In silico analysis of the sequence features responsible for alternatively spliced introns in the model green alga Chlamydomonas reinhardtii.

Alternatively spliced introns are the ones that are usually spliced but can be occasionally retained in a transcript isoform. They are the most frequently used alternative splice form in plants (~50% of alternative splicing events). Chlamydomonas reinhardtii, a unicellular alga, is a good model to understand alternative splicing (AS) in plants from an evolutionary perspective as it diverged from land plants a billion years ago. Using over 7 million cDNA sequences from both pyrosequencing and Sanger sequencing, we found that a much higher percentage of genes (~20% of multi-exon genes) undergo AS than previously reported (3-5%). We found a full component of SR and SR-like proteins possibly involved in AS. The most prevalent type of AS event (40%) was retention of introns, most of which were supported by multiple cDNA evidence (72%) while only 20% of them have coding capacity. By comparing retained and constitutive introns, we identified sequence features potentially responsible for the retention of introns, in the framework of an "intron definition" model for splicing. We find that retained introns tend to have a weaker 5' splice site, more Gs in their poly-pyrimidine tract and a lesser conservation of nucleotide 'C' at position -3 of the 3' splice site. In addition, the sequence motifs found in the potential branch-point region differed between retained and constitutive introns. Furthermore, the enrichment of G-triplets and C-triplets among the first and last 50 nt of the introns significantly differ between constitutive and retained introns. These could serve as intronic splicing enhancers. All the alternative splice forms can be accessed at http://bioinfolab.miamioh.edu/cgi-bin/PASA_r20140417/cgi-bin/status_report.cgi?db=Chre_AS .