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1.  GC content around splice sites affects splicing through pre-mRNA secondary structures 
BMC Genomics  2011;12:90.
Alternative splicing increases protein diversity by generating multiple transcript isoforms from a single gene through different combinations of exons or through different selections of splice sites. It has been reported that RNA secondary structures are involved in alternative splicing. Here we perform a genomic study of RNA secondary structures around splice sites in humans (Homo sapiens), mice (Mus musculus), fruit flies (Drosophila melanogaster), and nematodes (Caenorhabditis elegans) to further investigate this phenomenon.
We observe that GC content around splice sites is closely associated with the splice site usage in multiple species. RNA secondary structure is the possible explanation, because the structural stability difference among alternative splice sites, constitutive splice sites, and skipped splice sites can be explained by the GC content difference. Alternative splice sites tend to be GC-enriched and exhibit more stable RNA secondary structures in all of the considered species. In humans and mice, splice sites of first exons and long exons tend to be GC-enriched and hence form more stable structures, indicating the special role of RNA secondary structures in promoter proximal splicing events and the splicing of long exons. In addition, GC-enriched exon-intron junctions tend to be overrepresented in tissue-specific alternative splice sites, indicating the functional consequence of the GC effect. Compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched. We also found that the GC-content effect is much stronger than the nucleotide-order effect to form stable secondary structures.
All of these results indicate that GC content is related to splice site usage and it may mediate the splicing process through RNA secondary structures.
PMCID: PMC3041747  PMID: 21281513
2.  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.
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.
PMCID: PMC1500816  PMID: 16839192
3.  Analysis of the role of Caenorhabditis elegans GC-AG introns in regulated splicing 
Nucleic Acids Research  2002;30(15):3360-3367.
GC-AG introns represent 0.7% of total human pre-mRNA introns. To study the function of GC-AG introns in splicing regulation, 196 cDNA-confirmed GC-AG introns were identified in Caenorhabditis elegans. These represent 0.6% of the cDNA- confirmed intron data set for this organism. Eleven of these GC-AG introns are involved in alternative splicing. In a comparison of the genomic sequences of homologous genes between C.elegans and Caenorhabditis briggsae for 26 GC-AG introns, the C at the +2 position is conserved in only five of these introns. A system to experimentally test the function of GC-AG introns in alternative splicing was developed. Results from these experiments indicate that the conserved C at the +2 position of the tenth intron of the let-2 gene is essential for developmentally regulated alternative splicing. This C allows the splice donor to function as a very weak splice site that works in balance with an alternative GT splice donor. A weak GT splice donor can functionally replace the GC splice donor and allow for splicing regulation. These results indicate that while the majority of GC-AG introns appear to be constitutively spliced and have no evolutionary constraints to prevent them from being GT-AG introns, a subset of GC-AG introns is involved in alternative splicing and the C at the +2 position of these introns can have an important role in splicing regulation.
PMCID: PMC137088  PMID: 12140320
4.  Intron-exon structures of eukaryotic model organisms. 
Nucleic Acids Research  1999;27(15):3219-3228.
To investigate the distribution of intron-exon structures of eukaryotic genes, we have constructed a general exon database comprising all available intron-containing genes and exon databases from 10 eukaryotic model organisms: Homo sapiens, Mus musculus, Gallus gallus, Rattus norvegicus, Arabidopsis thaliana, Zea mays, Schizosaccharomyces pombe, Aspergillus, Caenorhabditis elegans and Drosophila. We purged redundant genes to avoid the possible bias brought about by redundancy in the databases. After discarding those questionable introns that do not contain correct splice sites, the final database contained 17 102 introns, 21 019 exons and 2903 independent or quasi-independent genes. On average, a eukaryotic gene contains 3.7 introns per kb protein coding region. The exon distribution peaks around 30-40 residues and most introns are 40-125 nt long. The variable intron-exon structures of the 10 model organisms reveal two interesting statistical phenomena, which cast light on some previous speculations. (i) Genome size seems to be correlated with total intron length per gene. For example, invertebrate introns are smaller than those of human genes, while yeast introns are shorter than invertebrate introns. However, this correlation is weak, suggesting that other factors besides genome size may also affect intron size. (ii) Introns smaller than 50 nt are significantly less frequent than longer introns, possibly resulting from a minimum intron size requirement for intron splicing.
PMCID: PMC148551  PMID: 10454621
5.  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.
PMCID: PMC1877881  PMID: 17530930
6.  Phylogenetically widespread alternative splicing at unusual GYNGYN donors 
Genome Biology  2006;7(7):R65.
