*Article* **Mitochondrial mRNA Processing in the Chlorophyte Alga** *Pediastrum duplex* **and Streptophyte Alga** *Chara vulgaris* **Reveals an Evolutionary Branch in Mitochondrial mRNA Processing**

**Grayson C. R. Proulex 1,†, Marcus J. Meade 1,†, Kalina M. Manoylov <sup>2</sup> and A. Bruce Cahoon 1,\***


**Abstract:** Mitochondria carry the remnant of an ancestral bacterial chromosome and express those genes with a system separate and distinct from the nucleus. Mitochondrial genes are transcribed as poly-cistronic primary transcripts which are post-transcriptionally processed to create individual translationally competent mRNAs. Algae post-transcriptional processing has only been explored in *Chlamydomonas reinhardtii* (Class: Chlorophyceae) and the mature mRNAs are different than higher plants, having no 5- UnTranslated Regions (UTRs), much shorter and more variable 3- UTRs and polycytidylated mature mRNAs. In this study, we analyzed transcript termini using circular RT-PCR and PacBio Iso-Seq to survey the 3 and 5- UTRs and termini for two green algae, *Pediastrum duplex* (Class: Chlorophyceae) and *Chara vulgaris* (Class: Charophyceae). This enabled the comparison of processing in the chlorophyte and charophyte clades of green algae to determine if the differences in mitochondrial mRNA processing pre-date the invasion of land by embryophytes. We report that the 5 mRNA termini and non-template 3 termini additions in *P. duplex* resemble those of *C. reinhardtii*, suggesting a conservation of mRNA processing among the chlorophyceae. We also report that *C. vulgaris* mRNA UTRs are much longer than chlorophytic examples, lack polycytidylation, and are polyadenylated similar to embryophytes. This demonstrates that some mitochondrial mRNA processing events diverged with the split between chlorophytic and streptophytic algae.

**Keywords:** mitochondria; RNA processing; algal evolution; circular RNA; polycytidylation; PacBio Iso-Seq
