GAn Unusual RNA Trans-Splicing Typeare included (Fig. 1B). The length of oligoadenylation observed for all taxa and all cox3 products is similar, typically ranging from 12?19 nucleotides. For cox3H1-6 this is sufficient to span the respective coding gaps between exons. The sequence termini of cox3 precursor transcripts and positions of oligoadenylation seen in the cRT-PCR data are corroborated by available transcriptome data. For example, the cox3H1-6 oligoadenylation sites (Fig. 1B) are identical in K. veneficum EST sequences [17], and from Symbiodinium sp. eight ESTs precisely match the POR8 cox3H7 59 22948146 sequence (accessions; FE537727, FE537728, FE537811, FE537812, FE537869, FE537870, FE538147, FE538148). We did, however, recover some cRT-PCR data that showed some termini variation (Data S1). In K. veneficum cox3H7, two of six independent cRT-PCR products bore an additional 15 nucleotides at the 59 terminus (UUCCAAGAAAAGCCU). This extra tag lacks any complementarity with cox3 coding sequence, BLAST searches did not recover matches to K. veneficum mitochondrial genomic sequence [17], and RT-PCR could not reproduce a cox3H7 fragment linked to this extension. Similarly, in Symbiodinium sp., one of seven cox3H7 amplicons is 59 truncated by 10 nucleotides, relative to the other six sequences. These data are consistent with previous evidence of dinoflagellate mitochondrial transcripts occasionally occurring either fused to unrelated sequence, or truncated [23], and likely represent non-functional transcript species. In A. carterae, of three cox3H1-6 cRT-PCR amplicons, one lacks an oligo-A tail, and another is oligoadenylated one nucleotide earlier (c.f. Fig. 1B). Neither of these two variations would directly affect the sequence of complete cox3 as they occur downstream of the splice site, and therefore such variation in A. carterae might be tolerated. Post-transcriptional RNA end capping has been described in some dinoflagellate organelles, but we observe no evidence of such modification to any of the cox3 transcripts. In the deep-branching dinoflagellate Oxyrrhis marinus 59 capping by addition of 8? U nucleotides to mitochondrial protein-encoding transcripts has been reported, and in dinoflagellate plastids mRNAs gain 39 polyuridine tracts of up to 40 nucleotides after transcription [24?6]. Both of these additions are detectible by cRT-PCR [24,26], but were not observed in cox3 transcripts for any of the taxa examined. Further capping reactions that modify the 59-phosphate group on RNA molecules, such as the modified guanine nucleotide added 1516647 to the 59 end of most eukaryotic nuclear transcripts [27], would prevent RNA ligation and detection by cRT-PCR. While such capping is not known from either bacteria or mitochondria, it remains possible that further cox3 transcript species might exist in addition to those MedChemExpress JI 101 detected by cRT-PCR and transcriptomics approaches. To examine the relative abundance of cox3H1-6 and cox3H7 transcripts in comparison to full-length cox3 in dinoflagellate mitochondria, we performed Northern blot analysis of K. veneficum total RNA. Probes were made corresponding to either cox3H1-6 or cox3H7. Each would therefore detect the respective precursor and also the full-length cox3 transcript, enabling relative steady-state quantitation of these species before and after splicing. Indeed, two bands were detected in Northern blots for each probe, and in each case these bands corresponded in size to the respective precursor and full-len.GAn Unusual RNA Trans-Splicing Typeare included (Fig. 1B). The length of oligoadenylation observed for all taxa and all cox3 products is similar, typically ranging from 12?19 nucleotides. For cox3H1-6 this is sufficient to span the respective coding gaps between exons. The sequence termini of cox3 precursor transcripts and positions of oligoadenylation seen in the cRT-PCR data are corroborated by available transcriptome data. For example, the cox3H1-6 oligoadenylation sites (Fig. 1B) are identical in K. veneficum EST sequences [17], and from Symbiodinium sp. eight ESTs precisely match the cox3H7 59 22948146 sequence (accessions; FE537727, FE537728, FE537811, FE537812, FE537869, FE537870, FE538147, FE538148). We did, however, recover some cRT-PCR data that showed some termini variation (Data S1). In K. veneficum cox3H7, two of six independent cRT-PCR products bore an additional 15 nucleotides at the 59 terminus (UUCCAAGAAAAGCCU). This extra tag lacks any complementarity with cox3 coding sequence, BLAST searches did not recover matches to K. veneficum mitochondrial genomic sequence [17], and RT-PCR could not reproduce a cox3H7 fragment linked to this extension. Similarly, in Symbiodinium sp., one of seven cox3H7 amplicons is 59 truncated by 10 nucleotides, relative to the other six sequences. These data are consistent with previous evidence of dinoflagellate mitochondrial transcripts occasionally occurring either fused to unrelated sequence, or truncated [23], and likely represent non-functional transcript species. In A. carterae, of three cox3H1-6 cRT-PCR amplicons, one lacks an oligo-A tail, and another is oligoadenylated one nucleotide earlier (c.f. Fig. 1B). Neither of these two variations would directly affect the sequence of complete cox3 as they occur downstream of the splice site, and therefore such variation in A. carterae might be tolerated. Post-transcriptional RNA end capping has been described in some dinoflagellate organelles, but we observe no evidence of such modification to any of the cox3 transcripts. In the deep-branching dinoflagellate Oxyrrhis marinus 59 capping by addition of 8? U nucleotides to mitochondrial protein-encoding transcripts has been reported, and in dinoflagellate plastids mRNAs gain 39 polyuridine tracts of up to 40 nucleotides after transcription [24?6]. Both of these additions are detectible by cRT-PCR [24,26], but were not observed in cox3 transcripts for any of the taxa examined. Further capping reactions that modify the 59-phosphate group on RNA molecules, such as the modified guanine nucleotide added 1516647 to the 59 end of most eukaryotic nuclear transcripts [27], would prevent RNA ligation and detection by cRT-PCR. While such capping is not known from either bacteria or mitochondria, it remains possible that further cox3 transcript species might exist in addition to those detected by cRT-PCR and transcriptomics approaches. To examine the relative abundance of cox3H1-6 and cox3H7 transcripts in comparison to full-length cox3 in dinoflagellate mitochondria, we performed Northern blot analysis of K. veneficum total RNA. Probes were made corresponding to either cox3H1-6 or cox3H7. Each would therefore detect the respective precursor and also the full-length cox3 transcript, enabling relative steady-state quantitation of these species before and after splicing. Indeed, two bands were detected in Northern blots for each probe, and in each case these bands corresponded in size to the respective precursor and full-len.