Computational and experimental evidence is given for alternative splicing at the unusual GYNGYN motif in several species, enabling in most cases subtle protein variations.
Splice donor sites have a highly conserved GT or GC dinucleotide and an extended intronic consensus sequence GTRAGT that reflects the sequence complementarity to the U1 snRNA. Here, we focus on unusual donor sites with the motif GYNGYN (Y stands for C or T; N stands for A, C, G, or T).
While only one GY functions as a splice donor for the majority of these splice sites in human, we provide computational and experimental evidence that 110 (1.3%) allow alternative splicing at both GY donors. The resulting splice forms differ in only three nucleotides, which results mostly in the insertion/deletion of one amino acid. However, we also report the insertion of a stop codon in four cases. Investigating what distinguishes alternatively from not alternatively spliced GYNGYN donors, we found differences in the binding to U1 snRNA, a strong correlation between U1 snRNA binding strength and the preferred donor, over-represented sequence motifs in the adjacent introns, and a higher conservation of the exonic and intronic flanks between human and mouse. Extending our genome-wide analysis to seven other eukaryotic species, we found alternatively spliced GYNGYN donors in all species from mouse to Caenorhabditis elegans and even in Arabidopsis thaliana. Experimental verification of a conserved GTAGTT donor of the STAT3 gene in human and mouse reveals a remarkably similar ratio of alternatively spliced transcripts in both species.
In contrast to alternative splicing in general, GYNGYN donors in addition to NAGNAG acceptors enable subtle protein variations.
PMCID: PMC1779574  PMID: 16869967
7.  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.
PMCID: PMC3250501  PMID: 22235189
8.  Noisy Splicing Drives mRNA Isoform Diversity in Human Cells 
PLoS Genetics  2010;6(12):e1001236.
While the majority of multiexonic human genes show some evidence of alternative splicing, it is unclear what fraction of observed splice forms is functionally relevant. In this study, we examine the extent of alternative splicing in human cells using deep RNA sequencing and de novo identification of splice junctions. We demonstrate the existence of a large class of low abundance isoforms, encompassing approximately 150,000 previously unannotated splice junctions in our data. Newly-identified splice sites show little evidence of evolutionary conservation, suggesting that the majority are due to erroneous splice site choice. We show that sequence motifs involved in the recognition of exons are enriched in the vicinity of unconserved splice sites. We estimate that the average intron has a splicing error rate of approximately 0.7% and show that introns in highly expressed genes are spliced more accurately, likely due to their shorter length. These results implicate noisy splicing as an important property of genome evolution.
Author Summary
Most human genes are split into pieces, such that the protein-coding parts (exons) are separated in the genome by large tracts of non-coding DNA (introns) that must be transcribed and spliced out to create a functional transcript. Variation in splicing reactions can create multiple transcripts from the same gene, yet the function for many of these alternative transcripts is unknown. In this study, we show that many of these transcripts are due to splicing errors which are not preserved over evolutionary time. We estimate that the error rate in the splicing of an intron is about 0.7% and demonstrate that there are two major types of splicing error: errors in the recognition of exons and errors in the precise choice of splice site. These results raise the possibility that variation in levels of alternative splicing across species may in part be to variation in splicing error rate.
PMCID: PMC3000347  PMID: 21151575
9.  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.
PMCID: PMC1876488  PMID: 17530917
10.  Oriented Scanning Is the Leading Mechanism Underlying 5′ Splice Site Selection in Mammals 
PLoS Genetics  2006;2(9):e138.
Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5′ splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7) a 37-bp VNTR minisatellite whose first element spans the exon7–IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 5′ pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 5′ splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 5′ splice site, rather than repressing the 3′ following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 5′ pseudo-sites are functional, but do not compete with the authentic donor splice site. Moreover, we show that the selection of the 5′ splice site follows a scanning-type mechanism, precluding competition with other functional 5′ pseudo-sites available on immediate sequence context downstream of the activated one. In addition, 5′ pseudo-sites with an increased complementarity to U1snRNA up to 91% do not compete with the identified scanning mechanism. Altogether, these findings, which unveil a cell type–independent 5′−3′-oriented scanning process for accurate recognition of the authentic 5′ splice site, reconciliate apparently contradictory observations by establishing a hierarchy of competitiveness among the determinants involved in 5′ splice site selection.
