Genome-wide analysis of alternative splicing in Volvox carteri

Background: Alternative splicing is an essential mechanism for increasing transcriptome and proteome diversity in eukaryotes. Particularly in multicellular eukaryotes, this mechanism is involved in the regulation of developmental and physiological processes like growth, differentiation and signal transduction. Results: Here we report the genome-wide analysis of alternative splicing in the multicellular green alga Volvox carteri. The bioinformatic analysis of 132,038 expressed sequence tags (ESTs) identified 580 alternative splicing events in a total of 426 genes. The predominant type of alternative splicing in Volvox is intron retention (46.5%) followed by alternative 5' (17.9%) and 3' (21.9%) splice sites and exon skipping (9.5%). Our analysis shows that in Volvox at least similar to 2.9% of the intron-containing genes are subject to alternative splicing. Considering the total number of sequenced ESTs, the Volvox genome seems to provide more favorable conditions (e.g., regarding length and GC content of introns) for the occurrence of alternative splicing than the genome of its close unicellular relative Chlamydomonas. Moreover, many randomly chosen alternatively spliced genes of Volvox do not show alternative splicing in Chlamydomonas. Since the Volvox genome contains about the same number of protein-coding genes as the Chlamydomonas genome (similar to 14,500 protein-coding genes), we assumed that alternative splicing may play a key role in generation of genomic diversity, which is required to evolve from a simple one-cell ancestor to a multicellular organism with differentiated cell types (Mol Biol Evol 31: 1402-1413, 2014). To confirm the alternative splicing events identified by bioinformatic analysis, several genes with different types of alternatively splicing have been selected followed by experimental verification of the predicted splice variants by RT-PCR. Conclusions: The results show that our approach for prediction of alternative splicing events in Volvox was accurate and reliable. Moreover, quantitative real-time RT-PCR appears to be useful in Volvox for analyses of relationships between the appearance of specific alternative splicing variants and different kinds of physiological, metabolic and developmental processes as well as responses to environmental changes.