Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii

Abstract

Background: Genome-wide computational analysis of alternative splicing (AS) in several flowering plants has revealed that pre-mRNAs from about 30% of genes undergo AS. Chlamydomonas, a simple unicellular green alga, is part of the lineage that includes land plants. However, it diverged from land plants about one billion years ago. Hence, it serves as a good model system to study alternative splicing in early photosynthetic eukaryotes, to obtain insights into the evolution of this process in plants, and to compare splicing in simple unicellular photosynthetic and non-photosynthetic eukaryotes. We performed a global analysis of alternative splicing in Chlamydomonas reinhardtii using its recently completed genome sequence and all available ESTs and cDNAs.

Results: Our analysis of AS using BLAT and a modified version of the Sircah tool revealed AS of 498 transcriptional units with 611 events, representing about 3% of the total number of genes. As in land plants, intron retention is the most prevalent form of AS. Retained introns and skipped exons tend to be shorter than their counterparts in constitutively spliced genes. The splice site signals in all types of AS events are weaker than those in constitutively spliced genes. Furthermore, in alternatively spliced genes, the prevalent splice form has a stronger splice site signal than the non-prevalent form. Analysis of constitutively spliced introns revealed an over-abundance of motifs with simple repetitive elements in comparison to introns involved in intron retention. In almost all cases, AS results in a truncated ORF, leading to a coding sequence that is around 50% shorter than the prevalent splice form. Using RT-PCR we verified AS of two genes and show that they produce more isoforms than indicated by EST data. All cDNA/EST alignments and splice graphs are provided in a website at http://combi.cs.colostate.edu/as/chlamy.

Conclusions: The extent of AS in Chlamydomonas that we observed is much smaller than observed in land plants, but is much higher than in simple unicellular heterotrophic eukaryotes. The percentage of different alternative splicing events is similar to flowering plants. Prevalence of constitutive and alternative splicing in Chlamydomonas, together with its simplicity, many available public resources, and well developed genetic and molecular tools for this organism make it an excellent model system to elucidate the mechanisms involved in regulated splicing in photosynthetic eukaryotes.

Figure 1

Example splice graph. Shown are two splice graphs along with the relevant EST evidence for the gene fgenesh2_pg.C_scaffold_39000087, which exhibits intron retention and Alt3' (top), and for the gene estExt_fgenesh2_kg.C_380020, which exhibits intron retention, exon skipping, and Alt3' (bottom). Figures were generated by Sircah as part of our pipeline.

Figure 2

Comparison of splice site motifs for prevalent and non-prevalent splice forms. WebLogo [58] images of 5' and 3' splice site motifs for the prevalent and non-prevalent Alt5' and Alt3' splice forms. In the case of Alt5' there is a difference between the prevalent and non-prevalent forms only in the 5' splice site, and similarly for Alt3', there is a difference only in the 3' splice site.

Figure 3

Analysis of ornithine decarboxylase 1 (ODC1) and asparagine synthase (ASyn) splice variants in Chlamydomonas using RT-PCR. A) DNAase-treated RNA was used in PCR with ODC1 primers. B) cDNA amplified with ODC1 primers. An asterisk indicates the spliced form for the full-length protein. Numbers on the right indicate amplified product size in Kb. C) cDNA amplified with ASyn primers. Numbers on the right indicate amplified product size in Kb. D) Diagram showing splicing events in six splice variants (left) and predicted proteins (right) for ODC1. Gene is indicated on top and all six splice variants are shown under the gene. Black boxes indicate constitutively spliced exons and red boxes indicate the included regions in different isoforms. Asterisk indicates the position of translation termination codon. Isoforms 1 to 6 correspond to the bottom to top bands in 3B. The number next to each predicted protein indicates the length of the protein. Conserved signature motifs in ODC1 are represented as green boxes in the full-length protein. Red, blue and magenta colors in truncated proteins represent amino acids unique to them. E) Diagram showing splicing events in four splice variants (left) and predicted proteins (right) for the gene ASyn. The representation of the gene and its splice variants is the same as in D. A conserved domain in ASyn is shown in pink. The green region in isoform 4 represents a unique sequence.