Typically, mammalian genes contain coding sequences (exons) separated by non-coding sequences (introns). Introns are removed during pre-mRNA splicing. The accurate recognition of introns during splicing is essential, as any abnormality in that process will generate abnormal mRNAs that can cause diseases. Understanding the mechanisms of accurate splice site selection is of prime interest to life scientists. Exon–intron borders (splice sites) are defined by short sequences that are poorly conserved. The strength of any splice sequence can be assessed by its degree of homology with a splice site consensus sequence. Within exons and introns, several sequences can match with this consensus as well as or better than the splice sites. Using a system in which a splice site sequence is repeated several times in the intron, the authors showed that linear 5′−3′ search is a leading mechanism underlying splice site selection. This scanning mechanism is cell type–independent, and only the most upstream splice site of all the series is selected, even if splice sites with a better match to the consensus are in the vicinity. These findings reconciliate contradictory observations and establish a hierarchy among the determinants involved in splice site selection.
PMCID: PMC1557585  PMID: 16948532
11.  Splice site prediction with quadratic discriminant analysis using diversity measure 
Nucleic Acids Research  2003;31(21):6214-6220.
Based on the conservation of nucleotides at splicing sites and the features of base composition and base correlation around these sites we use the method of increment of diversity combined with quadratic discriminant analysis (IDQD) to study the dependence structure of splicing sites and predict the exons/introns and their boundaries for four model genomes: Caenorhabditis elegans, Arabidopsis thaliana, Drosophila melanogaster and human. The comparison of compositional features between two sequences and the comparison of base dependencies at adjacent or non-adjacent positions of two sequences can be integrated automatically in the increment of diversity (ID). Eight feature variables around a potential splice site are defined in terms of ID. They are integrated in a single formal framework given by IDQD. In our calculations 7 (8) base region around the donor (acceptor) sites have been considered in studying the conservation of nucleotides and sequences of 48 bp on either side of splice sites have been used in studying the compositional and base-correlating features. The windows are enlarged to 16 (donor), 29 (acceptor) and 80 bp (either side) to improve the prediction for human splice sites. The prediction capability of the present method is comparable with the leading splice site detector—GeneSplicer.
PMCID: PMC275452  PMID: 14576308
12.  CELF Family RNA–Binding Protein UNC-75 Regulates Two Sets of Mutually Exclusive Exons of the unc-32 Gene in Neuron-Specific Manners in Caenorhabditis elegans 
PLoS Genetics  2013;9(2):e1003337.
An enormous number of alternative pre–mRNA splicing patterns in multicellular organisms are coordinately defined by a limited number of regulatory proteins and cis elements. Mutually exclusive alternative splicing should be strictly regulated and is a challenging model for elucidating regulation mechanisms. Here we provide models of the regulation of two sets of mutually exclusive exons, 4a–4c and 7a–7b, of the Caenorhabditis elegans uncoordinated (unc)-32 gene, encoding the a subunit of V0 complex of vacuolar-type H+-ATPases. We visualize selection patterns of exon 4 and exon 7 in vivo by utilizing a trio and a pair of symmetric fluorescence splicing reporter minigenes, respectively, to demonstrate that they are regulated in tissue-specific manners. Genetic analyses reveal that RBFOX family RNA–binding proteins ASD-1 and FOX-1 and a UGCAUG stretch in intron 7b are involved in the neuron-specific selection of exon 7a. Through further forward genetic screening, we identify UNC-75, a neuron-specific CELF family RNA–binding protein of unknown function, as an essential regulator for the exon 7a selection. Electrophoretic mobility shift assays specify a short fragment in intron 7a as the recognition site for UNC-75 and demonstrate that UNC-75 specifically binds via its three RNA recognition motifs to the element including a UUGUUGUGUUGU stretch. The UUGUUGUGUUGU stretch in the reporter minigenes is actually required for the selection of exon 7a in the nervous system. We compare the amounts of partially spliced RNAs in the wild-type and unc-75 mutant backgrounds and raise a model for the mutually exclusive selection of unc-32 exon 7 by the RBFOX family and UNC-75. The neuron-specific selection of unc-32 exon 4b is also regulated by UNC-75 and the unc-75 mutation suppresses the Unc phenotype of the exon-4b-specific allele of unc-32 mutants. Taken together, UNC-75 is the neuron-specific splicing factor and regulates both sets of the mutually exclusive exons of the unc-32 gene.
