Overlapping splicing regulatory motifs--combinatorial effects on splicing.

Regulation of splicing in eukaryotes occurs through the coordinated action of multiple splicing factors. Exons and introns contain numerous putative binding sites for splicing regulatory proteins. Regulation of splicing is presumably achieved by the combinatorial output of the binding of splicing factors to the corresponding binding sites. Although putative regulatory sites often overlap, no extensive study has examined whether overlapping regulatory sequences provide yet another dimension to splicing regulation. Here we analyzed experimentally-identified splicing regulatory sequences using a computational method based on the natural distribution of nucleotides and splicing regulatory sequences. We uncovered positive and negative interplay between overlapping regulatory sequences. Examination of these overlapping motifs revealed a unique spatial distribution, especially near splice donor sites of exons with weak splice donor sites. The positively selected overlapping splicing regulatory motifs were highly conserved among different species, implying functionality. Overall, these results suggest that overlap of two splicing regulatory binding sites is an evolutionary conserved widespread mechanism of splicing regulation. Finally, over-abundant motif overlaps were experimentally tested in a reporting minigene revealing that overlaps may facilitate a mode of splicing that did not occur in the presence of only one of the two regulatory sequences that comprise it.

Insights into the connection between cancer and alternative splicing.

Computational and experimental evidence has revealed that cancerous cells express transcript variants that are abnormally spliced, suggesting that mRNAs are more frequently alternatively spliced in cancerous tissues than in normal ones. We show that cancerous tissues exhibit lower levels of alternative splicing than do normal tissues. Moreover, we found that the distribution of types of alternative splicing differs between cancerous and normal tissues. We further show evidence suggesting that the lower levels of alternative splicing in cancerous tissues might be a result of disruption of splicing regulatory proteins.

Intronic Alus influence alternative splicing.

Examination of the human transcriptome reveals higher levels of RNA editing than in any other organism tested to date. This is indicative of extensive double-stranded RNA (dsRNA) formation within the human transcriptome. Most of the editing sites are located in the primate-specific retrotransposed element called Alu. A large fraction of Alus are found in intronic sequences, implying extensive Alu-Alu dsRNA formation in mRNA precursors. Yet, the effect of these intronic Alus on splicing of the flanking exons is largely unknown. Here, we show that more Alus flank alternatively spliced exons than constitutively spliced ones; this is especially notable for those exons that have changed their mode of splicing from constitutive to alternative during human evolution. This implies that Alu insertions may change the mode of splicing of the flanking exons. Indeed, we demonstrate experimentally that two Alu elements that were inserted into an intron in opposite orientation undergo base-pairing, as evident by RNA editing, and affect the splicing patterns of a downstream exon, shifting it from constitutive to alternative. Our results indicate the importance of intronic Alus in influencing the splicing of flanking exons, further emphasizing the role of Alus in shaping of the human transcriptome.

Alu exonization events reveal features required for precise recognition of exons by the splicing machinery.

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.

The "alternative" choice of constitutive exons throughout evolution.

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.

Different levels of alternative splicing among eukaryotes.

Alternative splicing increases transcriptome and proteome diversification. Previous analyses aiming at comparing the rate of alternative splicing between different organisms provided contradicting results. These contradicting results were attributed to the fact that both analyses were dependent on the expressed sequence tag (EST) coverage, which varies greatly between the tested organisms. In this study we compare the level of alternative splicing among eight different organisms. By employing an EST independent approach we reveal that the percentage of genes and exons undergoing alternative splicing is higher in vertebrates compared with invertebrates. We also find that alternative exons of the skipping type are flanked by longer introns compared to constitutive ones, whereas alternative 5' and 3' splice sites events are generally not. In addition, although the regulation of alternative splicing and sizes of introns and exons have changed during metazoan evolution, intron retention remained the rarest type of alternative splicing, whereas exon skipping is more prevalent and exhibits a slight increase, from invertebrates to vertebrates. The difference in the level of alternative splicing suggests that alternative splicing may contribute greatly to the mammal higher level of phenotypic complexity, and that accumulation of introns confers an evolutionary advantage as it allows increasing the number of alternative splicing forms.

Alternative splicing and disease.

Splicing is a molecular mechanism, by which introns are removed from an mRNA precursor and exons are ligated to form a mature mRNA. Mutations that cause defects in the splicing mechanism are known to be responsible for many diseases, including cystic fibrosis and familial dysautonomia. If mutations that cause defects in splicing are responsible for such severe deleterious phenotypic differences, it is possible that mutations in splicing are also responsible for mildly deleterious phenotypic differences. Although deleterious mutations are rapidly eliminated from the population by purifying selection, the selection against mild deleterious effects is not as strong. Since mildly deleterious mutations have a chance of surviving natural selection, we might be mistakenly referring to these mutations as neutral variation between individuals. Splicing has also been shown to be seriously affected in cancer. Examination of cancerous tissues revealed alterations in expression levels of genes involved in mRNA processing and also a slight reduction in the level of exon skipping--the most common form of alternative splicing in humans. This implies that defects in genes involved in the regulation of splicing in cancerous tissues affect the delicate regulation of the inclusion level of alternatively skipped exons, shifting their mode of splicing back to constitutive. It may be that splicing silencers play a more prominent role in alternative splicing regulation than previously anticipated.

Differential GC content between exons and introns establishes distinct strategies of splice-site recognition.

