Probabilistic models of biological enzymatic polymerization

Hampton, Marshall; Galey, Miranda; Smoniewski, Clara M; Zimmer, Sara L.

doi:10.1371/journal.pone.0244858

Cited by 3 publications

(4 citation statements)

References 33 publications

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“…Two enzymes add nucleotides to mRNA 3′ ends: KPAP1 adds As and KRET1 adds Us. To investigate how A and U addition may be different for circRNA and total RNA, we used hidden Markov modelling (HMM) 19 . To define the number of nucleotide addition ‘states’ required for HMM, the two types of mRNA tails on kinetoplastid mitochondrial mRNAs must be considered.…”

Section: Resultsmentioning

confidence: 99%

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

Smoniewski

Borujeni

Petersen

et al. 2023

Sci Rep

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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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Section: Resultsmentioning

confidence: 99%

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

Smoniewski

Borujeni

Petersen

et al. 2023

Sci Rep

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“…Hidden Markov modelling (HMM) was performed as described in (Gazestani et al, 2018) and (Hampton et al, 2021). To ensure consistency, models were trained hierarchically, with additional randomized states added one at a time after fully training the submodel, using the Baum-Welch algorithm.…”

Section: Hidden Markov Modellingmentioning

confidence: 99%

“…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.…”

Section: Hidden Markov Modelling -Kpap1 and Kpap2mentioning

confidence: 99%

“…Trypanosome mitochondrial 3 ′ nonencoded tails have both lifestage and transcript-specific characteristics, such as their length profile and A/U composition, though how this specificity is achieved is unknown (Gazestani et al, 2016;Gazestani et al, 2018). Our previous work that examined homopolymer lengths in tails showed that there is potentially more variation in Ahomopolymers than U-homopolymers (Hampton et al, 2021). This suggests that differential action of the KPAPs may be responsible for transcript and life stage specific tail characteristics.…”

Section: Introductionmentioning

confidence: 99%

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

Smoniewski,

Mirzavand Borujeni,

Hampton

et al. 2024

Front. Parasitol.

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RNA-specific nucleotidyltransferases (rNTrs) add nontemplated nucleotides to the 3′ end of RNA. Two noncanonical rNTRs that are thought to be poly(A) polymerases (PAPs) have been identified in the mitochondria of trypanosomes – KPAP1 and KPAP2. KPAP1 is the primary polymerase that adds adenines (As) to trypanosome mitochondrial mRNA 3′ tails, while KPAP2 is a non-essential putative polymerase whose role in the mitochondria is ambiguous. Here, we elucidate the effects of manipulations of KPAP1 and KPAP2 on the 5′ and 3′ termini of transcripts and their 3′ tails. Using glycerol gradients followed by immunoblotting, we present evidence that KPAP2 is found in protein complexes of up to about 1600 kDa. High-throughput sequencing of mRNA termini showed that KPAP2 overexpression subtly changes an edited transcript’s 3′ tails, though not in a way consistent with general PAP activity. Next, to identify possible roles of posttranslational modifications on KPAP1 regulation, we mutated two KPAP1 arginine methylation sites to either mimic methylation or hypomethylation. We assessed their effect on 3′ mRNA tail characteristics and found that the two mutants generally had opposing effects, though some of these were transcript-specific. We present results suggesting that while methylation increases KPAP1 substrate binding and/or initial nucleotide additions, unmethylated KPAP1is more processive. We also present a comprehensive review of UTR termini, and evidence that tail addition activity may change as mRNA editing is initiated. Together, this work furthers our understanding of the role of KPAP1 and KPAP2 on trypanosome mitochondrial mRNA 3′ tail addition, as well as provides more information on mRNA termini processing in general.

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

Smoniewski

Borujeni

Petersen

et al. 2023

Preprint

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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 anin vitrocircularization step normally required to produce PCR products. Starting from totalin vitrocircularized RNA andin vivocircRNA, 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.

show abstract

Probabilistic models of biological enzymatic polymerization

Cited by 3 publications

References 33 publications

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

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

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

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

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