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1.  Intronic Alternative Splicing Regulators Identified by Comparative Genomics in Nematodes 
PLoS Computational Biology  2006;2(7):e86.
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.
Synopsis
Alternative splicing of precursor messenger RNA is a process by which multiple protein isoforms are generated from a single gene. As many as 60% of human genes are processed in this manner, creating tissue-specific isoforms of proteins that may be a key factor in regulating the complexity of our physiology. One of the major challenges to understanding this process is to identify the sequences on the precursor messenger RNA responsible for splicing regulation. Some of these regulatory sequences occur in regions that are spliced out (called introns). This study tested the hypothesis that there should be evolutionary pressure to maintain these intronic regulatory sequences, even though intron sequence is non-coding and rapidly diverges between species. The authors employed a genomic alignment of two roundworms, Caenorhabditis elegans and Caenorhabditis briggsae, to investigate the regulation of alternative splicing. By examining evolutionarily conserved stretches of introns flanking alternatively spliced exons, the authors identified and functionally confirmed splicing regulatory sequences. Many of the top scoring sequences match known mammalian regulators, suggesting the alternative splicing regulatory mechanism is conserved across all metazoans. Other sequences were not previously identified in mammals and may represent new alternative splicing regulatory elements in higher organisms or ones that may be specific to worms.
doi:10.1371/journal.pcbi.0020086
PMCID: PMC1500816  PMID: 16839192
2.  Evolutionary conservation analysis increases the colocalization of predicted exonic splicing enhancers in the BRCA1 gene with missense sequence changes and in-frame deletions, but not polymorphisms 
Breast Cancer Research  2005;7(6):R929-R939.
Introduction
Aberrant pre-mRNA splicing can be more detrimental to the function of a gene than changes in the length or nature of the encoded amino acid sequence. Although predicting the effects of changes in consensus 5' and 3' splice sites near intron:exon boundaries is relatively straightforward, predicting the possible effects of changes in exonic splicing enhancers (ESEs) remains a challenge.
Methods
As an initial step toward determining which ESEs predicted by the web-based tool ESEfinder in the breast cancer susceptibility gene BRCA1 are likely to be functional, we have determined their evolutionary conservation and compared their location with known BRCA1 sequence variants.
Results
Using the default settings of ESEfinder, we initially detected 669 potential ESEs in the coding region of the BRCA1 gene. Increasing the threshold score reduced the total number to 464, while taking into consideration the proximity to splice donor and acceptor sites reduced the number to 211. Approximately 11% of these ESEs (23/211) either are identical at the nucleotide level in human, primates, mouse, cow, dog and opossum Brca1 (conserved) or are detectable by ESEfinder in the same position in the Brca1 sequence (shared). The frequency of conserved and shared predicted ESEs between human and mouse is higher in BRCA1 exons (2.8 per 100 nucleotides) than in introns (0.6 per 100 nucleotides). Of conserved or shared putative ESEs, 61% (14/23) were predicted to be affected by sequence variants reported in the Breast Cancer Information Core database. Applying the filters described above increased the colocalization of predicted ESEs with missense changes, in-frame deletions and unclassified variants predicted to be deleterious to protein function, whereas they decreased the colocalization with known polymorphisms or unclassified variants predicted to be neutral.
Conclusion
In this report we show that evolutionary conservation analysis may be used to improve the specificity of an ESE prediction tool. This is the first report on the prediction of the frequency and distribution of ESEs in the BRCA1 gene, and it is the first reported attempt to predict which ESEs are most likely to be functional and therefore which sequence variants in ESEs are most likely to be pathogenic.
doi:10.1186/bcr1324
PMCID: PMC1410749  PMID: 16280041
3.  Intronic motif pairs cooperate across exons to promote pre-mRNA splicing 
Genome Biology  2010;11(8):R84.
Background
A very early step in splice site recognition is exon definition, a process that is as yet poorly understood. Communication between the two ends of an exon is thought to be required for this step. We report genome-wide evidence for exons being defined through the combinatorial activity of motifs located in flanking intronic regions.
Results
Strongly co-occurring motifs were found to specifically reside in four intronic regions surrounding a large number of human exons. These paired motifs occur around constitutive and alternative exons but not pseudo exons. Most co-occurring motifs are limited to intronic regions within 100 nucleotides of the exon. They are preferentially associated with weaker exons. Their pairing is conserved in evolution and they exhibit a lower frequency of single nucleotide polymorphism when paired. Paired motifs display specificity with respect to distance from the exon borders and in constitutive versus alternative splicing. Many resemble binding sites for heterogeneous nuclear ribonucleoproteins. Specific pairs are associated with tissue-specific genes, the higher expression of which coincides with that of the pertinent RNA binding proteins. Tested pairs acted synergistically to enhance exon inclusion, and this enhancement was found to be exon-specific.
Conclusions
The exon-flanking sequence pairs identified here by genomic analysis promote exon inclusion and may play a role in the exon definition step in pre-mRNA splicing. We propose a model in which multiple concerted interactions are required between exonic sequences and flanking intronic sequences to effect exon definition.
doi:10.1186/gb-2010-11-8-r84
PMCID: PMC2945786  PMID: 20704715
4.  Differentiated evolutionary rates in alternative exons and the implications for splicing regulation 
Background
Alternatively spliced exons play an important role in the diversification of gene function in most metazoans and are highly regulated by conserved motifs in exons and introns. Two contradicting properties have been associated to evolutionary conserved alternative exons: higher sequence conservation and higher rate of non-synonymous substitutions, relative to constitutive exons. In order to clarify this issue, we have performed an analysis of the evolution of alternative and constitutive exons, using a large set of protein coding exons conserved between human and mouse and taking into account the conservation of the transcript exonic structure. Further, we have also defined a measure of the variation of the arrangement of exonic splicing enhancers (ESE-conservation score) to study the evolution of splicing regulatory sequences. We have used this measure to correlate the changes in the arrangement of ESEs with the divergence of exon and intron sequences.