During evolution segments of homeothermic genomes underwent a GC content increase. Our analyses reveal that two exon-intron architectures have evolved from an ancestral state of low GC content exons flanked by short introns with a lower GC content. One group underwent a GC content elevation that abolished the differential exon-intron GC content, with introns remaining short. The other group retained the overall low GC content as well as the differential exon-intron GC content, and is associated with longer introns. We show that differential exon-intron GC content regulates exon inclusion level in this group, in which disease-associated mutations often lead to exon skipping. This group's exons also display higher nucleosome occupancy compared to flanking introns and exons of the other group, thus "marking" them for spliceosomal recognition. Collectively, our results reveal that differential exon-intron GC content is a previously unidentified determinant of exon selection and argue that the two GC content architectures reflect the two mechanisms by which splicing signals are recognized: exon definition and intron definition.

The role of transposable elements in the evolution of non-mammalian vertebrates and invertebrates.

BACKGROUND: Transposable elements (TEs) have played an important role in the diversification and enrichment of mammalian transcriptomes through various mechanisms such as exonization and intronization (the birth of new exons/introns from previously intronic/exonic sequences, respectively), and insertion into first and last exons. However, no extensive analysis has compared the effects of TEs on the transcriptomes of mammals, non-mammalian vertebrates and invertebrates. RESULTS: We analyzed the influence of TEs on the transcriptomes of five species, three invertebrates and two non-mammalian vertebrates. Compared to previously analyzed mammals, there were lower levels of TE introduction into introns, significantly lower numbers of exonizations originating from TEs and a lower percentage of TE insertion within the first and last exons. Although the transcriptomes of vertebrates exhibit significant levels of exonization of TEs, only anecdotal cases were found in invertebrates. In vertebrates, as in mammals, the exonized TEs are mostly alternatively spliced, indicating that selective pressure maintains the original mRNA product generated from such genes. CONCLUSIONS: Exonization of TEs is widespread in mammals, less so in non-mammalian vertebrates, and very low in invertebrates. We assume that the exonization process depends on the length of introns. Vertebrates, unlike invertebrates, are characterized by long introns and short internal exons. Our results suggest that there is a direct link between the length of introns and exonization of TEs and that this process became more prevalent following the appearance of mammals.

Alternative splicing: current perspectives.

Alternative splicing is a well-characterized mechanism by which multiple transcripts are generated from a single mRNA precursor. By allowing production of several protein isoforms from one pre-mRNA, alternative splicing contributes to proteomic diversity. But what do we know about the origin of this mechanism? Do the same evolutionary forces apply to alternatively and constitutively splice exons? Do similar forces act on all types of alternative splicing? Are the products generated by alternative splicing functional? Why is "improper" recognition of exons and introns allowed by the splicing machinery? In this review, we summarize the current knowledge regarding these issues from an evolutionary perspective.

Alternative approach to a heavy weight problem.

Obesity is reaching epidemic proportions in developed countries and represents a significant risk factor for hypertension, heart disease, diabetes, and dyslipidemia. Splicing mutations constitute at least 14% of disease-causing mutations, thus implicating polymorphisms that affect splicing as likely candidates for disease susceptibility. A recent study suggested that genes associated with obesity were significantly enriched for rare nucleotide variants. Here, we examined these variants and revealed that they are located near splice junctions and tend to affect exonic splicing regulatory sequences. We also show that the majority of the exons that harbor these SNPs are constitutively spliced, yet they exhibit weak splice sites, typical to alternatively spliced exons, and are hence suboptimal for recognition by the splicing machinery and prone to become alternatively spliced. Using ex vivo assays, we tested a few representative variants and show that they indeed affect splicing by causing a shift from a constitutive to an alternative pattern, suggesting a possible link between extreme body mass index and abnormal splicing patterns.

Towards alignment independent quantitative assessment of homology detection.

Identification of homologous proteins provides a basis for protein annotation. Sequence alignment tools reliably identify homologs sharing high sequence similarity. However, identification of homologs that share low sequence similarity remains a challenge. Lowering the cutoff value could enable the identification of diverged homologs, but also introduces numerous false hits. Methods are being continuously developed to minimize this problem. Estimation of the fraction of homologs in a set of protein alignments can help in the assessment and development of such methods, and provides the users with intuitive quantitative assessment of protein alignment results. Herein, we present a computational approach that estimates the amount of homologs in a set of protein pairs. The method requires a prevalent and detectable protein feature that is conserved between homologs. By analyzing the feature prevalence in a set of pairwise protein alignments, the method can estimate the number of homolog pairs in the set independently of the alignments' quality. Using the HomoloGene database as a standard of truth, we implemented this approach in a proteome-wide analysis. The results revealed that this approach, which is independent of the alignments themselves, works well for estimating the number of homologous proteins in a wide range of homology values. In summary, the presented method can accompany homology searches and method development, provides validation to search results, and allows tuning of tools and methods.

Discovering hidden viral piracy.

MOTIVATION: Viruses and developers of anti-inflammatory therapies share a common interest in proteins that manipulate the immune response. Large double-stranded DNA viruses acquire host proteins to evade host defense mechanisms. Hence, viral pirated proteins may have a therapeutic potential. Although dozens of viral piracy events have already been identified, we hypothesized that sequence divergence impedes the discovery of many others. RESULTS: We developed a method to assess the number of viral/human homologs and discovered that at least 917 highly diverged homologs are hidden in low-similarity alignment hits that are usually ignored. However, these low-similarity homologs are masked by many false alignment hits. We therefore applied a filtering method to increase the proportion of viral/human homologous proteins. The homologous proteins we found may facilitate functional annotation of viral and human proteins. Furthermore, some of these proteins play a key role in immune modulation and are therefore therapeutic protein candidates.