Efficient in vivo manipulation of alternative pre-mRNA splicing events using antisense morpholinos in mice

Mammalian pre-mRNA alternative splicing mechanisms are typically studied using artificial minigenes in cultured cells, conditions that may not accurately reflect the physiological context of either the pre-mRNA or the splicing machinery. Here, we describe a strategy to investigate splicing of normal endogenous full-length pre-mRNAs under physiological conditions in live mice. This approach employs antisense vivo-morpholinos (vMOs) to mask cis-regulatory sequences or to disrupt splicing factor expression, allowing functional evaluation of splicing regulation in vivo. We applied this strategy to gain mechanistic insight into alternative splicing events involving exons 2 and 16 (E2 and E16) that control the structure and function of cytoskeletal protein 4.1R. In several mouse tissues, inclusion of E16 was substantially inhibited by interfering with a splicing enhancer mechanism using a target protector morpholino that blocked Fox2-dependent splicing enhancers in intron 16 or a splice-blocking morpholino that disrupted Fox2 expression directly. For E2, alternative 3'-splice site choice is coordinated with upstream promoter use across a long 5'-intron such that E1A splices almost exclusively to the distal acceptor (E2dis). vMOs were used to test the in vivo relevance of a deep intron element previously proposed to determine use of E2dis via a two-step intrasplicing model. Two independent vMOs designed against this intronic regulatory element inhibited intrasplicing, robustly switching E1A splicing to the proximal acceptor (E2prox). This finding strongly supports the in vivo physiological relevance of intrasplicing. vMOs represent a powerful tool for alternative splicing studies in vivo and may facilitate exploration of alternative splicing networks in vivo.     

Visualization of alternative splicing in vivo

The analysis of sequences required for alternative splicing of mRNA has predominantly been performed using cell culture systems. However, the phenotype of cultured cells is almost invariably different from that of cells in the intact animal. It is therefore possible that there are significant differences in the regulation of specific splicing reactions in vivo compared to in cell culture. Here, we describe methods for the visualization and analysis of alternative splicing in vivo using transgenic mice. These methods allow for the analysis of the temporal and tissue-specific regulation of alternative splicing both visually and quantitatively.     

Xenopus as a model to study alternative splicing in vivo

An increasing number of genes are being identified for which the corresponding mRNAs contain different combinations of the encoded exons. This highly regulated exon choice, or alternative splicing, is often tissue-specific and potentially could differentially affect cellular functions. Alternative splicing is therefore not only a means to increase the coding capacity of the genome, but also to regulate gene expression during differentiation or development. To both evaluate the importance for cellular functions and define the regulatory pathways of alternative splicing, it is necessary to progress from the in vitro or ex vivo experimental models actually used towards in vivo whole-animal studies. We present here the amphibian, Xenopus, as an experimental model highly amenable for such studies. The various experimental approaches that can be used with Xenopus oocytes and embryos to characterize regulatory sequence elements and factors are presented and the advantages and drawbacks of these approaches are discussed. Finally, the real possibilities for large-scale identification of mRNAs containing alternatively spliced exons, the tissue-specific patterns of exon usage and the way in which these patterns are modified by perturbing the relative amount of splicing factors are discussed.     

Alternative splicing of U12-dependent introns in vivo responds to purine-rich enhancers.

Alternative splicing increases the coding capacity of genes through the production of multiple protein isoforms by the conditional use of splice sites and exons. Many alternative splice sites are regulated by the presence of purine-rich splicing enhancer elements (ESEs) located in the downstream exon. Although the role of ESEs in alternative splicing of the major class U2-dependent introns is well established, no alternatively spliced minor class U12-dependent introns have so far been described. Although in vitro studies have shown that ESEs can stimulate splicing of individual U12-dependent introns, there is no direct evidence that the U12-dependent splicing system can respond to ESEs in vivo. To investigate the ability of U12-dependent introns to use alternative splice sites and to respond to ESEs in an in vivo context, we have constructed two sets of artificial minigenes with alternative splicing pathways and evaluated the effects of ESEs on their alternative splicing patterns. In minigenes with alternative U12-dependent 3' splice sites, a purine-rich ESE promotes splicing to the immediately upstream 3' splice site. As a control, a mutant ESE has no stimulatory effect. In minigene constructs with two adjacent U12-dependent introns, the predominant in vivo splicing pattern results in the skipping of the internal exon. Insertion of a purine-rich ESE into the internal exon promotes the inclusion of the internal exon. These results show that U12-dependent introns can participate in alternative splicing pathways and that U12-dependent splice sites can respond to enhancer elements in vivo.     