Results
We find evidence for a relation between the lack of conservation of the exonic structure and the weakening of the sequence evolutionary constraints in alternative and constitutive exons. Exons in transcripts with non-conserved exonic structures have higher synonymous (dS) and non-synonymous (dN) substitution rates than exons in conserved structures. Moreover, alternative exons in transcripts with non-conserved exonic structure are the least constrained in sequence evolution, and at high EST-inclusion levels they are found to be very similar to constitutive exons, whereas alternative exons in transcripts with conserved exonic structure have a dS significantly lower than average at all EST-inclusion levels. We also find higher conservation in the arrangement of ESEs in constitutive exons compared to alternative ones. Additionally, the sequence conservation at flanking introns remains constant for constitutive exons at all ESE-conservation values, but increases for alternative exons at high ESE-conservation values.
Conclusion
We conclude that most of the differences in dN observed between alternative and constitutive exons can be explained by the conservation of the transcript exonic structure. Low dS values are more characteristic of alternative exons with conserved exonic structure, but not of those with non-conserved exonic structure. Additionally, constitutive exons are characterized by a higher conservation in the arrangement of ESEs, and alternative exons with an ESE-conservation similar to that of constitutive exons are characterized by a conservation of the flanking intron sequences higher than average, indicating the presence of more intronic regulatory signals.
doi:10.1186/1471-2148-6-50
PMCID: PMC1543662  PMID: 16792801
5.  Global control of aberrant splice-site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition 
Nucleic Acids Research  2007;35(19):6399-6413.
Auxiliary splicing signals play a major role in the regulation of constitutive and alternative pre-mRNA splicing, but their relative importance in selection of mutation-induced cryptic or de novo splice sites is poorly understood. Here, we show that exonic sequences between authentic and aberrant splice sites that were activated by splice-site mutations in human disease genes have lower frequencies of splicing enhancers and higher frequencies of splicing silencers than average exons. Conversely, sequences between authentic and intronic aberrant splice sites have more enhancers and less silencers than average introns. Exons that were skipped as a result of splice-site mutations were smaller, had lower SF2/ASF motif scores, a decreased availability of decoy splice sites and a higher density of silencers than exons in which splice-site mutation activated cryptic splice sites. These four variables were the strongest predictors of the two aberrant splicing events in a logistic regression model. Elimination or weakening of predicted silencers in two reporters consistently promoted use of intron-proximal splice sites if these elements were maintained at their original positions, with their modular combinations producing expected modification of splicing. Together, these results show the existence of a gradient in exon and intron definition at the level of pre-mRNA splicing and provide a basis for the development of computational tools that predict aberrant splicing outcomes.
doi:10.1093/nar/gkm680
PMCID: PMC2095810  PMID: 17881373
6.  ESEfinder: a web resource to identify exonic splicing enhancers 
Nucleic Acids Research  2003;31(13):3568-3571.
Point mutations frequently cause genetic diseases by disrupting the correct pattern of pre-mRNA splicing. The effect of a point mutation within a coding sequence is traditionally attributed to the deduced change in the corresponding amino acid. However, some point mutations can have much more severe effects on the structure of the encoded protein, for example when they inactivate an exonic splicing enhancer (ESE), thereby resulting in exon skipping. ESEs also appear to be especially important in exons that normally undergo alternative splicing. Different classes of ESE consensus motifs have been described, but they are not always easily identified. ESEfinder (http://exon.cshl.edu/ESE/) is a web-based resource that facilitates rapid analysis of exon sequences to identify putative ESEs responsive to the human SR proteins SF2/ASF, SC35, SRp40 and SRp55, and to predict whether exonic mutations disrupt such elements.
PMCID: PMC169022  PMID: 12824367
7.  Pre-mRNA Secondary Structures Influence Exon Recognition 
PLoS Genetics  2007;3(11):e204.
The secondary structure of a pre-mRNA influences a number of processing steps including alternative splicing. Since most splicing regulatory proteins bind to single-stranded RNA, the sequestration of RNA into double strands could prevent their binding. Here, we analyzed the secondary structure context of experimentally determined splicing enhancer and silencer motifs in their natural pre-mRNA context. We found that these splicing motifs are significantly more single-stranded than controls. These findings were validated by transfection experiments, where the effect of enhancer or silencer motifs on exon skipping was much more pronounced in single-stranded conformation. We also found that the structural context of predicted splicing motifs is under selection, suggesting a general importance of secondary structures on splicing and adding another level of evolutionary constraints on pre-mRNAs. Our results explain the action of mutations that affect splicing and indicate that the structural context of splicing motifs is part of the mRNA splicing code.
Author Summary
Almost all human protein-coding genes contain several exons and introns. Prior to translation, introns have to be removed and exons have to be joined, which happens in a processing step called splicing that generates the mature mRNA. For most genes, certain exons can be either included or excluded from the mature mRNA. It is currently not fully understood which signals are needed to accurately recognize the boundaries of exons in the intron-containing primary transcript. As in transcriptional regulation, enhancer and silencer sequence motifs are crucial for the correct recognition of exons. Splicing regulatory proteins identify these motifs in a sequence-specific manner. In general, these proteins bind to single-stranded RNA. Here, we analyzed local secondary structures of primary transcripts and found that known splicing motifs are preferentially located in a single-stranded context. Experimental tests demonstrated that motifs in single-stranded contexts have a stronger effect on splice site selection than those located in double-stranded regions. These results help to understand the action of human mutations that change the splicing pattern and indicate that local pre-mRNA secondary structures influence exon recognition.
doi:10.1371/journal.pgen.0030204
PMCID: PMC2077896  PMID: 18020710
8.  Depletion of TDP 43 overrides the need for exonic and intronic splicing enhancers in the human apoA-II gene 
Nucleic Acids Research  2005;33(18):6000-6010.
