Gth cox3 (Fig. 2, arrowheads). The two bands detected by cox3H1-6 are of approximately equal abundance, whereas the cox3H7 precursor band is even more abundant than the full-length band detected by this probe. Together, these Northern blots indicate that rather than precursor transcripts being very minor components of the 
total RNA pool, they are present in similar amounts to full length cox3 mRNA. The high relative abundance of precursors suggests either a slow rate of trans-splicing, or a regulated process that maintains a large pool of precursors. Wetested to see if compounds that are known to perturb mitochondrial electron transport (antimycin A and Salicylhydroxamic Acid (SHAM) [28]), would lead to changes in the relative 
abundances of cox3 precursors, but found no evidence of such regulation in these experiments (not shown). A further result of the Northern blots was lack of evidence of additional cox3 size species as prevalent transcripts. SPDB web polycistronic transcript sequence has previously been detected in dinoflagellate mitochondria [17,23,29,30], and generation of large transcripts from few promoters is quite common in mtDNAs where large precursor RNA molecules are processed to generate individual gene transcripts [31]. If cox3 precursor transcripts are similarly generated by processing large polycistronic transcripts, then processing to the final precise lengths must be fast enough that little intermediate is evident by Northern blot detection or sequencing methods described above. Alternatively, it is possible that the cox3 precursors could be transcribed as their final lengths; we presently have no data that can discern between these scenarios. A consequence of abundant precursor transcripts is that these would need to be excluded from the downstream Anlotinib site expression machinery, namely translation. However, we detected no obvious differentiation of precursor versus complete transcript, such as post-transcriptional modifications or oligoadenylated tail length differences, that might distinguish precursors from mature transcripts ready for translation. The function of oligo-adenylation in dinoflagellate mitochondria is unknown (other than its inclusion in cox3 splice products), but it is consistently present in mitochondrial transcripts of both dinoflagellates and apicomplexans suggesting it does not serve as a cue for mRNA degradation as for some other organelle systems [18,29,32?4]. While RNA editing is a necessary process of mRNA maturation in dinoflagellate mitochondria [35,36], we have previously shown that K. veneficum cox3H1-6 precursors are fully edited [17]. Instances of minor incomplete editing were observed in some of these cox3H1-6 transcripts, however this was also seen for cob transcripts (which are not trans-spliced), and appears to be a general feature of RNA editing [17]. It is, therefore, unclear how the abundant presence ofFigure 2. Northern blot analysis of K. veneficum cox3H1-6, cox3H7 and full-length cox3 transcripts. Total K. veneficum RNA was hybridized with either a probe corresponding to the cox3H1-6 or cox3H7 sequence. Bands observed correspond in size to the precursor molecules cox3H1-6 (,745 nt) and cox3H7 (,136 nt), along with full length cox3 (,872 nt) (note: predicted RNA lengths include oligoadenylation tails). doi:10.1371/journal.pone.0056777.gAn Unusual RNA Trans-Splicing Typethese immature transcripts 23115181 is managed. One possibility is that the precursor transcripts might be translated into partial Cox3.Gth cox3 (Fig. 2, arrowheads). The two bands detected by cox3H1-6 are of approximately equal abundance, whereas the cox3H7 precursor band is even more abundant than the full-length band detected by this probe. Together, these Northern blots indicate that rather than precursor transcripts being very minor components of the total RNA pool, they are present in similar amounts to full length cox3 mRNA. The high relative abundance of precursors suggests either a slow rate of trans-splicing, or a regulated process that maintains a large pool of precursors. Wetested to see if compounds that are known to perturb mitochondrial electron transport (antimycin A and Salicylhydroxamic Acid (SHAM) [28]), would lead to changes in the relative abundances of cox3 precursors, but found no evidence of such regulation in these experiments (not shown). A further result of the Northern blots was lack of evidence of additional cox3 size species as prevalent transcripts. Polycistronic transcript sequence has previously been detected in dinoflagellate mitochondria [17,23,29,30], and generation of large transcripts from few promoters is quite common in mtDNAs where large precursor RNA molecules are processed to generate individual gene transcripts [31]. If cox3 precursor transcripts are similarly generated by processing large polycistronic transcripts, then processing to the final precise lengths must be fast enough that little intermediate is evident by Northern blot detection or sequencing methods described above. Alternatively, it is possible that the cox3 precursors could be transcribed as their final lengths; we presently have no data that can discern between these scenarios. A consequence of abundant precursor transcripts is that these would need to be excluded from the downstream expression machinery, namely translation. However, we detected no obvious differentiation of precursor versus complete transcript, such as post-transcriptional modifications or oligoadenylated tail length differences, that might distinguish precursors from mature transcripts ready for translation. The function of oligo-adenylation in dinoflagellate mitochondria is unknown (other than its inclusion in cox3 splice products), but it is consistently present in mitochondrial transcripts of both dinoflagellates and apicomplexans suggesting it does not serve as a cue for mRNA degradation as for some other organelle systems [18,29,32?4]. While RNA editing is a necessary process of mRNA maturation in dinoflagellate mitochondria [35,36], we have previously shown that K. veneficum cox3H1-6 precursors are fully edited [17]. Instances of minor incomplete editing were observed in some of these cox3H1-6 transcripts, however this was also seen for cob transcripts (which are not trans-spliced), and appears to be a general feature of RNA editing [17]. It is, therefore, unclear how the abundant presence ofFigure 2. Northern blot analysis of K. veneficum cox3H1-6, cox3H7 and full-length cox3 transcripts. Total K. veneficum RNA was hybridized with either a probe corresponding to the cox3H1-6 or cox3H7 sequence. Bands observed correspond in size to the precursor molecules cox3H1-6 (,745 nt) and cox3H7 (,136 nt), along with full length cox3 (,872 nt) (note: predicted RNA lengths include oligoadenylation tails). doi:10.1371/journal.pone.0056777.gAn Unusual RNA Trans-Splicing Typethese immature transcripts 23115181 is managed. One possibility is that the precursor transcripts might be translated into partial Cox3.