Intronic sequences flanking alternatively spliced exons are conserved between human and mouse

Comparison of the sequences of mouse and human genomes revealed a surprising number of nonexonic, nonexpressed conserved sequences, for which no function could be assigned. To study the possible correlation between these conserved intronic sequences and alternative splicing regulation, we developed a method to identify exons that are alternatively spliced in both human and mouse. We compiled two exon sets: one of alternatively spliced conserved exons and another of constitutively spliced conserved exons. We found that 77% of the conserved alternatively spliced exons were flanked on both sides by long conserved intronic sequences. In comparison, only 17% of the conserved constitutively spliced exons were flanked by such conserved intronic sequences. The average length of the conserved intronic sequences was 103 bases in the upstream intron and 94 bases in the downstream intron. The average identity levels in the immediately flanking intronic sequences were 88% and 80% for the upstream and downstream introns, respectively, higher than the conservation levels of 77% that were measured in promoter regions. Our results suggest that the function of many of the intronic sequence blocks that are conserved between human and mouse is the regulation of alternative splicing.     

Evolution of exon-intron structure and alternative splicing in fruit flies and malarial mosquito genomes

Comparative analysis of alternative splicing of orthologous genes from fruit flies (Drosophila melanogaster and Drosophila pseudoobscura) and mosquito (Anopheles gambiae) demonstrated that both in the fruit fly genes and in fruit fly-mosquito comparisons, constitutive exons and splicing sites are more conserved than alternative ones. While >97% of constitutive D. melanogaster exons are conserved in D. pseudoobscura, only approximately 80% of alternative exons are conserved. Similarly, 77% of constitutive fruit fly exons are conserved in the mosquito genes, compared with <50% of alternative exons. Internal alternatives are more conserved than terminal ones. Retained introns are the least conserved, alternative acceptor sites are slightly more conserved than donor sites, and mutually exclusive exons are almost as conserved as constitutive exons. Cassette and mutually exclusive exons experience almost no intron insertions. We also observed cases of interconversion of various elementary alternatives, e.g., transformation of cassette exons into alternative sites. These results agree with the observations made earlier in human-mouse comparisons and demonstrate that the phenomenon of relatively low conservation of alternatively spliced regions may be universal, as it has been observed in different taxonomic groups (mammals and insects) and at various evolutionary distances.     

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.     

STAR family RNA-binding protein ASD-2 regulates developmental switching of mutually exclusive alternative splicing in vivo

Alternative splicing of pre-mRNAs greatly contributes to the spatiotemporal diversity of gene expression in metazoans. However, the molecular basis of developmental regulation and the precise sequence of alternative pre-mRNA processing in vivo are poorly understood. In the present study, we focus on the developmental switching of the mutually exclusive alternative splicing of the let-2 gene of Caenorhabditis elegans from the exon 9 form in embryos to the exon 10 form in adults. By visualizing the usage of the let-2 mutually exclusive exons through differential expression of green fluorescent protein (GFP) and red fluorescent protein (RFP), we isolated several switching-defective mutants and identified the alternative splicing defective-2 (asd-2) gene, encoding a novel member of the evolutionarily conserved STAR (signal transduction activators of RNA) family of RNA-binding proteins. Comparison of the amounts of partially spliced let-2 RNAs in synchronized wild-type and asd-2 mutant worms suggested that either of the introns downstream from the let-2 mutually exclusive exons is removed prior to the removal of the upstream ones, and that asd-2 promotes biased excision of intron 10 in the late larval stages. We propose that the developmental switching between alternative sequences of intron removal determines the ratio between the mature let-2 mRNA isoforms.     

In NF1, CFTR, PER3, CARS and SYT7, alternatively included exons show higher conservation of surrounding intron sequences than constitutive exons

It is still not fully understood to what extent intronic sequences contribute to the regulation of the different forms of alternative splicing. We are interested in the regulation of alternative cassette exon events, such as exon inclusion and exon skipping. We investigated these events by comparative genomic analysis of human and mouse in five experimentally well-characterized genes, neurofibromatosis 1 (NF1), cystic fibrosis transmembrane conductance regulator (CFTR), period 3 (PER3), cysteinyl-tRNA synthetase (CARS) and synaptotagmin 7 (SYT7). In NF1, high intron identity around the 52 constitutive and four alternatively skipped NF1 exons is restricted to the close vicinity of the exons. In contrast, we found on average high conservation of intron sequences over 300 base pairs up- and downstream of the five alternatively included NF1 exons. The investigation of alternatively included exons in CFTR, PER3, CARS and SYT7 supported this finding. In contrast, the mean intron identities around the alternatively skipped exons in CTFR and NF1 do not differ considerably from those around the constitutive exons. In these genes, the difference in intron conservation could point to a difference between the regulation of alternative exon inclusion and alternative exon skipping or constitutive exon splicing. Additional genome-wide investigations are necessary to elucidate to what extent our finding can be generalized.