Global regulation of alternative splicing during myogenic differentiation

Recent genome-wide analyses have elucidated the extent of alternative splicing (AS) in mammals, often focusing on comparisons of splice isoforms between differentiated tissues. However, regulated splicing changes are likely to be important in biological transitions such as cellular differentiation, or response to environmental stimuli. To assess the extent and significance of AS in myogenesis, we used splicing-sensitive microarray analysis of differentiating C2C12 myoblasts. We identified 95 AS events that undergo robust splicing transitions during C2C12 differentiation. More than half of the splicing transitions are conserved during differentiation of avian myoblasts, suggesting the products and timing of transitions are functionally significant. The majority of splicing transitions during C2C12 differentiation fall into four temporal patterns and were dependent on the myogenic program, suggesting that they are integral components of myogenic differentiation. Computational analyses revealed enrichment of many sequence motifs within the upstream and downstream intronic regions near the alternatively spliced regions corresponding to binding sites of splicing regulators. Western analyses demonstrated that several splicing regulators undergo dynamic changes in nuclear abundance during differentiation. These findings show that within a developmental context, AS is a highly regulated and conserved process, suggesting a major role for AS regulation in myogenic differentiation.     

Recognition of unknown conserved alternatively spliced exons

The split structure of most mammalian protein-coding genes allows for the potential to produce multiple different mRNA and protein isoforms from a single gene locus through the process of alternative splicing (AS). We propose a computational approach called UNCOVER based on a pair hidden Markov model to discover conserved coding exonic sequences subject to AS that have so far gone undetected. Applying UNCOVER to orthologous introns of known human and mouse genes predicts skipped exons or retained introns present in both species, while discriminating them from conserved noncoding sequences. The accuracy of the model is evaluated on a curated set of genes with known conserved AS events. The prediction of skipped exons in the approximately 1% of the human genome represented by the ENCODE regions leads to more than 50 new exon candidates. Five novel predicted AS exons were validated by RT-PCR and sequencing analysis of 15 introns with strong UNCOVER predictions and lacking EST evidence. These results imply that a considerable number of conserved exonic sequences and associated isoforms are still completely missing from the current annotation of known genes. UNCOVER also identifies a small number of candidates for conserved intron retention.     

Alternative splicing regulation at tandem 3' splice sites

Alternative splicing (AS) constitutes a major mechanism creating protein diversity in humans. Previous bioinformatics studies based on expressed sequence tag and mRNA data have identified many AS events that are conserved between humans and mice. Of these events, ~25% are related to alternative choices of 3′ and 5′ splice sites. Surprisingly, half of all these events involve 3′ splice sites that are exactly 3 nt apart. These tandem 3′ splice sites result from the presence of the NAGNAG motif at the acceptor splice site, recently reported to be widely spread in the human genome. Although the NAGNAG motif is common in human genes, only a small subset of sites with this motif is confirmed to be involved in AS. We examined the NAGNAG motifs and observed specific features such as high sequence conservation of the motif, high conservation of ~30 bp at the intronic regions flanking the 3′ splice site and overabundance of cis-regulatory elements, which are characteristic of alternatively spliced tandem acceptor sites and can distinguish them from the constitutive sites in which the proximal NAG splice site is selected. Our findings imply that AS at tandem splice sites and constitutive splicing of the distal NAG are highly regulated.     

Structural implication of splicing stochastics

Even though nearly every human gene has at least one alternative splice form, very little is so far known about the structure and function of resulting protein products. It is becoming increasingly clear that a significant fraction of all isoforms are products of noisy selection of splice sites and thus contribute little to actual functional diversity, and may potentially be deleterious. In this study, we examine the impact of alternative splicing on protein sequence and structure in three datasets: alternative splicing events conserved across multiple species, alternative splicing events in genes that are strongly linked to disease and all observed alternative splicing events. We find that the vast majority of all alternative isoforms result in unstable protein conformations. In contrast to that, the small subset of isoforms conserved across species tends to maintain protein structural integrity to a greater extent. Alternative splicing in disease-associated genes produces unstable structures just as frequently as all other genes, indicating that selection to reduce the effects of alternative splicing on this set is not especially pronounced. Overall, the properties of alternative spliced proteins are consistent with the outcome of noisy selection of splice sites by splicing machinery.     

Alternative splicing and the steady-state ratios of mRNA isoforms generated by it are under strong stabilizing selection in Caenorhabditis elegans

Evolutionary studies indicate that a high proportion of alternative splicing (AS) events are species-specific; just 28% of minor-form alternatively spliced exons are conserved between mice and humans. We employed a splicing-sensitive microarray to study the evolution of allele-specific AS in nematodes. We compared splicing levels among five distinct Caenorhabditis elegans lines. Our results indicate that AS is less variable between natural isolates (NIs) from England, Hawaii, and Australia than when compared with mutation accumulation lines (6% vs. 21%, respectively, vary compared with N2). This suggests that strong stabilizing selection shapes the evolution of the ratios of isoforms generated by AS in C. elegans. When we analyzed some of the splicing changes between the NIs, we found examples of changes in both cis and trans that lead to alterations in gene-specific AS. This indicates that both these mechanisms for changing AS are employed along the path toward speciation in nematodes.