Exon 3 of the human apolipoprotein A-II (apoA-II) gene is efficiently included in the mRNA although its acceptor site is significantly weak because of a peculiar (GU)16 tract instead of a canonical polypyrimidine tract within the intron 2/exon 3 junction. Our previous studies demonstrated that the SR proteins ASF/SF2 and SC35 bind specifically an exonic splicing enhancer (ESE) within exon 3 and promote exon 3 splicing. In the present study, we show that the ESE is necessary only in the proper context. In addition, we have characterized two novel sequences in the flanking introns that modulate apoA-II exon 3 splicing. There is a G-rich element in intron 2 that interacts with hnRNPH1 and inhibits exon 3 splicing. The second is a purine rich region in intron 3 that binds SRp40 and SRp55 and promotes exon 3 inclusion in mRNA. We have also found that the (GU) repeats in the apoA-II context bind the splicing factor TDP-43 and interfere with exon 3 definition. Significantly, blocking of TDP-43 expression by small interfering RNA overrides the need for all the other cis-acting elements making exon 3 inclusion constitutive even in the presence of disrupted exonic and intronic enhancers. Altogether, our results suggest that exonic and intronic enhancers have evolved to balance the negative effects of the two silencers located in intron 2 and hence rescue the constitutive exon 3 inclusion in apoA-II mRNA.
doi:10.1093/nar/gki897
PMCID: PMC1270946  PMID: 16254078
9.  Structure and Sequence of the Human Fast Skeletal Troponin T (TNNT3) Gene: Insight Into the Evolution of the Gene and the Origin of the Developmentally Regulated Isoforms 
We describe the cloning, sequencing and structure of the human fast skeletal troponin T (TNNT3) gene located on chromosome 11p15.5. The single-copy gene encodes 19 exons and 18 introns. Eleven of these exons, 1–3, 9–15 and 18, are constitutively spliced, whereas exons 4–8 are alternatively spliced. The gene contains an additional subset of developmentally regulated and alternatively spliced exons, including a foetal exon located between exon 8 and 9 and exon 16 or α (adult) and 17 or β (foetal and neonatal). Exon phasing suggests that the majority of the alternatively spliced exons located at the 5′ end of the gene may have evolved as a result of exon shuffling, because they are of the same phase class. In contrast, the 3′ exons encoding an evolutionarily conserved heptad repeat domain, shared by both TnT and troponin I (TnI), may be remnants of an ancient ancestral gene. The sequence of the 5′ flanking region shows that the putative promoter contains motifs including binding sites for MyoD, MEF-2 and several transcription factors which may play a role in transcriptional regulation and tissue-specific expression of TnT. The coding region of TNNT3 exhibits strong similarity to the corresponding rat sequence. However, unlike the rat TnT gene, TNNT3 possesses two repeat regions of CCA and TC. The exclusive presence of these repetitive elements in the human gene indicates divergence in the evolutionary dynamics of mammalian TnT genes. Homologous muscle-specific splicing enhancer motifs are present in the introns upstream and downstream of the foetal exon, and may play a role in the developmental pattern of alternative splicing of the gene. The genomic correlates of TNNT3 are relevant to our understanding of the evolution and regulation of expression of the gene, as well as the structure and function of the protein isoforms. The nucleotide sequence of TNNT3 has been submitted to EMBL/GenBank under Accession No. AF026276.
doi:10.1002/cfg.343
PMCID: PMC2447309  PMID: 18629027
10.  Alternative Splicing of RNA Triplets Is Often Regulated and Accelerates Proteome Evolution 
PLoS Biology  2012;10(1):e1001229.
Inclusion or exclusion of single codons at the splice acceptor site of mammalian genes is regulated in a tissue-specific manner, is strongly conserved, and is associated with local accelerated protein evolution.
Thousands of human genes contain introns ending in NAGNAG (N any nucleotide), where both NAGs can function as 3′ splice sites, yielding isoforms that differ by inclusion/exclusion of three bases. However, few models exist for how such splicing might be regulated, and some studies have concluded that NAGNAG splicing is purely stochastic and nonfunctional. Here, we used deep RNA-Seq data from 16 human and eight mouse tissues to analyze the regulation and evolution of NAGNAG splicing. Using both biological and technical replicates to estimate false discovery rates, we estimate that at least 25% of alternatively spliced NAGNAGs undergo tissue-specific regulation in mammals, and alternative splicing of strongly tissue-specific NAGNAGs was 10 times as likely to be conserved between species as was splicing of non-tissue-specific events, implying selective maintenance. Preferential use of the distal NAG was associated with distinct sequence features, including a more distal location of the branch point and presence of a pyrimidine immediately before the first NAG, and alteration of these features in a splicing reporter shifted splicing away from the distal site. Strikingly, alignments of orthologous exons revealed a ∼15-fold increase in the frequency of three base pair gaps at 3′ splice sites relative to nearby exon positions in both mammals and in Drosophila. Alternative splicing of NAGNAGs in human was associated with dramatically increased frequency of exon length changes at orthologous exon boundaries in rodents, and a model involving point mutations that create, destroy, or alter NAGNAGs can explain both the increased frequency and biased codon composition of gained/lost sequence observed at the beginnings of exons. This study shows that NAGNAG alternative splicing generates widespread differences between the proteomes of mammalian tissues, and suggests that the evolutionary trajectories of mammalian proteins are strongly biased by the locations and phases of the introns that interrupt coding sequences.
