Genomic overview of mRNA 5'-leader trans-splicing in the ascidian Ciona intestinalis

Abstract

Although spliced leader (SL) trans-splicing in the chordates was discovered in the tunicate Ciona intestinalis there has been no genomic overview analysis of the extent of trans-splicing or the make-up of the trans-spliced and non-trans-spliced gene populations of this model organism. Here we report such an analysis for Ciona based on the oligo-capping full-length cDNA approach. We randomly sampled 2078 5'-full-length ESTs representing 668 genes, or 4.2% of the entire genome. Our results indicate that Ciona contains a single major SL, which is efficiently trans-spliced to mRNAs transcribed from a specific set of genes representing approximately 50% of the total number of expressed genes, and that individual trans-spliced mRNA species are, on average, 2-3-fold less abundant than non-trans-spliced mRNA species. Our results also identify a relationship between trans-splicing status and gene functional classification; ribosomal protein genes fall predominantly into the non-trans-spliced category. In addition, our data provide the first evidence for the occurrence of polycistronic transcription in Ciona. An interesting feature of the Ciona polycistronic transcription units is that the great majority entirely lack intercistronic sequences.

Figure 1

Distances between 5′ ends of genomically-mapped full-length ESTs and the nearest end of their 5′-neighbouring gene models, plotted in 100 base-windows. (A) SL-full-length ESTs whose 5′-neighbouring gene is transcribed in the same direction (group I), (B) SL-full-length ESTs whose 5′-neighbouring gene is transcribed in the opposite direction (group II), (C) non-SL-full-length ESTs whose 5′-neighbouring gene is transcribed in the same direction (group III) and (D) non-SL-full-length ESTs whose 5′-neighbouring gene is transcribed in the opposite direction (group IV). Note that only in (A) (group I) are gene pairs separated by <100 bases (first data point) well-represented. The number of gene pairs representing each distance interval is reported as a percentage of the total number of gene pairs in each group.

Figure 2

Dicistronic and moncistronic conventional ESTs, and SL-full-length ESTs, representing operon 41. The top part of the figure is a schematic depiction of the genomic DNA in terms of the intron/exon (lines/boxes) structures for two adjacent genes. The genes, coding for a protein similar to hypothetical proteins in other animals and a GTP-binding nuclear protein Ran, are immediately adjacent, so they are shown on separate lines for clarity. Protein-coding and non-coding regions are shown by grey and green. Below the genomic DNA depiction are diagrams showing conventional EST cDNA clones aligning with exons in this region. Each cDNA clone is represented by two EST sequencing runs, a 5′-EST (red) and a 3′ EST (blue), which are joined by dashed lines and which in some cases overlap. The first cDNA clone depicted, citb076c21, is a dicistronic transcript whose 5′-EST corresponds to the upstream gene and whose 3′ EST corresponds to the downstream gene (intron sequences were not present in the ESTs). An additional cDNA clone, cima003i16, was similar. Other cDNA clones depicted represent mature monocistronic mRNAs corresponding to either the upstream or downstream gene. The bottom depiction represents SL-full-length ESTs corresponding to the downstream gene, showing it to be trans-spliced. The genomic juxtaposition of these two cistrons is not due to an artifact of genome assembly, because eight raw whole-genome shotgun reads, of which three are depicted by black arrows at the bottom, can be aligned across the intercistron boundary.

Figure 3

Cistrons in Ciona candidate operons are immediately juxtaposed with no intervening DNA. Genomic sequences (assembly version 1.0) and coordinates are shown on the top line of each panel, with scaffold name and nucleotide position indicated. ESTs mapping to this site are shown below; conventional ESTs for the upstream genes (lower case roman letters, poly(A)—10 residues shown—shaded in red) and SL-full-length ESTs for the downstream genes (lower case italic letters, SL sequence shaded in grey). The intercistron boundaries are indicated by arrows. In addition to the two examples shown here, 16 similarly-organized operons are shown in Supplementary Figure S3.