Author Summary
Tissue-specific and mutually exclusive alternative pre–mRNA splicing is a challenging model for elucidating regulation mechanisms. We previously demonstrated that evolutionarily conserved RBFOX family RNA–binding proteins ASD-1 and FOX-1 and a muscle-specific RNA–binding protein SUP-12 cooperatively direct muscle-specific selection of exon 5B of the C. elegans egl-15 gene. Here we demonstrate that two sets of mutually exclusive exons, 4a–4c and 7a–7b, of the unc-32 gene are regulated in tissue-specific manners and that ASD-1 and FOX-1, expressed in a variety of tissues, can regulate the neuron-specific selection of unc-32 exon 7a in combination with the neuron-specific CELF family RNA–binding protein UNC-75. We determine the cis-elements for the RBFOX family and UNC-75, which separately reside in intron 7b and intron 7a, respectively. By analyzing the partially spliced RNA species, we propose the orders of intron removal and the sites of action for the RBFOX family and UNC-75 in the mutually exclusive selection of exon 7a and exon 7b. We also demonstrate that UNC-75 regulates the neuron-specific selection of exon 4b and propose the models of the mutually exclusive selection of exons 4a, 4b, and 4c. These studies thus provide novel modes of regulation for tissue-specific and mutually exclusive alternative splicing in vivo.
PMCID: PMC3585155  PMID: 23468662
13.  Analysis of canonical and non-canonical splice sites in mammalian genomes 
Nucleic Acids Research  2000;28(21):4364-4375.
A set of 43 337 splice junction pairs was extracted from mammalian GenBank annotated genes. Expressed sequence tag (EST) sequences support 22 489 of them. Of these, 98.71% contain canonical dinucleotides GT and AG for donor and acceptor sites, respectively; 0.56% hold non-canonical GC-AG splice site pairs; and the remaining 0.73% occurs in a lot of small groups (with a maximum size of 0.05%). Studying these groups we observe that many of them contain splicing dinucleotides shifted from the annotated splice junction by one position. After close examination of such cases we present a new classification consisting of only eight observed types of splice site pairs (out of 256 a priori possible combinations). EST alignments allow us to verify the exonic part of the splice sites, but many non-canonical cases may be due to intron sequencing errors. This idea is given substantial support when we compare the sequences of human genes having non-canonical splice sites deposited in GenBank by high throughput genome sequencing projects (HTG). A high proportion (156 out of 171) of the human non-canonical and EST-supported splice site sequences had a clear match in the human HTG. They can be classified after corrections as: 79 GC-AG pairs (of which one was an error that corrected to GC-AG), 61 errors that were corrected to GT-AG canonical pairs, six AT-AC pairs (of which two were errors that corrected to AT-AC), one case was produced from non-existent intron, seven cases were found in HTG that were deposited to GenBank and finally there were only two cases left of supported non-canonical splice sites. If we assume that approximately the same situation is true for the whole set of annotated mammalian non-canonical splice sites, then the 99.24% of splice site pairs should be GT-AG, 0.69% GC-AG, 0.05% AT-AC and finally only 0.02% could consist of other types of non-canonical splice sites. We analyze several characteristics of EST-verified splice sites and build weight matrices for the major groups, which can be incorporated into gene prediction programs. We also present a set of EST-verified canonical splice sites larger by two orders of magnitude than the current one (22 199 entries versus ~600) and finally, a set of 290 EST-supported non-canonical splice sites. Both sets should be significant for future investigations of the splicing mechanism.
PMCID: PMC113136  PMID: 11058137
14.  Conserved RNA structures in the non-canonical Hac1/Xbp1 intron 
RNA Biology  2011;8(4):552-556.
The unconventional splicing of Hac1 by the ribonuclease Ire1 is a key event in the activation of the unfolded protein response (UPR) in Saccharomyces cerevisiae. This splicing is independent of the spliceosome and is mediated by a secondary structure at the intron-exon boundaries of the mRNA. Similar unconventional splicing was also described for the gene Xbp1 in human, mouse, Caenorhabditis elegans and Drosophila melanogaster, and for Hac1 in five other fungi. We used reported RNA structures to build a multiple sequence alignment and the Infernal package to search for homologous structures. We identified homologous non-canonical intron structures in 128 out of 156 searched eukaryotic genomes. Our results show that the sequence of the Hac1/Xbp1 intron is highly conserved only around the splice sites recognized by Ire1. The consensus structure of the Hac1/Xbp1 mRNA is well conserved in Fungi and Metazoa and resembles structures previously described. We show that a typical Hac1/Xbp1 intron is very short, only 20–26 bases, whereas yeast species have a long intron (>100 bases). We identified six species with unambiguous Hac1/Xbp1 homologs that have lost the non-canonical intron structure. We propose that these species use a different mechanism to regulate the UPR.
PMCID: PMC3225973  PMID: 21593604
unfolded protein response; splicing; RNA structure; intron; HAC1; XBP1
15.  Newly evolved introns in human retrogenes provide novel insights into their evolutionary roles 
Retrogenes generally do not contain introns. However, in some instances, retrogenes may recruit internal exonic sequences as introns, which is known as intronization. A retrogene that undergoes intronization is a good model with which to investigate the origin of introns. Nevertheless, previously, only two cases in vertebrates have been reported.