Author Summary
In order to translate a gene into protein, all of the non-coding regions (introns) need to be removed from the transcript and the coding regions (exons) stitched back together to make an mRNA. Most human genes are alternatively spliced, allowing the selection of different combinations of exons to produce multiple distinct mRNAs and proteins. Many types of alternative splicing are known to play crucial roles in biological processes including cell fate determination, tumor metabolism, and apoptosis. In this study, we investigated a form of alternative splicing in which competing adjacent 3′ splice sites (or splice acceptor sites) generate mRNAs differing by just an RNA triplet, the size of a single codon. This mode of alternative splicing, known as NAGNAG splicing, affects thousands of human genes and has been known for a decade, but its potential regulation, physiological importance, and conservation across species have been disputed. Using high-throughput sequencing of cDNA (“RNA-Seq”) from human and mouse tissues, we found that single-codon splicing often shows strong tissue specificity. Regulated NAGNAG alternative splice sites are selectively conserved between human and mouse genes, suggesting that they are important for organismal fitness. We identified features of the competing splice sites that influence NAGNAG splicing, and validated their effects in cultured cells. Furthermore, we found that this mode of splicing is associated with accelerated and highly biased protein evolution at exon boundaries. Taken together, our analyses demonstrate that the inclusion or exclusion of RNA triplets at exon boundaries can be effectively regulated by the splicing machinery, and highlight an unexpected connection between RNA processing and protein evolution.
doi:10.1371/journal.pbio.1001229
PMCID: PMC3250501  PMID: 22235189
11.  Depolarization and CaM Kinase IV Modulate NMDA Receptor Splicing through Two Essential RNA Elements 
PLoS Biology  2007;5(2):e40.
Alternative splicing controls the activity of many proteins important for neuronal excitation, but the signal-transduction pathways that affect spliced isoform expression are not well understood. One particularly interesting system of alternative splicing is exon 21 (E21) of the NMDA receptor 1 (NMDAR1 E21), which controls the trafficking of NMDA receptors to the plasma membrane and is repressed by Ca++/calmodulin-dependent protein kinase (CaMK) IV signaling. Here, we characterize the splicing of NMDAR1 E21. We find that E21 splicing is reversibly repressed by neuronal depolarization, and we identify two RNA elements within the exon that function together to mediate the inducible repression. One of these exonic elements is similar to an intronic CaMK IV–responsive RNA element (CaRRE) originally identified in the 3′ splice site of the BK channel STREX exon, but not previously observed within an exon. The other element is a new RNA motif. Introduction of either of these two motifs, called CaRRE type 1 and CaRRE type 2, into a heterologous constitutive exon can confer CaMK IV–dependent repression on the new exon. Thus, either exonic CaRRE can be sufficient for CaMK IV–induced repression. Single nucleotide scanning mutagenesis defined consensus sequences for these two CaRRE motifs. A genome-wide motif search and subsequent RT-PCR validation identified a group of depolarization-regulated alternative exons carrying CaRRE consensus sequences. Many of these exons are likely to alter neuronal function. Thus, these two RNA elements define a group of co-regulated splicing events that respond to a common stimulus in neurons to alter their activity.
Alternative splicing of NMDA receptor 1 exon 21 is reversibly repressed by depolarization in a CaMK IV-dependent manner in neurons. This suggests splicing is finely tuned by dynamic activity inputs.
Author Summary
Multiple mechanisms direct changes in neuronal activity in response to external stimuli, ranging from short-acting modifications of membrane proteins to longer-acting changes in gene expression. A frequently regulated step in gene expression is the pre-mRNA splicing reaction in which the inclusion of exons (protein-coding sequences) or the position of splice sites produces alternatively spliced mRNA isoforms encoding functionally different proteins. Here, we study splicing of the NMDA receptor, which responds to the neurotransmitter glutamate to modify neuronal activity. We show that the splicing of an important exon (E21) in the NMDA receptor subunit NR1 mRNA is repressed by cell depolarization and activation of the intracellular signaling molecule, CaMK IV. We find that this splicing repression is mediated by two regulatory sequences within the exon itself. One sequence is similar to a previously described regulatory element that had not been known to function in an exon. The other is a new element. The characterization of these elements as a family of degenerate sequences allowed the identification of a group of exons sharing responsiveness to cell depolarization and CamK IV. These results define a new set of gene expression changes that may occur in modulating neuronal activity.
doi:10.1371/journal.pbio.0050040
PMCID: PMC1790950  PMID: 17298178
12.  The “Alternative” Choice of Constitutive Exons throughout Evolution 
PLoS Genetics  2007;3(11):e203.
Alternative cassette exons are known to originate from two processes—exonization of intronic sequences and exon shuffling. Herein, we suggest an additional mechanism by which constitutively spliced exons become alternative cassette exons during evolution. We compiled a dataset of orthologous exons from human and mouse that are constitutively spliced in one species but alternatively spliced in the other. Examination of these exons suggests that the common ancestors were constitutively spliced. We show that relaxation of the 5′ splice site during evolution is one of the molecular mechanisms by which exons shift from constitutive to alternative splicing. This shift is associated with the fixation of exonic splicing regulatory sequences (ESRs) that are essential for exon definition and control the inclusion level only after the transition to alternative splicing. The effect of each ESR on splicing and the combinatorial effects between two ESRs are conserved from fish to human. Our results uncover an evolutionary pathway that increases transcriptome diversity by shifting exons from constitutive to alternative splicing.
Author Summary
Alternative splicing is believed to play a major role in the creation of transcriptomic diversification leading to higher order of organismal complexity, especially in mammals. As much as 80% of human genes generate more than one type of mRNA by alternative splicing. Thus, alternative splicing can bridge the low number of protein coding genes (∼24,500) and the total number of proteins generated in the human proteome (∼90,000). The correlation between the higher order of phenotypic diversity and alternative splicing was recently demonstrated and thus the origin of alternative splicing is of great interest. There are currently two models regarding the origin of alternatively spliced exons—exonization of intronic sequences and exon shuffling. According to these two mechanisms, a protein-coding gene was first established and only then a new alternative exon appeared within it or was added to the gene. Our current study provides evidences for a new mechanism indicating that during evolution constitutively spliced exons became alternatively spliced. Large-scale bioinformatic analyses reveal the magnitude of this process and experimental validation systems provide insights into its mechanisms.
doi:10.1371/journal.pgen.0030203
PMCID: PMC2077895  PMID: 18020709
13.  Unusual Intron Conservation near Tissue-Regulated Exons Found by Splicing Microarrays 
Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5′ splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families.
