Cross-validated methods for promoter/transcription start site mapping in SL trans-spliced genes, established using the Ciona intestinalis troponin I gene.

In conventionally-expressed eukaryotic genes, transcription start sites (TSSs) can be identified by mapping the mature mRNA 5'-terminal sequence onto the genome. However, this approach is not applicable to genes that undergo pre-mRNA 5'-leader trans-splicing (SL trans-splicing) because the original 5'-segment of the primary transcript is replaced by the spliced leader sequence during the trans-splicing reaction and is discarded. Thus TSS mapping for trans-spliced genes requires different approaches. We describe two such approaches and show that they generate precisely agreeing results for an SL trans-spliced gene encoding the muscle protein troponin I in the ascidian tunicate chordate Ciona intestinalis. One method is based on experimental deletion of trans-splice acceptor sites and the other is based on high-throughput mRNA 5'-RACE sequence analysis of natural RNA populations in order to detect minor transcripts containing the pre-mRNA's original 5'-end. Both methods identified a single major troponin I TSS located ∼460 nt upstream of the trans-splice acceptor site. Further experimental analysis identified a functionally important TATA element 31 nt upstream of the start site. The two methods employed have complementary strengths and are broadly applicable to mapping promoters/TSSs for trans-spliced genes in tunicates and in trans-splicing organisms from other phyla.

SL RNA genes of the ascidian tunicates Ciona intestinalis and Ciona savignyi.

We characterized by bioinformatics the trans-spliced leader donor RNA (SL RNA) genes of two ascidians, Ciona intestinalis and Ciona savignyi. The Ciona intestinalis genome contains approximately 670 copies of the SL RNA gene, principally on a 264-bp tandemly repeated element. Fluorescent in-situ hybridization mapped most of the repeats to a single site on the short arm of chromosome 8. The Ciona intestinalis genome also contains approximately 100 copies of a >3.6-kb element that carries 1) an SL RNA-related sequence (possible a pseudogene) and 2) genes for the U6 snRNA and a histone-like protein. The Ciona savignyi genome contains two SL RNA gene classes having the same SL sequence as Ciona intestinalis but differing in the intron-like segments. These reside in similar but distinct repeat units of 575 bp ( approximately 410 copies) and 552 bp ( approximately 250 copies) that are arranged as separate tandem repeats. In neither Ciona species is the 5S RNA gene present within the SL RNA gene repeat unit. Although the number of SL RNA genes is similar, there is little sequence similarity between the intestinalis and savignyi repeat units, apart from the region encoding the SL RNA itself. This suggests that cis-regulatory elements involved in transcription and 3'-end processing are likely to be present within the transcribed region. The genomes of both Ciona species also include > 100 dispersed short elements containing the 16-nt SL sequence and up to 6 additional nucleotides of the SL RNA sequence.