Abstract
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.
Highlights
One of the interesting lessons learned from the analysis of the human genome is that we may possess fewer than 25,000 genes [1]
In order to identify alternatively spliced cassette exons in the C. elegans genome, we used the Intronerator [24] to generate an initial set of 1,471 putative alternatively spliced genes. We did this by aligning over 200,000 expressed sequence tag (EST) and mRNAs to the C. elegans genome and identifying regions where the alignments are consistent with more than one way to process a gene
We saw evidence of ESTs that showed internal deletions at short direct repeats. These indicated the potential for intron removal, but upon further inspection, these did not meet the criteria of introns and were likely the result of cloning artifacts of the ESTs
Summary
One of the interesting lessons learned from the analysis of the human genome is that we may possess fewer than 25,000 genes [1]. One mechanism to dramatically increase the complexity of the human proteome from this lower-thanexpected number of genes is to allow some genes to encode multiple proteins. This process can be accomplished by alternative precursor messenger RNA (pre-mRNA) splicing. Alternative splicing events can be regulated in tissue-specific, developmental, and hormone-responsive manners, providing additional mechanisms for the regulation of gene expression [6,7]. Understanding alternative splicing and its regulation is a key component to understanding metazoan genomes
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