In this study, we systematically screened the human (Homo sapiens) genome for retrogenes that evolved introns and analyzed their patterns in structure, expression and origin. In total, we identified nine intron-containing retrogenes. Alignment of pairs of retrogenes and their parents indicated that, in addition to intronization (five cases), retrogenes also may have gained introns by insertion of external sequences into the genes (one case) or reversal of the orientation of transcription (three cases). Interestingly, many intronizations were promoted not by base substitutions but by cryptic splice sites, which were silent in the parental genes but active in the retrogenes. We also observed that the majority of introns generated by intronization did not involve frameshifts.
Intron gains in retrogenes are not as rare as previously thought. Furthermore, diverse mechanisms may lead to intron creation in retrogenes. The activation of cryptic splice sites in the intronization of retrogenes may be triggered by the change of gene structure after retroposition. A high percentage of non-frameshift introns in retrogenes may be because non-frameshift introns do not dramatically affect host proteins. Introns generated by intronization in human retrogenes are generally young, which is consistent with previous findings for Caenorhabditis elegans. Our results provide novel insights into the evolutionary role of introns.
PMCID: PMC3565874  PMID: 22839428
16.  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.
PMCID: PMC2077895  PMID: 18020709
17.  AU-rich intronic elements affect pre-mRNA 5' splice site selection in Drosophila melanogaster. 
Molecular and Cellular Biology  1993;13(12):7689-7697.
cis-spliced nuclear pre-mRNA introns found in a variety of organisms, including Tetrahymena thermophila, Drosophila melanogaster, Caenorhabditis elegans, and plants, are significantly richer in adenosine and uridine residues than their flanking exons are. The functional significance of this intronic AU richness, however, has been demonstrated only in plant nuclei. In these nuclei, 5' and 3' splice sites are selected in part by their positions relative to AU-rich elements spread throughout the length of an intron. Because of this position-dependent selection scheme, a 5' splice site at the normal (+1) exon-intron boundary having only three contiguous consensus nucleotides can compete effectively with an enhanced exonic site (-57E) having nine consensus nucleotides and outcompete an enhanced site (+106E) embedded within the AU-rich intron. To determine whether transitions from AU-poor exonic sequences to AU-rich intronic sequences influence 5' splice site selection in other organisms, alleles of the pea rbcS3A1 intron were expressed in Drosophila Schneider 2 cells, and their splicing patterns were compared with those in tobacco nuclei. We demonstrate that this heterologous transcript can be accurately spliced in transfected Drosophila nuclei and that a +1 G-to-A knockout mutation at the normal splice site activates the same three cryptic 5' splice sites as in tobacco. Enhancement of the exonic (-57) and intronic (+106) sites to consensus splice sites indicates that potential splice sites located in the upstream exon or at the 5' exon-intron boundary are preferred in Drosophila cells over those embedded within AU-rich intronic sequences. In contrast to tobacco, in which the activities of two competing 5' splice sites upstream of the AU-rich intron are modulated by their proximity to the AU transition point, D. melanogaster utilizes the upstream site which has a higher proportion of consensus nucleotides. The enhanced version of the cryptic intronic site is efficiently selected in D. melanogaster when the normal +1 site is weakened or discrete AU-rich elements upstream of the +106E site are disrupted. Selection of this internal site in tobacco requires more drastic disruption of these motifs. We conclude that 5' splice site selection in Drosophila nuclei is influenced by the intrinsic strengths of competing sites and by the presence of AU-rich intronic elements but to a different extent than in tobacco.
PMCID: PMC364840  PMID: 8246985
18.  SpliceDB: database of canonical and non-canonical mammalian splice sites 
Nucleic Acids Research  2001;29(1):255-259.
