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Species within the green algal order Cladophorales have an unconventional plastome structure where individual coding regions or small numbers of genes occur as linear single-stranded DNAs folded into hairpin structures. Another group of photosynthetic organisms with an equivalently reduced chloroplast genome are the peridinin dinoflagellates of the Alveolata eukaryotic lineage whose plastomes are mini-circles carrying one or a few genes required for photosynthesis. One unusual aspect of the Alveolata is the polyuridylylation of mRNA 30 ends among peridinin dinoflagellates and the chromerid algae. This study was conducted to understand if an unconventional highly reduced plastome structure co-occurs with unconventional RNA processing. To address this, the 50 and 30 mRNA termini of the known chloroplast genes of Pithophora roettleri (order Cladophorales) were analyzed for evidence of post-transcriptional processing. Circular Reverse Transcriptase PCR (cRT-PCR) followed by deep sequencing of the amplicons was used to analyze 50 and 30 mRNA termini. Evidence of several processing events were collected, most notably the 30 termini of six of the eight genes were polyuridylylated, which has not been reported for any lineage outside of the Alveolata. Other processing events include poly(A) and heteropolymeric 30 additions, 50 primary transcript start sites, as well as the presence of circularized RNAs. Five other species representing other green algal lineages were also tested and poly(U) additions appear to be limited to the order Cladophorales. These results demonstrate that chloroplast mRNA polyuridylylation is not the sole provenance of photosynthetic alveolates and may have convergently evolved in two distinct photosynthetic lineages. 

Species within the green algal order Cladophorales have an unconventional plastome structure where individual coding regions or small numbers of genes occur as linear single-stranded DNAs folded into hairpin structures. Another group of photosynthetic organisms with an equivalently reduced chloroplast genome are the peridinin dinoflagellates of the Alveolata eukaryotic lineage whose plastomes are mini-circles carrying one or a few genes required for photosynthesis. One unusual aspect of the Alveolata is the polyuridylylation of mRNA 30 ends among peridinin dinoflagellates and the chromerid algae. This study was conducted to understand if an unconventional highly reduced plastome structure co-occurs with unconventional RNA processing. To address this, the 50 and 30 mRNA termini of the known chloroplast genes of Pithophora roettleri (order Cladophorales) were analyzed for evidence of post-transcriptional processing. Circular Reverse Transcriptase PCR (cRT-PCR) followed by deep sequencing of the amplicons was used to analyze 50 and 30 mRNA termini. Evidence of several processing events were collected, most notably the 30 termini of six of the eight genes were polyuridylylated, which has not been reported for any lineage outside of the Alveolata. Other processing events include poly(A) and heteropolymeric 30 additions, 50 primary transcript start sites, as well as the presence of circularized RNAs. Five other species representing other green algal lineages were also tested and poly(U) additions appear to be limited to the order Cladophorales. These results demonstrate that chloroplast mRNA polyuridylylation is not the sole provenance of photosynthetic alveolates and may have convergently evolved in two distinct photosynthetic lineages. 

The relatively recent focus on the widespread occurrence and abundance of circular RNAs (circRNA) in the human cell nucleus has sparked an intensive interest in their existence and possible roles in cell gene expression and physiology. The presence of circRNAs in mammalian mitochondria, however, has been under-explored. Mitochondrial mRNAs differ from those produced from nuclear genes because they lack introns and are transcribed as poly-cistronic transcripts that are endonucleolytically cleaved, leaving transcripts with very small 5′ and 3′ UTRs. Circular RNAs have been identified in the semi-autonomous organelles of single-celled organisms and plants but their purpose has not been clearly demonstrated. The goal of our project was to test the hypothesis, processed mRNAs are circularized in vertebrate mitochondria as a necessary RNA processing step prior to translation. Mitochondrial mRNAs were isolated from the human cell line HEK293 and evidence of circularization sought by treating RNA with RNAse-R and then amplifying putative 3′-5′ junction sites. Sequence results demonstrated the occurrence of mRNA circularization within each coding region of the mitochondrial genome. However, in most cases the circRNAs carried coding regions that had been truncated, suggesting they were not translatable. Quantification of the circularized versions of the mRNAs revealed they comprise a small portion (~10%) of the total mRNA. These findings demonstrate that mRNA circularization occurs in mammalian mitochondria but it does not appear to play a role in making translatable mRNAs.