Circular mitochondrial-encoded mRNAs are a distinct subpopulation of mitochondrial mRNA in Trypanosoma brucei

Abstract

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.

Manipulation of mitochondrial poly(A) polymerase family proteins in Trypanosoma brucei impacts mRNA termini processing

“…CircTAIL-seq was performed as described in (Gazestani et al, 2016) and modified as described in (Smoniewski et al, 2023). Illumina primers and PCR conditions are listed in Supplementary Table S2.…”

“…Supplementary Table S3 lists the numbers of raw reads for each sequence file and, following processing, the number of merged reads utilized for downstream analysis of tails and UTR lengths. Processing was performed as in (Smoniewski et al, 2023). Read diversity was assessed and is presented in Supplementary Table S4.…”

“…To further investigate how A and U addition differ when the KPAPs are manipulated, we used hidden Markov models (HMMs), as previously implemented to describe tail addition et al, 2016; Gazestani et al, 2018;Hampton et al, 2021;Smoniewski et al, 2023). We use HMMs to elucidate how the polymerases are acting during in-and ex-tail addition, as they are very sensitive to differences between libraries.…”