Conditional knockout mice to study alternative splicing in vivo

Analysis of genomes has revealed that the total number of human genes is comparable to those of simpler organisms, and thus, the number of genes does not correlate with the complexity and functional diversity of different organisms. Multiple mechanisms, including alternative splicing, are believed to contribute to the molecular complexity in higher eukaryotes. Given the fact that more than half of human genes undergo alternative splicing, however, little is known about the biological relevance of most alternative splicing events and their regulatory mechanisms. Recent work has highlighted the power of reverse genetic approaches in addressing regulated splicing in animal models. Here, we focus on the conditional knockout approach adapted for splicing research with the intention to provide a general guide to the generation of mouse models to study regulated splicing in development and disease.     

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.     

A serine–arginine-rich (SR) splicing factor modulates alternative splicing of over a thousand genes in Toxoplasma gondii

Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii, and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine–rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii. Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes.     

Finding signals that regulate alternative splicing in the post-genomic era

Alternative splicing of pre-mRNAs is central to the generation of diversity from the relatively small number of genes in metazoan genomes. Auxiliary cis elements and trans-acting factors are required for the recognition of constitutive and alternatively spliced exons and their inclusion in pre-mRNA. Here, we discuss the regulatory elements that direct alternative splicing and how genome-wide analyses can aid in their identification.     

Discovery and analysis of evolutionarily conserved intronic splicing regulatory elements

Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs). Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5′ and 94% altered 3′ splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%–45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%–50% of ISREs were enriched near alternatively spliced (AS) exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events.     

Computational identification of protein coding potential of conserved sequence tags through cross-species evolutionary analysis

The identification of conserved sequence tags (CSTs) through comparative genome analysis may reveal important regulatory elements involved in shaping the spatio-temporal expression of genetic information. It is well known that the most significant fraction of CSTs observed in human–mouse comparisons correspond to protein coding exons, due to their strong evolutionary constraints. As we still do not know the complete gene inventory of the human and mouse genomes it is of the utmost importance to establish if detected conserved sequences are genes or not. We propose here a simple algorithm that, based on the observation of the specific evolutionary dynamics of coding sequences, efficiently discriminates between coding and non-coding CSTs. The application of this method may help the validation of predicted genes, the prediction of alternative splicing patterns in known and unknown genes and the definition of a dictionary of non-coding regulatory elements.     

Prediction and computer analysis of the exon-intron structure of human genes

This review of the original works on computer analysis of the human genome considers the development of methods to predict the exon-intron structure of genes and analysis of alternative splicing. Prediction of the gene structure is based on homology between the gene product and a known protein or between the genomic sequences of the gene and its homolog from another organism. The methods were tested and proved highly efficient. Human gene splicing was analyzed with original methods and EST databases. Genes with alternative splicing were for the first time shown to account for no less than 35% total genes. Alternative splicing was compared for the human and mouse genomes. Species-specific isoforms were demonstrated for 50% alternatively spliced genes (25% total genes).     

Bioinformatics analysis of alternative splicing

Over the past few years, the analysis of alternative splicing using bioinformatics has emerged as an important new field, and has significantly changed our view of genome function. One exciting front has been the analysis of microarray data to measure alternative splicing genome-wide. Pioneering studies of both human and mouse data have produced algorithms for discerning evidence of alternative splicing and clustering genes and samples by their alternative splicing patterns. Moreover, these data indicate the presence of alternative splice forms in up to 80 per cent of human genes. Comparative genomics studies in both mammals and insects have demonstrated that alternative splicing can in some cases be predicted directly from comparisons of genome sequences, based on heightened sequence conservation and exon length. Such studies have also provided new insights into the connection between alternative splicing and a variety of evolutionary processes such as Alu-based exonisation, exon creation and loss. A number of groups have used a combination of bioinformatics, comparative genomics and experimental validation to identify new motifs for splice regulatory factors, analyse the balance of factors that regulate alternative splicing, and propose a new mechanism for regulation based on the interaction of alternative splicing and nonsense-mediated decay. Bioinformatics studies of the functional impact of alternative splicing have revealed a wide range of regulatory mechanisms, from NAGNAG sites that add a single amino acid; to short peptide segments that can play surprisingly complex roles in switching protein conformation and function (as in the Piccolo C2A domain); to events that entirely remove a specific protein interaction domain or membrane anchoring domain. Common to many bioinformatics studies is a new emphasis on graph representations of alternative splicing structures, which have many advantages for analysis.     

Genome-wide survey of alternative splicing in the grass Brachypodium distachyon: a emerging model biosystem for plant functional genomics

A draft sequence of the genome of Brachypodium distachyon, the emerging grass model, was recently released. This represents a unique opportunity to determine its functional diversity compared to the genomes of other model species. Using homology mapping of assembled expressed sequence tags with chromosome scale pseudomolecules, we identified 128 alternative splicing events in B. distachyon. Our study identified that retention of introns is the major type of alternative splicing events (53%) in this plant and highlights the prevalence of splicing site recognition for definition of introns in plants. We have analyzed the compositional profiles of exon-intron junctions by base-pairing nucleotides with U1 snRNA which serves as a model for describing the possibility of sequence conservation. The alternative splicing isoforms identified in this study are novel and represent one of the potentially biologically significant means by which B. distachyon controls the function of its genes. Our observations serve as a basis to understand alternative splicing events of cereal crops with more complex genomes, like wheat or barley.     