Synopsis
Alternative splicing expands the protein-coding potential of genes and genomes. RNAs copied from a gene can be spliced differently to produce distinct proteins under regulatory influences that arise during development or upon environmental change. These authors present a global analysis of alternative splicing in the mouse, using microarray measurements of splicing from 22 adult tissues. The ability to measure thousands of splicing events across the genome in many tissues has allowed the capture of co-regulated sets of exons whose inclusion in mRNA occurs preferentially in a given set of tissues. An examination of the sequences associated with exons whose expression is regulated in brain or muscle as compared to other tissues reveals extreme conservation of intron sequences nearby the regulated exon. These conserved regions contain sequence motifs likely to contribute to the regulation of alternative splicing in brain and muscle cells. The availability of global gene expression data with splicing level resolution should spur the development of computational methods for detecting and predicting alternative splicing and its regulation. In addition, the authors make strong predictions for biological experiments leading to the identification of components and their mechanisms of action in the regulation of splicing during mammalian development.
doi:10.1371/journal.pcbi.0020004
PMCID: PMC1331982  PMID: 16424921
14.  Context-dependent robustness to 5′ splice site polymorphisms in human populations 
Human Molecular Genetics  2010;20(6):1084-1096.
There has been growing evidence for extensive diversity of alternative splicing in human populations. Genetic variants within the 5′ splice site can cause splicing differences among human individuals and constitute an important class of human disease mutations. In this study, we explored whether natural variations of splicing could reveal important signals of 5′ splice site recognition. In seven lymphoblastoid cell lines of Asian, European and African ancestry, we identified 1174 single nucleotide polymorphisms (SNPs) within the consensus 5′ splice site. We selected 129 SNPs predicted to significantly alter the splice site activity, and quantitatively examined their splicing impact in the seven individuals. Surprisingly, outside of the essential GT dinucleotide position, only ∼14% of the tested SNPs altered splicing. Bioinformatic and minigene analyses identified signals that could modify the impact of 5′ splice site polymorphisms, most notably a strong 3′ splice site and the presence of intronic motifs downstream of the 5′ splice site. Strikingly, we found that the poly-G run, a known intronic splicing enhancer, was the most significantly enriched motif downstream of exons unaffected by 5′ splice site SNPs. In TRIM62, the upstream 3′ splice site and downstream intronic poly-G runs functioned redundantly to protect an exon from its 5′ splice site polymorphism. Collectively, our study reveals widespread context-dependent robustness to 5′ splice site polymorphisms in human transcriptomes. Consequently, certain exons are more susceptible to 5′ splice site mutations. Additionally, our work demonstrates that genetic diversity of alternative splicing can provide significant insights into the splicing code of mammalian cells.
doi:10.1093/hmg/ddq553
PMCID: PMC3043661  PMID: 21224255
15.  Alu Exonization Events Reveal Features Required for Precise Recognition of Exons by the Splicing Machinery 
PLoS Computational Biology  2009;5(3):e1000300.
Despite decades of research, the question of how the mRNA splicing machinery precisely identifies short exonic islands within the vast intronic oceans remains to a large extent obscure. In this study, we analyzed Alu exonization events, aiming to understand the requirements for correct selection of exons. Comparison of exonizing Alus to their non-exonizing counterparts is informative because Alus in these two groups have retained high sequence similarity but are perceived differently by the splicing machinery. We identified and characterized numerous features used by the splicing machinery to discriminate between Alu exons and their non-exonizing counterparts. Of these, the most novel is secondary structure: Alu exons in general and their 5′ splice sites (5′ss) in particular are characterized by decreased stability of local secondary structures with respect to their non-exonizing counterparts. We detected numerous further differences between Alu exons and their non-exonizing counterparts, among others in terms of exon–intron architecture and strength of splicing signals, enhancers, and silencers. Support vector machine analysis revealed that these features allow a high level of discrimination (AUC = 0.91) between exonizing and non-exonizing Alus. Moreover, the computationally derived probabilities of exonization significantly correlated with the biological inclusion level of the Alu exons, and the model could also be extended to general datasets of constitutive and alternative exons. This indicates that the features detected and explored in this study provide the basis not only for precise exon selection but also for the fine-tuned regulation thereof, manifested in cases of alternative splicing.
Author Summary
A typical human gene consists of 9 exons around 150 nucleotides in length, separated by introns that are ∼3,000 nucleotides long. The challenge of the splicing machinery is to precisely identify and ligate the exons, while removing the introns. We aimed to understand how the splicing machinery meets this momentous challenge, based on Alu exonization events. Alus are transposable elements, of which approximately one million copies exist in the human genome, a large portion of which within introns. Throughout evolution, some intronic Alus accumulated mutations and became recognized by the splicing machinery as exons, a process termed exonization. Such Alus remain highly similar to their non-exonizing counterparts but are perceived as different by the splicing machinery. By comparing exonizing Alus to their non-exonizing counterparts, we were able to identify numerous features in which they differ and which presumably lead to the recognition only of the former by the splicing machinery. Our findings reveal insights regarding the role of local RNA secondary structures, exon–intron architecture constraints, and splicing regulatory signals. We integrated these features in a computational model, which was able to successfully mimic the function of the splicing machinery and discriminate between true Alu exons and their intronic counterparts, highlighting the functional importance of these features.
doi:10.1371/journal.pcbi.1000300
PMCID: PMC2639721  PMID: 19266014
16.  Inference of Splicing Regulatory Activities by Sequence Neighborhood Analysis 
PLoS Genetics  2006;2(11):e191.
Sequence-specific recognition of nucleic-acid motifs is critical to many cellular processes. We have developed a new and general method called Neighborhood Inference (NI) that predicts sequences with activity in regulating a biochemical process based on the local density of known sites in sequence space. Applied to the problem of RNA splicing regulation, NI was used to predict hundreds of new exonic splicing enhancer (ESE) and silencer (ESS) hexanucleotides from known human ESEs and ESSs. These predictions were supported by cross-validation analysis, by analysis of published splicing regulatory activity data, by sequence-conservation analysis, and by measurement of the splicing regulatory activity of 24 novel predicted ESEs, ESSs, and neutral sequences using an in vivo splicing reporter assay. These results demonstrate the ability of NI to accurately predict splicing regulatory activity and show that the scope of exonic splicing regulatory elements is substantially larger than previously anticipated. Analysis of orthologous exons in four mammals showed that the NI score of ESEs, a measure of function, is much more highly conserved above background than ESE primary sequence. This observation indicates a high degree of selection for ESE activity in mammalian exons, with surprisingly frequent interchangeability between ESE sequences.
