Control Mechanisms of the Holo-Editosome in Trypanosomes

Cruz‐Reyes, Jorge; Mooers, Blaine H. M.; Kumar, Vikas; Doharey, Pawan Kumar; Meehan, Joshua; Chaparro, Luenn

doi:10.1007/978-3-319-78190-7_5

Cited by 7 publications

(5 citation statements)

References 48 publications

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“…Our structural searches of the OB fold-like domain in REH2 using Phyre2 [20] found the best agreements of the REH2 OB fold with structures of yeast ADP-bound Prp43p helicase and Drosophila RNA helicase MLE in a complex with RNA and ADP-Alf4 [4, 16, 18, 21]. Prp43p is a bi-functional helicase in mRNA splicing and ribosome biogenesis [15, 22].…”

Section: Resultsmentioning

confidence: 99%

“…Mutation of some of these residues impaired RNA binding by MLE in vitro or the proper localization of MLE in chromosomes [18]. Sequence alignments and homology models indicated that the residues H1032 and K1033 in MLE likely represent residues H1998 and R1999 in the predicted β3−β4 loop in the OB fold in REH2 (Fig 2D; S4 Fig)[21]. The H1998E mutant appeared to retain a normal association with H2 F1 but exhibited a moderate decrease in association with GAP1.…”

Section: Resultsmentioning

confidence: 99%

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Protein features for assembly of the RNA editing helicase 2 subcomplex (REH2C) in Trypanosome holo-editosomes

et al. 2019

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Uridylate insertion/deletion RNA editing in Trypanosoma brucei is a complex system that is not found in humans, so there is interest in targeting this system for drug development. This system uses hundreds of small non-coding guide RNAs (gRNAs) to modify the mitochondrial mRNA transcriptome. This process occurs in holo-editosomes that assemble several macromolecular trans factors around mRNA including the RNA-free RNA editing core complex (RECC) and auxiliary ribonucleoprotein (RNP) complexes. Yet, the regulatory mechanisms of editing remain obscure. The enzymatic accessory RNP complex, termed the REH2C, includes mRNA substrates and products, the multi-domain 240 kDa RNA Editing Helicase 2 (REH2) and an intriguing 8-zinc finger protein termed REH2-Associated Factor 1 ( H2 F1). Both of these proteins are essential in editing. REH2 is a member of the DExH/RHA subfamily of RNA helicases with a conserved C-terminus that includes a regulatory OB-fold domain. In trypanosomes, H2 F1 recruits REH2 to the editing apparatus, and H2 F1 downregulation causes REH2 fragmentation. Our systematic mutagenesis dissected determinants in REH2 and H2 F1 for the assembly of REH2C, the stability of REH2, and the RNA-mediated association of REH2C with other editing trans factors. We identified functional OB-fold amino acids in eukaryotic DExH/RHA helicases that are conserved in REH2 and that impact the assembly and interactions of REH2C. H2 F1 upregulation stabilized REH2 in vivo . Mutation of the core cysteines or basic amino acids in individual zinc fingers affected the stabilizing property of H2 F1 but not its interactions with other examined editing components. This result suggests that most, if not all, fingers may contribute to REH2 stabilization. Finally, a recombinant REH2 (240 kDa) established that the full-length protein is a bona fide RNA helicase with ATP-dependent unwinding activity. REH2 is the only DExH/RHA-type helicase in kinetoplastid holo-editosomes.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Protein features for assembly of the RNA editing helicase 2 subcomplex (REH2C) in Trypanosome holo-editosomes

et al. 2019

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“…3 ). Similarly, the knockdown of core editing proteins in RECC and RECC reduces the accumulation of fully edited transcripts in PCF and BSF (5,73). The cumulative loss in total edits (canonical and non-canonical) at most editing sites likely causes a net loss in full editing in these knockdowns.…”

Section: Discussionmentioning

confidence: 99%

KREH2 helicase represses ND7 mRNA editing in procyclic-stageTrypanosoma bruceiby opposite modulation of canonical and “moonlighting” gRNA utilization creating a proposed mRNA structure