A database (SpliceDB) of known mammalian splice site sequences has been developed. We extracted 43 337 splice pairs from mammalian divisions of the gene-centered Infogene database, including sites from incomplete or alternatively spliced genes. Known EST sequences supported 22 815 of them. After discarding sequences with putative errors and ambiguous location of splice junctions the verified dataset includes 22 489 entries. Of these, 98.71% contain canonical GT–AG junctions (22 199 entries) and 0.56% have non-canonical GC–AG splice site pairs. The remainder (0.73%) occurs in a lot of small groups (with a maximum size of 0.05%). We especially studied non-canonical splice sites, which comprise 3.73% of GenBank annotated splice pairs. EST alignments allowed us to verify only the exonic part of splice sites. To check the conservative dinucleotides we compared sequences of human non-canonical splice sites with sequences from the high throughput genome sequencing project (HTG). Out of 171 human non-canonical and EST-supported splice pairs, 156 (91.23%) had a clear match in the human HTG. They can be classified after sequence analysis as: 79 GC–AG pairs (of which one was an error that corrected to GC–AG), 61 errors corrected to GT–AG canonical pairs, six AT–AC pairs (of which two were errors corrected to AT–AC), one case was produced from a non-existent intron, seven cases were found in HTG that were deposited to GenBank and finally there were only two other cases left of supported non-canonical splice pairs. The information about verified splice site sequences for canonical and non-canonical sites is presented in SpliceDB with the supporting evidence. We also built weight matrices for the major splice groups, which can be incorporated into gene prediction programs. SpliceDB is available at the computational genomic Web server of the Sanger Centre: and at
PMCID: PMC29840  PMID: 11125105
19.  Identification and characterization of NAGNAG alternative splicing in the moss Physcomitrella patens 
BMC Plant Biology  2010;10:76.
Alternative splicing (AS) involving tandem acceptors that are separated by three nucleotides (NAGNAG) is an evolutionarily widespread class of AS, which is well studied in Homo sapiens (human) and Mus musculus (mouse). It has also been shown to be common in the model seed plants Arabidopsis thaliana and Oryza sativa (rice). In one of the first studies involving sequence-based prediction of AS in plants, we performed a genome-wide identification and characterization of NAGNAG AS in the model plant Physcomitrella patens, a moss.
Using Sanger data, we found 295 alternatively used NAGNAG acceptors in P. patens. Using 31 features and training and test datasets of constitutive and alternative NAGNAGs, we trained a classifier to predict the splicing outcome at NAGNAG tandem splice sites (alternative splicing, constitutive at the first acceptor, or constitutive at the second acceptor). Our classifier achieved a balanced specificity and sensitivity of ≥ 89%. Subsequently, a classifier trained exclusively on data well supported by transcript evidence was used to make genome-wide predictions of NAGNAG splicing outcomes. By generation of more transcript evidence from a next-generation sequencing platform (Roche 454), we found additional evidence for NAGNAG AS, with altogether 664 alternative NAGNAGs being detected in P. patens using all currently available transcript evidence. The 454 data also enabled us to validate the predictions of the classifier, with 64% (80/125) of the well-supported cases of AS being predicted correctly.
NAGNAG AS is just as common in the moss P. patens as it is in the seed plants A. thaliana and O. sativa (but not conserved on the level of orthologous introns), and can be predicted with high accuracy. The most informative features are the nucleotides in the NAGNAG and in its immediate vicinity, along with the splice sites scores, as found earlier for NAGNAG AS in animals. Our results suggest that the mechanism behind NAGNAG AS in plants is similar to that in animals and is largely dependent on the splice site and its immediate neighborhood.
PMCID: PMC3095350  PMID: 20426810
20.  Large introns in relation to alternative splicing and gene evolution: a case study of Drosophila bruno-3 
BMC Genetics  2009;10:67.
Alternative splicing (AS) of maturing mRNA can generate structurally and functionally distinct transcripts from the same gene. Recent bioinformatic analyses of available genome databases inferred a positive correlation between intron length and AS. To study the interplay between intron length and AS empirically and in more detail, we analyzed the diversity of alternatively spliced transcripts (ASTs) in the Drosophila RNA-binding Bruno-3 (Bru-3) gene. This gene was known to encode thirteen exons separated by introns of diverse sizes, ranging from 71 to 41,973 nucleotides in D. melanogaster. Although Bru-3's structure is expected to be conducive to AS, only two ASTs of this gene were previously described.
Cloning of RT-PCR products of the entire ORF from four species representing three diverged Drosophila lineages provided an evolutionary perspective, high sensitivity, and long-range contiguity of splice choices currently unattainable by high-throughput methods. Consequently, we identified three new exons, a new exon fragment and thirty-three previously unknown ASTs of Bru-3. All exon-skipping events in the gene were mapped to the exons surrounded by introns of at least 800 nucleotides, whereas exons split by introns of less than 250 nucleotides were always spliced contiguously in mRNA. Cases of exon loss and creation during Bru-3 evolution in Drosophila were also localized within large introns. Notably, we identified a true de novo exon gain: exon 8 was created along the lineage of the obscura group from intronic sequence between cryptic splice sites conserved among all Drosophila species surveyed. Exon 8 was included in mature mRNA by the species representing all the major branches of the obscura group. To our knowledge, the origin of exon 8 is the first documented case of exonization of intronic sequence outside vertebrates.