Synopsis
Gene expression involves a series of steps in which specific short DNA or RNA segments are recognized by nucleic acid–binding proteins. One step that is particularly prominent and complex in humans and other vertebrates is the removal of introns and the ligation of exons in the process of pre-mRNA splicing. To better understand the sequences in exons that regulate this process, the authors have developed a method termed Neighborhood Inference that predicts the splicing regulatory activity of RNA segments based on the known splicing enhancer or silencer activity of other segments that have closely neighboring sequences. This method is applied to predict hundreds of new exonic splicing regulatory elements, as well as splicing-neutral sequences. A number of these predictions were validated experimentally, indicating that the number of exonic splicing regulatory sequences is larger than previously suspected. Neighborhood Inference scoring is also used to show that selection on exonic splicing enhancers (ESEs) frequently allows conversion of one ESE sequence to another over evolutionary time periods, suggesting that ESEs are, to at least some degree, interchangeable in constitutively spliced exons. The methods described may also find application in the study of other biomolecular processes that involve sequence-specific nucleic acid–binding proteins.
doi:10.1371/journal.pgen.0020191
PMCID: PMC1657047  PMID: 17121466
17.  Computational analysis of splicing errors and mutations in human transcripts 
BMC Genomics  2008;9:13.
Background
Most retained introns found in human cDNAs generated by high-throughput sequencing projects seem to result from underspliced transcripts, and thus they capture intermediate steps of pre-mRNA splicing. On the other hand, mutations in splice sites cause exon skipping of the respective exon or activation of pre-existing cryptic sites. Both types of events reflect properties of the splicing mechanism.
Results
The retained introns were significantly shorter than constitutive ones, and skipped exons are shorter than exons with cryptic sites. Both donor and acceptor splice sites of retained introns were weaker than splice sites of constitutive introns. The authentic acceptor sites affected by mutations were significantly weaker in exons with activated cryptic sites than in skipped exons. The distance from a mutated splice site to the nearest equivalent site is significantly shorter in cases of activated cryptic sites compared to exon skipping events. The prevalence of retained introns within genes monotonically increased in the 5'-to-3' direction (more retained introns close to the 3'-end), consistent with the model of co-transcriptional splicing. The density of exonic splicing enhancers was higher, and the density of exonic splicing silencers lower in retained introns compared to constitutive ones and in exons with cryptic sites compared to skipped exons.
Conclusion
Thus the analysis of retained introns in human cDNA, exons skipped due to mutations in splice sites and exons with cryptic sites produced results consistent with the intron definition mechanism of splicing of short introns, co-transcriptional splicing, dependence of splicing efficiency on the splice site strength and the density of candidate exonic splicing enhancers and silencers. These results are consistent with other, recently published analyses.
doi:10.1186/1471-2164-9-13
PMCID: PMC2234086  PMID: 18194514
18.  Exon creation and establishment in human genes 
Genome Biology  2008;9(9):R141.
A comparative genomics study of alternatively spliced exons showing that the relative local abundance of splicing regulatory motifs influences splicing decisions in humans.
Background
A large proportion of species-specific exons are alternatively spliced. In primates, Alu elements play a crucial role in the process of exon creation but many new exons have appeared through other mechanisms. Despite many recent studies, it is still unclear which are the splicing regulatory requirements for de novo exonization and how splicing regulation changes throughout an exon's lifespan.
Results
Using comparative genomics, we have defined sets of exons with different evolutionary ages. Younger exons have weaker splice-sites and lower absolute values for the relative abundance of putative splicing regulators between exonic and adjacent intronic regions, indicating a less consolidated splicing regulation. This relative abundance is shown to increase with exon age, leading to higher exon inclusion. We show that this local difference in the density of regulators might be of biological significance, as it outperforms other measures in real exon versus pseudo-exon classification. We apply this new measure to the specific case of the exonization of anti-sense Alu elements and show that they are characterized by a general lack of exonic splicing silencers.
Conclusions
Our results suggest that specific sequence environments are required for exonization and that these can change with time. We propose a model of exon creation and establishment in human genes, in which splicing decisions depend on the relative local abundance of regulatory motifs. Using this model, we provide further explanation as to why Alu elements serve as a major substrate for exon creation in primates. Finally, we discuss the benefits of integrating such information in gene prediction.
doi:10.1186/gb-2008-9-9-r141
PMCID: PMC2592719  PMID: 18811936
19.  The strength of the HIV-1 3' splice sites affects Rev function 
Retrovirology  2006;3:89.
Background
The HIV-1 Rev protein is a key component in the early to late switch in HIV-1 splicing from early intronless (e.g. tat, rev) to late intron-containing Rev-dependent (e.g. gag, vif, env) transcripts. Previous results suggested that cis-acting sequences and inefficient 5' and 3' splice sites are a prerequisite for Rev function. However, we and other groups have shown that two of the HIV-1 5' splice sites, D1 and D4, are efficiently used in vitro and in vivo. Here, we focus on the efficiency of the HIV-1 3' splice sites taking into consideration to what extent their intrinsic efficiencies are modulated by their downstream cis-acting exonic sequences. Furthermore, we delineate their role in RNA stabilization and Rev function.