Meehan,

Ivens,

Grote

et al. 2024

Preprint

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Unknown factors regulate mitochondrial U-insertion/deletion (U-indel) RNA editing in procyclic-form (PCF) and bloodstream-form (BSF) trypanosomes. This editing is directed by anti-sense gRNAs, creates canonical protein-encoding mRNAs, and may developmentally control respiration. Canonical editing by housekeeping cognate gRNAs occurs amid massive non-canonical editing of unclear sources and biological significance. We found PCF-specific repression in mRNA ND7 that involves novel modulation of gRNA usage by RNA helicase KREH2. At a major early checkpoint near the mRNA 3' terminus, KREH2 promotes the usage of a non-cognate "terminator" gRNA over a cognate gRNA. At this early checkpoint, terminator-directed editing installs a structural element in 30% of the ND7 transcriptome, which potentially occludes the mRNA 3' terminus and represses further editing. BSF-to-PCF differentiation in vitro recreated this form of negative control. Remarkably, KREH2-RNAi knockdown relieved repression by reverting the native usage of cognate/non-cognate gRNAs at the major early checkpoint. ND7 transcripts that lacked terminator-directed editing at the early checkpoint exhibited similar negative editing control along the mRNA sequence, suggesting that KREH2 modulates guiding fidelity globally. Thus, KREH2 is a key protein in ND7 developmental editing control involving modulation of gRNA selection and RNA structure installed by a regulatory terminator gRNA.

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“…The mechanism of this RNA editing is the co-transcriptional addition of specific nucleotides to the 3′ end of the nascent RNA transcript at editing sites by the mitochondrial RNA polymerase [ 21 , 22 ]. This is in contrast to post-transcriptional mitochondrial insertional editing systems which use antisense guide RNAs (gRNAs) to specify editing site location and insert nucleotides by breaking the RNA backbone at a specific location (RNA endonuclease), adding one or more non-templated nucleotides to the 3′ end of the cleaved RNA (terminal transferase), and then restoring the RNA backbone by RNA ligation (RNA ligase), reviewed in [ 23 ]. In P. polycephalum this co-transcriptional mechanism consists of the mitochondrial RNA polymerase proceeding along the template DNA strand, adding templated nucleotides during normal transcription until it arrives at an editing site.…”

Section: The Mitochondrial Dna Of P Polycephalummentioning

confidence: 99%

Genetic Diversity in the mtDNA of Physarum polycephalum

Hammar

Miller

2023

Genes

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The mtDNA of the myxomycete Physarum polycephalum can contain as many as 81 genes. These genes can be grouped in three different categories. The first category includes 46 genes that are classically found on the mtDNA of many organisms. However, 43 of these genes are cryptogenes that require a unique type of RNA editing (MICOTREM). A second category of gene is putative protein-coding genes represented by 26 significant open reading frames. However, these genes do not appear to be transcribed during the growth of the plasmodium and are currently unassigned since they do not have any apparent similarity to other classical mitochondrial protein-coding genes. The third category of gene is found in the mtDNA of some strains of P. polycephalum. These genes derive from a linear mitochondrial plasmid with nine significant, but unassigned, open reading frames which can integrate into the mitochondrial DNA by recombination. Here, we review the mechanism and evolution of the RNA editing necessary for cryptogene expression, discuss possible origins for the 26 unassigned open reading frames based on tentative identification of their protein product, and discuss the implications to mtDNA structure and replication of the integration of the linear mitochondrial plasmid.

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Control Mechanisms of the Holo-Editosome in Trypanosomes

Cited by 7 publications

References 48 publications

Protein features for assembly of the RNA editing helicase 2 subcomplex (REH2C) in Trypanosome holo-editosomes

Protein features for assembly of the RNA editing helicase 2 subcomplex (REH2C) in Trypanosome holo-editosomes

KREH2 helicase represses ND7 mRNA editing in procyclic-stageTrypanosoma bruceiby opposite modulation of canonical and “moonlighting” gRNA utilization creating a proposed mRNA structure

Genetic Diversity in the mtDNA of Physarum polycephalum

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