Transcript mapping and genome annotation of ascidian mtDNA using EST data

Abstract

Mitochondrial transcripts of two ascidian species were reconstructed through sequence assembly of publicly available ESTs resembling mitochondrial DNA sequences (mt-ESTs). This strategy allowed us to analyze processing and mapping of the mitochondrial transcripts and to investigate the gene organization of a previously uncharacterized mitochondrial genome (mtDNA). This new strategy would greatly facilitate the sequencing and annotation of mtDNAs. In Ciona intestinalis, the assembled mt-ESTs covered 22 mitochondrial genes ( approximately 12,000 bp) and provided the partial sequence of the mtDNA and the prediction of its gene organization. Such sequences were confirmed by amplification and sequencing of the entire Ciona mtDNA. For Halocynthia roretzi, for which the mtDNA sequence was already available, the inferred mt transcripts allowed better definition of gene boundaries (16S rRNA, ND1, ATP6, and tRNA-Ser genes) and the identification of a new gene (an additional Phe-tRNA). In both species, polycistronic and immature transcripts, creation of stop codons by polyadenylation, tRNA signal processing, and rRNA transcript termination signals were identified, thus suggesting that the main features of mitochondrial transcripts are conserved in Chordata.

Figure 1

Mapping of mt-like contigs/links on the complete mitochondrial genome of the corresponding organism, Ciona intestinalis (Ci) and Halocynthia roretzi (Hr). In both species, all genes are encoded on the same strand. Putative mitochondrial transcripts are indicated as RNAs and named in accordance with Tables 1 and 3. Links are connected by continuous green lines. Proposed modifications in the annotation of Halocynthia mtDNA are indicated as New. Partial genes are indicated as open boxes. Numbers below the mtDNA refer to the length (in base pairs) of the noncoding regions indicated in red. (ATP6) ATP synthase subunit 6; (8) ATP synthase subunit 8; (CO1–CO3) cytochrome c oxidase subunits; (Cyt b) cytochrome b; (ND1–ND6 and ND4L) NADH dehydrogenase subunits; (12S) small subunit ribosomal RNA; (16S) large subunit ribosomal RNA. tRNA genes, in yellow, are indicated by the amino acid transported. (L1) Leu (UUR); (L2) Leu (CUN); (S1) Ser (AGY); (S2) Ser (UCN); (G1) Gly (AGR); (G2) Gly (GGN); (M1) Met (AUG); (M2) Met (AUA).

Figure 2

Location of the poly(A) stretches of Halocynthia contigs on the corresponding mtDNA sequence, with numbers referred to the positions on the mtDNA (AB024528). Dashes indicate the poly(A) start site. Presence of genes and/or noncoding regions (Nc) downstream and upstream of such sites is indicated. All of the alternative poly(A) start sites found in the 16S rRNA contig are reported in the lower box, with numbers below arrows referring to the ESTs supporting that start site. (Lowercase) Noncoding region; (grey background) tRNA sequences; (boxed sequence) rRNA transcription termination box; (bold) putative start codons and stop codons.

Figure 3

Cloverleaf secondary structure of the annotated tRNA-Phe (Phe-1, location: 3694–3757) and the newly identified tRNA-Phe (Phe-2, location: 4400–4462) genes of Halocynthia roretzi mtDNA (AB024528). Nucleotide differences are reported in gray background.