可变剪切在植物成花转换中的作用

精确调控成花转换,确保植物在适宜环境下开花,对于植物的成功繁殖和物种繁衍至关重要。开花由多种分子机制在转录、转录后和蛋白质水平进行调控。可变剪切(AS)是一种普遍的转录后水平调控过程,可从单个基因产生多个转录本,从而丰富转录组和蛋白质组的多样性。大量研究表明,可变剪切在成花转换过程中发挥重要作用。根据发育和环境条件, AS能够影响mRNA的稳定性和/或蛋白亚型的功能,从而调控开花相关基因的功能转录本和/或功能蛋白水平。揭示成花相关pre-mRNA的AS作用将进一步增进人们对开花相关基因功能以及整个成花转换调控网络的认识。该文归纳了涉及成花转换的AS研究进展,并针对各个调控途径进行总结,以期为进一步研究植物AS和成花转换调控机制提供参考。     

Alternative splicing: a paradoxical qudo in eukaryotic genomes

One of the most remarkable observations stemming from the sequencing of genomes of diverse species is that the number of protein-coding genes in an organism does not correlate with its overall cellular complexity. Alternative splicing, a key mechanism for generating protein complexity, has been suggested as one of the major explanation for this discrepancy between the number of genes and genome complexity. Determining the extent and importance of alternative splicing required the confluence of critical advances in data acquisition, improved understanding of biological processes and the development of fast and accurate computational analysis tools. Although many model organisms have now been completely sequenced, we are still very far from understanding the exact frequency of alternative splicing from these sequenced genomes.This paper will highlight some recent progress and future challenges for functional genomics and bioinformatics in this rapidly developing area.     

Regulation of alternative splicing by reversible protein phosphorylation

The vast majority of human protein-coding genes are subject to alternative splicing, which allows the generation of more than one protein isoform from a single gene. Cells can change alternative splicing patterns in response to a signal, which creates protein variants with different biological properties. The selection of alternative splice sites is governed by the dynamic formation of protein complexes on the processed pre-mRNA. A unique set of these splicing regulatory proteins assembles on different pre-mRNAs, generating a "splicing" or "messenger ribonucleoprotein code" that determines exon recognition. By influencing protein/protein and protein/RNA interactions, reversible protein phosphorylation modulates the assembly of regulatory proteins on pre-mRNA and therefore contributes to the splicing code. Studies of the serine/arginine-rich protein class of regulators identified different kinases and protein phosphatase 1 as the molecules that control reversible phosphorylation, which controls not only splice site selection, but also the localization of serine/arginine-rich proteins and mRNA export. The involvement of protein phosphatase 1 explains why second messengers like cAMP and ceramide that control the activity of this phosphatase influence alternative splicing. The emerging mechanistic links between splicing regulatory proteins and known signal transduction pathways now allow in detail the understanding how cellular signals modulate gene expression by influencing alternative splicing. This knowledge can be applied to human diseases that are caused by the selection of wrong splice sites.     

Prediction and Computer Analysis of the Exon–Intron Structure of Human Genes

This review of the original works on computer analysis of the human genome considers (i) the development of methods to predict the exon–intron structure of genes and (ii) analysis of alternative splicing. Prediction of the gene structure is based on homology between the gene product and a known protein or between the genomic sequences of the gene and its homolog from another organism. The methods were tested and proved highly efficient. Human gene splicing was analyzed with original methods and EST databases. Genes with alternative splicing were for the first time shown to account for no less than 35% of total genes. Alternative splicing was compared for the human and mouse genomes. Species-specific isoforms were demonstrated for 50% of alternatively spliced genes (25% of total genes).     

RNA在RNA剪接中的功能：从催化到调控

对非编码RNA功能的认识是后基因组时代的一个研究焦点,本文主要介绍非编码RNA在RNA剪接中的催化和调控功能。在RNA加工过程中,三大类内含子的剪接都是由RNA成员主导。其中Ⅰ型和Ⅱ型内含子能催化自身的切除和外显子连接反应;而核mRNA内含子的剪接则由剪接体里的小核RNA主导。Ⅰ型和Ⅱ型内含子存在于细菌、低等真核细胞和植物的细胞器内;而真核细胞的核编码蛋白质基因内全部是核mRNA内含子,并且其数目随生物体的复杂性而显著升高。一个多内含子前体mRNA通过选择性剪接产生多种,甚至上万种不同的mRNA和蛋白质,对蛋白质组的复杂度和时空表达调控至关重要。选择性剪接调控由剪接调控蛋白特异识别和结合前体mRNA里所富含的顺式RNA调控元件完成的;系统认识这两者之间的对应关系是揭示基因组表达调控网络的一把钥匙。