatory factors which transiently co-assemble with the spliceosome. Both alternative and constitutive exons are recognized by multiple weak protein:RNA interactions and different exons differ in the interactions which are determinative for exon usage. Alternative exons are often regulated according to cell-specific patterns and regulation is mediated by specific sets of cis-acting elements and trans-acting factors. Transient expression of minigenes is a commonly used in vivo assay to identify the intrinsic features of a gene that control exon usage, identify specific cis-acting elements that control usage of constitutive and alternative exons, identify cis-acting elements that control cell-specific usage of alternative exons, and once regulatory elements have been identified, to identify the trans-acting factors that bind to these elements and modulate splicing. This chapter describes approaches and strategies for using minigenes to define the cis-acting elements that determine splice site usage and to identify and characterize the trans-acting factors that bind to these elements and regulate alternative splicing.