Results
In the presence of an efficient upstream 5' splice site the integrity of the 3' splice site is not essential for Rev function whereas an efficient 3' splice site impairs Rev function. The detrimental effect of a strong 3' splice site on the amount of Rev-dependent intron-containing HIV-1 glycoprotein coding (env) mRNA is not compensatable by weakening the strength of the upstream 5' splice site. Swapping the HIV-1 3' splice sites in an RRE-containing minigene, we found a 3' splice site usage which was variably dependent on the presence of the usual downstream exonic sequence. The most evident activation of 3' splice site usage by its usual downstream exonic sequence was observed for 3' splice site A1 which was turned from an intrinsic very weak 3' splice site into the most active 3' splice site, even abolishing Rev activity. Performing pull-down experiments with nuclear extracts of HeLa cells we identified a novel ASF/SF2-dependent exonic splicing enhancer (ESE) within HIV-1 exon 2 consisting of a heptameric sequence motif occurring twice (M1 and M2) within this short non-coding leader exon. Single point mutation of M1 within an infectious molecular clone is detrimental for HIV-1 exon 2 recognition without affecting Rev-dependent vif expression.
Conclusion
Under the conditions of our assay, the rate limiting step of retroviral splicing, competing with Rev function, seems to be exclusively determined by the functional strength of the 3' splice site. The bipartite ASF/SF2-dependent ESE within HIV-1 exon 2 supports cross-talk between splice site pairs across exon 2 (exon definition) which is incompatible with processing of the intron-containing vif mRNA. We propose that Rev mediates a switch from exon to intron definition necessary for the expression of all intron-containing mRNAs.
doi:10.1186/1742-4690-3-89
PMCID: PMC1697824  PMID: 17144911
20.  Identification of Evolutionarily Conserved Exons as Regulated Targets for the Splicing Activator Tra2β in Development 
PLoS Genetics  2011;7(12):e1002390.
Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10fl/fl; Nestin-Cretg/+). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.
Author Summary
Alternative splicing amplifies the informational content of the genome, making multiple mRNA isoforms from single genes. Tra2 proteins bind and activate alternative exons, and in mice Tra2β is essential for embryonic development through unknown target RNAs. Here we report the first target exons that are physiologically regulated by Tra2β in developing mice. Normal activation of these regulated exons depends on multiple Tra2β binding sites, and significant mis-regulation of these exons is observed during mouse development when Tra2β is removed. As expected, Tra2β activates splicing of some target exons through direct RNA binding via its RNA Recognition Motif. Surprisingly, for some exons Tra2β can also activate splicing independent of direct RNA binding through two domains enriched in arginine and serine residues (called RS domains). The N-terminal RS1 domain of Tra2β is absolutely essential for splicing activation of physiological target exons, explaining why this domain is conserved between vertebrates and invertebrates. Surprisingly, Tra2β proteins without RS1 operate as splicing repressors, suggesting the possibility that endogenous Tra2β protein isoforms may differentially regulate the same target exons.
doi:10.1371/journal.pgen.1002390
PMCID: PMC3240583  PMID: 22194695
21.  The Emergence of Alternative 3′ and 5′ Splice Site Exons from Constitutive Exons 
PLoS Computational Biology  2007;3(5):e95.
Alternative 3′ and 5′ splice site (ss) events constitute a significant part of all alternative splicing events. These events were also found to be related to several aberrant splicing diseases. However, only few of the characteristics that distinguish these events from alternative cassette exons are known currently. In this study, we compared the characteristics of constitutive exons, alternative cassette exons, and alternative 3′ss and 5′ss exons. The results revealed that alternative 3′ss and 5′ss exons are an intermediate state between constitutive and alternative cassette exons, where the constitutive side resembles constitutive exons, and the alternative side resembles alternative cassette exons. The results also show that alternative 3′ss and 5′ss exons exhibit low levels of symmetry (frame-preserving), similar to constitutive exons, whereas the sequence between the two alternative splice sites shows high symmetry levels, similar to alternative cassette exons. In addition, flanking intronic conservation analysis revealed that exons whose alternative splice sites are at least nine nucleotides apart show a high conservation level, indicating intronic participation in the regulation of their splicing, whereas exons whose alternative splice sites are fewer than nine nucleotides apart show a low conservation level. Further examination of these exons, spanning seven vertebrate species, suggests an evolutionary model in which the alternative state is a derivative of an ancestral constitutive exon, where a mutation inside the exon or along the flanking intron resulted in the creation of a new splice site that competes with the original one, leading to alternative splice site selection. This model was validated experimentally on four exons, showing that they indeed originated from constitutive exons that acquired a new competing splice site during evolution.
Author Summary
Alternative splicing is the mechanism that is responsible for the creation of multiple mRNA products from a single gene. It is considered a key player in genomic complexity achievement. Alternative 3′ and 5′ splicing events in which part of the exon is alternatively included or excluded in the mRNA constitute a significant part of all alternative splicing events, and yet little is known regarding their regulation mechanism and the evolutionary background that led to their creation. We show that alternative 3′ and 5′ splice site exons resemble constitutive exons. However, their alternative sequence resembles alternative cassette exons. Comparative genomics spanning seven vertebrate species suggests an evolutionary model in which the alternative state is a derivative of an ancestral constitutive exon, where a mutation inside the exon or along the flanking intron resulted in the creation of a new splice site that competes with the original one, leading to alternative splice site selection. This model was validated experimentally, showing that during evolution mutations shifted constitutive exons to undergo alternative 3′ and 5′ splicing.
doi:10.1371/journal.pcbi.0030095
PMCID: PMC1876488  PMID: 17530917
22.  Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements 
PLoS Genetics  2007;3(5):e85.
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.