Tra2β1蛋白在神经特异性的基因剪接中的功能

体外(in vitro)生化研究已证明哺乳动物Tra2蛋白是前体mRNA剪接的重要调控因子,但是,对该蛋白在in vivo条件下的剪接功能,尤其是在神经特异性基因剪接中的功能及其细胞特异性,目前所知甚少。本文采用in vivo分析模型,在COS-l和PFSK两种不同类型的细胞中,研究了两个神经特异性基因(GluR-B,SMN2)剪接的细胞特异性,同时分析了Tra2β1在这两个基因剪接中的功能及其细胞特异性。结果表明,在研究的两种细胞中,GluR-B和SMN2“小基因”的剪接均具有明显的细胞特异性;而Tra2β1蛋白的过量表达在这两种不同的细胞中对“小基因”的剪接有显著的相同倾向的调节作用,提示Tra2β1蛋白对该两个基因剪接的调节作用可能没有细胞特异性。     

Editing efficiency of a Drosophila gene correlates with a distant splice site selection

RNA editing and alternative splicing are two processes that increase protein diversity. The relationship between the two processes is not well understood. There are a few examples of correlations between editing and alternative splicing, but these are all nearby effects. A search for alternative splicing among 16 edited genes in Drosophila reveals two novel instances of alternative splicing. In one example where alternative splicing occurs downstream of editing, a strong correlation between editing efficiency and splice site selection is observed. In contrast, when editing occurs downstream of alternative splicing, no correlation is seen. These results suggest some models for the coupling of editing and splicing processes.     

Intronic alternative splicing regulators identified by comparative genomics in nematodes

Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high- scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (T)GCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis, (T)CTATC, is important for alternative splicing regulation of the unc-52 gene.     

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.     

Amount of RNA secondary structure required to induce an alternative splice.

We set up an alternative splicing system in vitro in which the relative amounts of two spliced RNAs, one containing and the other lacking a particular exon, were directly proportional to the length of an inverted repeat inserted into the flanking introns. We then used the system to measure the effect of intramolecular complementarity on alternative splicing in vivo. We found that an alternative splice was induced in vivo only when the introns contained more than approximately 50 nucleotides of perfect complementarity, that is, only when the secondary structure was much more stable than most if not all possible secondary structures in natural mRNA precursors. We showed further that intron insertions containing long complements to splice sites and a branch point inhibited splicing in vitro but not in vivo. These results raise the possibility that in cells most pre-mRNA secondary structures either are not maintained long enough to influence splicing choices, or never form at all.     

Developmentally induced, muscle-specific trans factors control the differential splicing of alternative and constitutive troponin T exons

Alternative RNA splicing is a ubiquitous process permitting single genes to encode multiple protein isoforms. Here we report experiments in which a gene construct, containing combinatorial Troponin T (TnT) exons that manifest an exceptional diversity of alternative splicing in vivo, has been transfected into muscle and nonmuscle cells. Analyses of the spliced RNAs show that the alternative TnT exons retain their capacity for differential splicing in the modified minigene context when introduced into a variety of nonmuscle and muscle cells. The patterns of alternative splicing differ depending on cell type. Only in differentiated myotubes are the alternative exons normally incorporated during splicing, reproducing their behavior in the native gene; they are excluded in nonmuscle cells and myoblasts that do not express the endogenous TnT. These results provide proof that trans factors required for correct alternative splicing are induced during myogenesis. Surprisingly, such factors are also required for the correct splicing of constitutive TnT exons.     

In vivo effects on intron retention and exon skipping by the U2AF large subunit and SF1/BBP in the nematode Caenorhabditis elegans

The in vivo analysis of the roles of splicing factors in regulating alternative splicing in animals remains a challenge. Using a microarray-based screen, we identified a Caenorhabditis elegans gene, tos-1, that exhibited three of the four major types of alternative splicing: intron retention, exon skipping, and, in the presence of U2AF large subunit mutations, the use of alternative 3' splice sites. Mutations in the splicing factors U2AF large subunit and SF1/BBP altered the splicing of tos-1. 3' splice sites of the retained intron or before the skipped exon regulate the splicing pattern of tos-1. Our study provides in vivo evidence that intron retention and exon skipping can be regulated largely by the identities of 3' splice sites.     

In vivo regulation of alternative pre-mRNA splicing by the Clk1 protein kinase.

Controlled expression of cellular and viral genes through alternative precursor messenger RNA (pre-mRNA) splicing requires serine/arginine-rich (SR) proteins. The Clk1 kinase, which phosphorylates SR proteins, is regulated through alternative splicing of the Clk1 pre-mRNA, yielding mRNAs encoding catalytically active and truncated inactive polypeptides (Clk1 and Clk1T, respectively). We present evidence that Clk1 and Clk1T proteins regulate the splicing of Clk1 and adenovirus pre-mRNAs in vivo. The peptide domain encoded by the alternatively spliced exon of Clk1 is essential for the regulatory activity of the Clk1 kinase. This is the first direct demonstration of an in vivo link between alternative splicing and protein kinase activity.     