We found that large introns can promote AS via exon-skipping and exon turnover during evolution likely due to frequent errors in their removal from maturing mRNA. Large introns could be a reservoir of genetic diversity, because they have a greater number of mutable sites than short introns. Taken together, gene structure can constrain and/or promote gene evolution.
PMCID: PMC2767349  PMID: 19840385
21.  Kassiopeia: a database and web application for the analysis of mutually exclusive exomes of eukaryotes 
BMC Genomics  2014;15:115.
Alternative splicing is an important process in higher eukaryotes that allows obtaining several transcripts from one gene. A specific case of alternative splicing is mutually exclusive splicing, in which exactly one exon out of a cluster of neighbouring exons is spliced into the mature transcript. Recently, a new algorithm for the prediction of these exons has been developed based on the preconditions that the exons of the cluster have similar lengths, sequence homology, and conserved splice sites, and that they are translated in the same reading frame.
In this contribution we introduce Kassiopeia, a database and web application for the generation, storage, and presentation of genome-wide analyses of mutually exclusive exomes. Currently, Kassiopeia provides access to the mutually exclusive exomes of twelve Drosophila species, the thale cress Arabidopsis thaliana, the flatworm Caenorhabditis elegans, and human. Mutually exclusive spliced exons (MXEs) were predicted based on gene reconstructions from Scipio. Based on the standard prediction values, with which 83.5% of the annotated MXEs of Drosophila melanogaster were reconstructed, the exomes contain surprisingly more MXEs than previously supposed and identified. The user can search Kassiopeia using BLAST or browse the genes of each species optionally adjusting the parameters used for the prediction to reveal more divergent or only very similar exon candidates.
We developed a pipeline to predict MXEs in the genomes of several model organisms and a web interface, Kassiopeia, for their visualization. For each gene Kassiopeia provides a comprehensive gene structure scheme, the sequences and predicted secondary structures of the MXEs, and, if available, further evidence for MXE candidates from cDNA/EST data, predictions of MXEs in homologous genes of closely related species, and RNA secondary structure predictions. Kassiopeia can be accessed at
PMCID: PMC3923563  PMID: 24507667
Mutually exclusive splicing; Database; Web application; Drosophila
22.  Accurate splice site prediction using support vector machines 
BMC Bioinformatics  2007;8(Suppl 10):S7.
For splice site recognition, one has to solve two classification problems: discriminating true from decoy splice sites for both acceptor and donor sites. Gene finding systems typically rely on Markov Chains to solve these tasks.
In this work we consider Support Vector Machines for splice site recognition. We employ the so-called weighted degree kernel which turns out well suited for this task, as we will illustrate in several experiments where we compare its prediction accuracy with that of recently proposed systems. We apply our method to the genome-wide recognition of splice sites in Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, Danio rerio, and Homo sapiens. Our performance estimates indicate that splice sites can be recognized very accurately in these genomes and that our method outperforms many other methods including Markov Chains, GeneSplicer and SpliceMachine. We provide genome-wide predictions of splice sites and a stand-alone prediction tool ready to be used for incorporation in a gene finder.
Data, splits, additional information on the model selection, the whole genome predictions, as well as the stand-alone prediction tool are available for download at .
PMCID: PMC2230508  PMID: 18269701
23.  The multiplicity of alternative splicing decisions in Caenorhabditis elegans is linked to specific intronic regulatory motifs and minisatellites 
BMC Genomics  2014;15(1):364.
Alternative splicing diversifies the pool of messenger RNA molecules encoded by individual genes. This diversity is particularly high when multiple splicing decisions cause a combinatorial arrangement of several alternate exons. We know very little on how the multiple decisions occurring during the maturation of single transcripts are coordinated and whether specific sequence elements might be involved.
Here, the Caenorhabditis elegans genome was surveyed in order to identify sequence elements that might play a specific role in the regulation of multiple splicing decisions. The introns flanking alternate exons in transcripts whose maturation involves multiple alternative splicing decisions were compared to those whose maturation involves a single decision. Fifty-eight penta-, hexa-, and hepta-meric elements, clustered in 17 groups, were significantly over-represented in genes subject to multiple alternative splicing decisions. Most of these motifs relate to known splicing regulatory elements and appear to be well conserved in the related species Caenorhabditis briggsae. The usage of specific motifs is not linked to the gene product function, but rather depends on the gene structure, since it is influenced by the distance separating the multiple splicing decision sites. Two of these motifs are part of the CeRep25B minisatellite, which is also over-represented at the vicinity of alternative splicing regions. Most of the remaining motifs are not part of repeated sequence elements, but tend to occur in specific heterologous pairs in genes subject to multiple alternative splicing decisions.
