RESUMEN

Since the first identification of circular RNA (circRNA) in viral-like systems, reports of circRNAs and their functions in various organisms, cell types, and organelles have greatly expanded. Here, we report the first evidence, to our knowledge, of circular mRNA in the mitochondrion of the eukaryotic parasite, Trypanosoma brucei. While using a circular RT-PCR technique developed to sequence mRNA tails of mitochondrial transcripts, we found that some mRNAs are circularized without an in vitro circularization step normally required to produce PCR products. Starting from total in vitro circularized RNA and in vivo circRNA, we high-throughput sequenced three transcripts from the 3' end of the coding region, through the 3' tail, to the 5' start of the coding region. We found that fewer reads in the circRNA libraries contained tails than in the total RNA libraries. When tails were present on circRNAs, they were shorter and less adenine-rich than the total population of RNA tails of the same transcript. Additionally, using hidden Markov modelling we determined that enzymatic activity during tail addition is different for circRNAs than for total RNA. Lastly, circRNA UTRs tended to be shorter and more variable than those of the same transcript sequenced from total RNA. We propose a revised model of Trypanosome mitochondrial tail addition, in which a fraction of mRNAs is circularized prior to the addition of adenine-rich tails and may act as a new regulatory molecule or in a degradation pathway.

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RESUMEN

Since the first identification of circular RNA (circRNA) in viral-like systems, reports of circRNAs and their functions in various organisms, cell types, and organelles have greatly expanded. Here, we report the first evidence of circular mRNA in the mitochondrion of the eukaryotic parasite, Trypanosoma brucei . While using a circular RT-PCR technique developed to sequence mRNA tails of mitochondrial transcripts, we found that some mRNAs are circularized without an in vitro circularization step normally required to produce PCR products. Starting from total in vitro circularized RNA and in vivo circRNA, we high-throughput sequenced three transcripts from the 3' end of the coding region, through the 3' tail, to the 5' start of the coding region. We found that fewer reads in the circRNA libraries contained tails than in the total RNA libraries. When tails were present on circRNAs, they were shorter and less adenine-rich than the total population of RNA tails of the same transcript. Additionally, using hidden Markov modelling we determined that enzymatic activity during tail addition is different for circRNAs than for total RNA. Lastly, circRNA UTRs tended to be shorter and more variable than those of the same transcript sequenced from total RNA. We propose a revised model of Trypanosome mitochondrial tail addition, in which a fraction of mRNAs is circularized prior to the addition of adenine-rich tails and may act as a new regulatory molecule or in a degradation pathway.

RESUMEN

The protozoan Trypanosoma cruzi has a complicated dual-host life cycle, and starvation can trigger transition from the replicating insect stage to the mammalian-infectious nonreplicating insect stage (epimastigote to trypomastigote differentiation). Abundance of some mature RNAs derived from its mitochondrial genome increase during culture starvation of T. cruzi for unknown reasons. Here, we examine T. cruzi mitochondrial gene expression in the mammalian intracellular replicating life stage (amastigote), and uncover implications of starvation-induced changes in gene expression. Mitochondrial RNA levels in general were found to be lowest in actively replicating amastigotes. We discovered that mitochondrial respiration decreases during starvation in insect stage cells, despite the previously observed increases in mitochondrial mRNAs encoding electron transport chain (ETC) components. Surprisingly, T. cruzi epimastigotes in replete medium grow at normal rates when we genetically compromised their ability to perform insertion/deletion editing and thereby generate mature forms of some mitochondrial mRNAs. However, these cells, when starved, were impeded in the epimastigote to trypomastigote transition. Further, they experience a short-flagella phenotype that may also be linked to differentiation. We hypothesize a scenario where levels of mature RNA species or editing in the single T. cruzi mitochondrion are linked to differentiation by a yet-unknown signaling mechanism.

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