Author Summary
During RNA splicing, sequences (introns) in a pre-mRNA are excised and discarded, and the remaining sequences (exons) are joined to form the mature RNA. Splicing is regulated not only by the binding of the basic splicing machinery to splice sites located at the exon–intron boundaries, but also by the combined effects of various other splicing factors that bind to a multitude of sequence elements located both in the exons as well as the flanking introns. Instances of alternative splicing, where usage of splice site(s) is incomplete or different between tissues, cell types, or lineages, can be created by the interaction of sequence elements and tissue, cell type, and stage-specific splicing factors. To better understand constitutive and alternative pre-mRNA splicing, the authors describe a comparative genomics approach, using available mammalian genomes, to systematically identify splicing regulatory elements located in the introns proximal to exons. A quarter of the elements were tested experimentally, and most of them altered splicing in human cells. The authors also showed that that the intronic elements are close to tissue-specific alternative exons and are more likely to be located in specific positions in the introns, suggestive of potential regulatory function. These elements are also frequently found in tissue-specific genes, suggesting a coupling between expression and alternative splicing of these genes. Finally, the authors propose a strategy using the elements to identify the binding sites of several splicing factors.
doi:10.1371/journal.pgen.0030085
PMCID: PMC1877881  PMID: 17530930
23.  Exonic Splicing Enhancer-Dependent Selection of the Bovine Papillomavirus Type 1 Nucleotide 3225 3′ Splice Site Can Be Rescued in a Cell Lacking Splicing Factor ASF/SF2 through Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway 
Journal of Virology  2003;77(3):2105-2115.
Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation-specific manner: the late leader 5′ splice site alternatively splices to a proximal 3′ splice site (at nucleotide 3225) to express L2 or to a distal 3′ splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF/SF2 (alternative splicing factor/splicing factor 2) binding sites, are located between the two 3′ splice sites and have been identified as regulating alternative 3′ splice site usage. The present report demonstrates for the first time that ASF/SF2 is required under physiological conditions for the expression of BPV-1 late RNAs and for selection of the proximal 3′ splice site for BPV-1 RNA splicing in DT40-ASF cells, a genetically engineered chicken B-cell line that expresses only human ASF/SF2 controlled by a tetracycline-repressible promoter. Depletion of ASF/SF2 from the cells by tetracycline greatly decreased viral RNA expression and RNA splicing at the proximal 3′ splice site while increasing use of the distal 3′ splice site in the remaining viral RNAs. Activation of cells lacking ASF/SF2 through anti-immunoglobulin M-B-cell receptor cross-linking rescued viral RNA expression and splicing at the proximal 3′ splice site and enhanced Akt phosphorylation and expression of the phosphorylated serine/arginine-rich (SR) proteins SRp30s (especially SC35) and SRp40. Treatment with wortmannin, a specific phosphatidylinositol 3-kinase/Akt kinase inhibitor, completely blocked the activation-induced activities. ASF/SF2 thus plays an important role in viral RNA expression and splicing at the proximal 3′ splice site, but activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells is mediated through the phosphatidylinositol 3-kinase/Akt pathway and is associated with the enhanced expression of other SR proteins.
doi:10.1128/JVI.77.3.2105-2115.2003
PMCID: PMC140879  PMID: 12525645
24.  Computational prediction of splicing regulatory elements shared by Tetrapoda organisms 
BMC Genomics  2009;10:508.
Background
Auxiliary splicing sequences play an important role in ensuring accurate and efficient splicing by promoting or repressing recognition of authentic splice sites. These cis-acting motifs have been termed splicing enhancers and silencers and are located both in introns and exons. They co-evolved into an intricate splicing code together with additional functional constraints, such as tissue-specific and alternative splicing patterns. We used orthologous exons extracted from the University of California Santa Cruz multiple genome alignments of human and 22 Tetrapoda organisms to predict candidate enhancers and silencers that have reproducible and statistically significant bias towards annotated exonic boundaries.
Results
A total of 2,546 Tetrapoda enhancers and silencers were clustered into 15 putative core motifs based on their Markov properties. Most of these elements have been identified previously, but 118 putative silencers and 260 enhancers (~15%) were novel. Examination of previously published experimental data for the presence of predicted elements showed that their mutations in 21/23 (91.3%) cases altered the splicing pattern as expected. Predicted intronic motifs flanking 3' and 5' splice sites had higher evolutionary conservation than other sequences within intronic flanks and the intronic enhancers were markedly differed between 3' and 5' intronic flanks.
Conclusion
Difference in intronic enhancers supporting 5' and 3' splice sites suggests an independent splicing commitment for neighboring exons. Increased evolutionary conservation for ISEs/ISSs within intronic flanks and effect of modulation of predicted elements on splicing suggest functional significance of found elements in splicing regulation. Most of the elements identified were shown to have direct implications in human splicing and therefore could be useful for building computational splicing models in biomedical research.
doi:10.1186/1471-2164-10-508
PMCID: PMC2777938  PMID: 19889216
25.  Regulation of Alternative Polyadenylation by U1 snRNPs and SRp20 
Molecular and Cellular Biology  1998;18(9):4977-4985.
Although considerable information is currently available about the factors involved in constitutive vertebrate polyadenylation, the factors and mechanisms involved in facilitating communication between polyadenylation and splicing are largely unknown. Even less is known about the regulation of polyadenylation in genes in which 3′-terminal exons are alternatively recognized. Here we demonstrate that an SR protein, SRp20, affects recognition of an alternative 3′-terminal exon via an effect on the efficiency of binding of a polyadenylation factor to an alternative polyadenylation site. The gene under study codes for the peptides calcitonin and calcitonin gene-related peptide. Its pre-mRNA is alternatively processed by the tissue-specific inclusion or exclusion of an embedded 3′-terminal exon, exon 4, via factors binding to an intronic enhancer element that contains both 3′ and 5′ splice site consensus sequence elements. In cell types that preferentially exclude exon 4, addition of wild-type SRp20 enhances exon 4 inclusion via recognition of the intronic enhancer. In contrast, in cell types that preferentially include exon 4, addition of a mutant form of SRp20 containing the RNA-binding domain but missing the SR domain inhibits exon 4 inclusion. Inhibition is likely at the level of polyadenylation, because the mutant SRp20 inhibits binding of CstF to the exon 4 poly(A) site. This is the first demonstration that an SR protein can influence alternative polyadenylation and suggests that this family of proteins may play a role in recognition of 3′-terminal exons and perhaps in the communication between polyadenylation and splicing.
PMCID: PMC109082  PMID: 9710581

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