The existence of specific intronic sequence elements linked to multiple alternative splicing decisions is intriguing and suggests that these elements might have some specialized regulatory role during splicing.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-364) contains supplementary material, which is available to authorized users.
PMCID: PMC4039745  PMID: 24884695
Alternate splice sites; Coordination of multiple choices; Regulatory elements; Worm; IMMAD; MASS; SASS
24.  The Caenorhabditis elegans Gene mfap-1 Encodes a Nuclear Protein That Affects Alternative Splicing 
PLoS Genetics  2012;8(7):e1002827.
RNA splicing is a major regulatory mechanism for controlling eukaryotic gene expression. By generating various splice isoforms from a single pre–mRNA, alternative splicing plays a key role in promoting the evolving complexity of metazoans. Numerous splicing factors have been identified. However, the in vivo functions of many splicing factors remain to be understood. In vivo studies are essential for understanding the molecular mechanisms of RNA splicing and the biology of numerous RNA splicing-related diseases. We previously isolated a Caenorhabditis elegans mutant defective in an essential gene from a genetic screen for suppressors of the rubberband Unc phenotype of unc-93(e1500) animals. This mutant contains missense mutations in two adjacent codons of the C. elegans microfibrillar-associated protein 1 gene mfap-1. mfap-1(n4564 n5214) suppresses the Unc phenotypes of different rubberband Unc mutants in a pattern similar to that of mutations in the splicing factor genes uaf-1 (the C. elegans U2AF large subunit gene) and sfa-1 (the C. elegans SF1/BBP gene). We used the endogenous gene tos-1 as a reporter for splicing and detected increased intron 1 retention and exon 3 skipping of tos-1 transcripts in mfap-1(n4564 n5214) animals. Using a yeast two-hybrid screen, we isolated splicing factors as potential MFAP-1 interactors. Our studies indicate that C. elegans mfap-1 encodes a splicing factor that can affect alternative splicing.
Author Summary
RNA splicing removes intervening intronic sequences from pre–mRNA transcripts and joins adjacent exonic sequences to generate functional messenger RNAs. The in vivo functions of numerous factors that regulate splicing remain to be understood. From a genetic screen for suppressors of the rubberband Unc phenotype caused by the Caenorhabditis elegans unc-93(e1500) mutation, we isolated a mutation that affects a highly conserved essential gene, mfap-1. MFAP-1 is a nuclear protein that is broadly expressed. MFAP-1 can affect the alternative splicing of tos-1, an endogenous reporter gene for splicing, and is required for the altered splicing at a cryptic 3′ splice site of tos-1. mfap-1 enhances the effects of the gene uaf-1 (splicing factor U2AF large subunit) in suppressing the rubberband Unc phenotype of unc-93(e1500) animals. Our studies provide in vivo evidence that MFAP-1 functions as a splicing factor.
PMCID: PMC3400559  PMID: 22829783
25.  Evolutionary dynamics of U12-type spliceosomal introns 
Many multicellular eukaryotes have two types of spliceosomes for the removal of introns from messenger RNA precursors. The major (U2) spliceosome processes the vast majority of introns, referred to as U2-type introns, while the minor (U12) spliceosome removes a small fraction (less than 0.5%) of introns, referred to as U12-type introns. U12-type introns have distinct sequence elements and usually occur together in genes with U2-type introns. A phylogenetic distribution of U12-type introns shows that the minor splicing pathway appeared very early in eukaryotic evolution and has been lost repeatedly.
We have investigated the evolution of U12-type introns among eighteen metazoan genomes by analyzing orthologous U12-type intron clusters. Examination of gain, loss, and type switching shows that intron type is remarkably conserved among vertebrates. Among 180 intron clusters, only eight show intron loss in any vertebrate species and only five show conversion between the U12 and the U2-type. Although there are only nineteen U12-type introns in Drosophila melanogaster, we found one case of U2 to U12-type conversion, apparently mediated by the activation of cryptic U12 splice sites early in the dipteran lineage. Overall, loss of U12-type introns is more common than conversion to U2-type and the U12 to U2 conversion occurs more frequently among introns of the GT-AG subtype than among introns of the AT-AC subtype. We also found support for natural U12-type introns with non-canonical terminal dinucleotides (CT-AC, GG-AG, and GA-AG) that have not been previously reported.
Although complete loss of the U12-type spliceosome has occurred repeatedly, U12 introns are extremely stable in some taxa, including eutheria. Loss of U12 introns or the genes containing them is more common than conversion to the U2-type. The degeneracy of U12-type terminal dinucleotides among natural U12-type introns is higher than previously thought.
PMCID: PMC2831892  PMID: 20163699

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