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Analysis of a botryllid enriched-full-length cDNA library: insight into the evolution of spliced leader trans-splicing in tunicates

Abstract

In some animals, mRNA may be modified after transcription by the addition of a 5′ spliced leader sequence. This is known as spliced leader (SL) trans-splicing, and is of uncertain function and evolutionary origin. Here, we report the identification of SL trans-splicing in the colonial ascidian Botryllus schlosseri. Combining our own expressed sequence tag (EST) data with additional data from GenBank, we identify the dominant spliced leader sequence and show it to be similar to that of other ascidians and to that of Oikopleura dioica, a basally diverging tunicate. Gene Ontology analysis of B. schlosseri ESTs with and without a 5′ spliced leader shows that genes encoding ribosomal proteins tend not to be trans-spliced, a character shared with the ascidian Ciona intestinalis. We also examine individual cases of genes that produce mRNAs that are SL trans-spliced in B. schlosseri but not in C. intestinalis. We conclude that SL trans-splicing evolved early in the tunicate lineage and shows stability over considerable evolutionary time. However, SL trans-splicing may be gained or lost in individual genes.

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Acknowledgements

The authors wish to thank Michael Kube, Richard Reinhardt and Sven Klages for the support in preparing the library during the NoE “Marine Genomics Europe” training course “Generation of cDNA Libraries by Primer Extension” (20 November–1 December, 2006) attended to one of us (FG) at the Max Planck Institute for Molecular Genetics, Berlin (Germany). We also thank two anonymous referees for their thoughtful comments. FG acknowledges the support of the Ministero della Università e Ricerca Scientifica e Tecnologica and the Università degli Studi di Padova. SMS acknowledges the support of the Royal Society and the British Council. The authors declare that the experiments comply with the current laws of the country in which they were performed. The authors declare that they have no conflict of interest. Additional data are available at http://www.fabiogasparini.org/Data/DGEsuppl/info.htm. (1) The 765 singleton clones and contigs derived from the assembled ESTs generated in this study; (2) accessions and sequences of the 4,477 B. schlosseri ESTs extracted from GenBank and used in this study; (3) dbEST and GenBank accession numbers of the 1,259 B. schlosseri ESTs generated in this study.

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Correspondence to Fabio Gasparini.

Additional information

Communicated by H. Nishida

Electronic supplementary materials

Below is the link to the electronic supplementary material.

ESM 1

The 765 singleton clones and contigs derived from the assembled ESTs generated in this study are available at: http://www.fabiogasparini.org/Data/DGEsuppl/info.htm. (TXT 0 kb)

ESM 2

B. schlosseri ESTs available in GenBank on 13 Oct 2009 with putaitive SL sequences marked in blue or green, and additional possible contaminant sequences as described in the text in purple. This subset includes only those without internal variants (PDF 96 kb)

ESM 3

B. schlosseri ESTs available in GenBank on 13 Oct 2009 with putative SL sequences marked in green, additional possible contaminant sequences as described in the text in purple, and internal variants marked in black. This subset includes only those with internal variants (PDF 26 kb)

ESM 4

B. schlosseri singleton clones and contigs from assembled library with putative SL sequences. This is a subset of Online Resource 1. Each sequence is in Fasta format with only the first 40 nucleotides of 5' region shown (TXT 9 kb)

ESM 5

B. schlosseri ESTs generated in this study with putative SL sequences. Each EST is in Fasta format with only the first 40 nucleotides of the 5' region shown (TXT 13 kb)

ESM 6

GOSSIP output of statistical analysis of association between annotations from the Gene Ontology and groups of genes. Test group, non trans-spliced members; reference group, trans-spliced members. Results with FDR < 0.01 are highlighted (PDF 416 kb)

ESM 7

GOSSIP output of statistical analysis of association between annotations from the Gene Ontology and groups of genes. Test group, trans-spliced members; reference group, non trans-spliced members (PDF 296 kb)

ESM 8

GOanna2ga gene association file format of trans-spliced annotated sequences obtained by converting the GOanna Excel spreadsheet output with GO annotations (XLS 51 kb)

ESM 9

GOanna2ga gene association file format of non-trans-spliced annotated sequences obtained by converting the GOanna Excel spreadsheet output with GO annotations (XLS 149 kb)

ESM 10

AmiGO graphs of ontological hierarchies obtained with the eight GO terms (encircled in red) significantly over-represented in the non-trans-spliced cDNA assembled library subset (PNG 4732 kb)

ESM 11

B. schlosseri sequences matching C. intestinalis KH gene models for which cRAR has been calculated by Matsumoto et al. (2010). Columns G to M show characters of the KH gene model predicted to be orthologous to the B. schlosseri sequence. These have only been calculated for genes with cRAR < 0.05 (XLS 45 kb)

ESM 12

B. schlosseri sequences matching C. intestinalis KH gene models for which cRAR is not calculated but for which evidence of SL trans-splicing comes from the raw data of Matsumoto et al. (2010; XLS 28 kb)

ESM 13

B. schlosseri sequences matching C. intestinalis KH gene models that were not detected in the tailbud stage sequencing data of Matsumoto et al. (2010; XLS 24 kb)

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Gasparini, F., Shimeld, S.M. Analysis of a botryllid enriched-full-length cDNA library: insight into the evolution of spliced leader trans-splicing in tunicates. Dev Genes Evol 220, 329–336 (2011). https://doi.org/10.1007/s00427-011-0351-y

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Keywords

  • Ascidian
  • Botryllus
  • Transcription
  • Splicing
  • Spliced leader