Cardiac tissue-specific repression of CELF activity disrupts alternative splicing and causes cardiomyopathy

Members of the CELF family of RNA binding proteins have been implicated in alternative splicing regulation in developing heart. Transgenic mice that express a nuclear dominant-negative CELF protein specifically in the heart (MHC-CELFDelta) develop cardiac hypertrophy and dilated cardiomyopathy with defects in alternative splicing beginning as early as 3 weeks after birth. MHC-CELFDelta mice exhibit extensive cardiac fibrosis, severe cardiac dysfunction, and premature death. Interestingly, the penetrance of the phenotype is greater in females than in males despite similar levels of dominant-negative expression, suggesting that there is sex-specific modulation of splicing activity. The cardiac defects in MHC-CELFdelta mice are directly attributable to reduced levels of CELF activity, as crossing these mice with mice overexpressing CUG-BP1, a wild-type CELF protein, rescues defects in alternative splicing, the severity and incidence of cardiac hypertrophy, and survival. We conclude that CELF protein activity is required for normal alternative splicing in the heart in vivo and that normal CELF-mediated alternative splicing regulation is in turn required for normal cardiac function.     

Role of the Modular Domains of SR Proteins in Subnuclear Localization and Alternative Splicing Specificity

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5′ splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.     

SRPK1 inhibition in vivo: modulation of VEGF splicing and potential treatment for multiple diseases

SRPK1 (serine-arginine protein kinase 1) is a protein kinase that specifically phosphorylates proteins containing serine-arginine-rich domains. Its substrates include a family of SR proteins that are key regulators of mRNA AS (alternative splicing). VEGF (vascular endothelial growth factor), a principal angiogenesis factor contains an alternative 3' splice site in the terminal exon that defines a family of isoforms with a different amino acid sequence at the C-terminal end, resulting in anti-angiogenic activity in the context of VEGF165-driven neovascularization. It has been shown recently in our laboratories that SRPK1 regulates the choice of this splice site through phosphorylation of the splicing factor SRSF1 (serine/arginine-rich splicing factor 1). The present review summarizes progress that has been made to understand how SRPK1 inhibition may be used to manipulate the balance of pro- and anti-angiogenic VEGF isoforms in animal models in vivo and therefore control abnormal angiogenesis and other pathophysiological processes in multiple disease states.     

Combinatorial control of signal-induced exon repression by hnRNP L and PSF

Cells can regulate their protein repertoire in response to extracellular stimuli via alternative splicing; however, the mechanisms controlling this process are poorly understood. The CD45 gene undergoes alternative splicing in response to T-cell activation to regulate T-cell function. The ESS1 splicing silencer in CD45 exon 4 confers basal exon skipping in resting T cells through the activity of hnRNP L and confers activation-induced exon skipping in T cells via previously unknown mechanisms. Here we have developed an in vitro splicing assay that recapitulates the signal-induced alternative splicing of CD45 and demonstrate that cellular stimulation leads to two changes to the ESS1-bound splicing regulatory complex. Activation-induced posttranslational modification of hnRNP L correlates with a modest increase in the protein's repressive activity. More importantly, the splicing factor PSF is recruited to the ESS1 complex in an activation-dependent manner and accounts for the majority of the signal-regulated ESS1 activity. The associations of hnRNP L and PSF with the ESS1 complex are largely independent of each other, but together these proteins account for the total signal-regulated change in CD45 splicing observed in vitro and in vivo. Such a combinatorial effect on splicing allows for precise regulation of signal-induced alternative splicing.     

The role of the polypyrimidine stretch at the SV40 early pre-mRNA 3'' splice site in alternative splicing.

We have studied the role in pre-mRNA splicing of the nucleotide sequence preceding the SV40 early region 3' splice site. Somewhat surprisingly, neither the pyrimidine at the highly conserved -3 position, nor the polypyrimidine stretch that extends from -5 to -15, relative to the 3' splice site, were found to be required for efficient splicing. Mutations that delete this region or create polypurine insertions at position -2 had no significant effects on the efficiency of SV40 early pre-mRNA splicing in vivo or in vitro. Interestingly, however, the pyrimidine content of this region had substantial effects on the alternative splicing pattern of this pre-mRNA in vivo. Mutations that increased the number of pyrimidine residues resulted in more efficient utilization of the large T antigen mRNA 5' splice site relative to the small t 5' splice site, while mutations that increased the purine content enhanced small t mRNA splicing. A possible molecular mechanism for these findings, as well as a model that proposes a role for the polypyrimidine stretch in alternative splicing, are discussed.