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Novel kinetoplast genome structure and RNA editing patterns in the trypanosomatid Vickermania

ABSTRACT: The trypanosomatid flagellates possess in their single mitochondrion a highly complex kinetoplast (k) DNA, which is composed of interlocked circular molecules of two types. Dozens of maxicircles represent a classical mitochondrial genome, and thousands of minicircles encode guide (g)RNAs, which direct the processive and essential uridine insertion/deletion mRNA editing of maxicircle transcripts. While the details of kDNA structure and this type of RNA editing are well-established, our knowledge mostly relies on a narrow foray of intensely studied human parasites of the genera Leishmania and Trypanosoma. Here, we analyzed kDNA, its expression, and RNA editing of two members of the poorly characterized genus Vickermania with very different cultivation histories. In both Vickermania species, the gRNA-containing HL-circles are atypically large with multiple gRNAs each. Examination of V. spadyakhi HL-circle loci revealed a massive redundancy of gRNAs relative to the editing needs. In comparison, the HL-circle repertoire of extensively cultivated V. ingenoplastis is greatly reduced. It correlates with V. ingenoplastis-specific loss of productive editing of transcripts encoding subunits of respiratory chain complex I and corresponding lack of complex I activity. This loss in a parasite already lacking genes for subunits of complexes III and IV suggests an apparent requirement for its mitochondrial ATP synthase to work in reverse to maintain membrane potential. In contrast, V. spadyakhi retains a functional complex I that allows ATP synthase to work in its standard direction.

INSTRUMENT(S): Orbitrap Exploris 240

ORGANISM(S): Vickermania

SUBMITTER: Peter Barath

LAB HEAD: Peter Barath

PROVIDER: PXD058243 | Pride | 2025-03-29

REPOSITORIES: Pride

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			Action	DRS
	20241112_Sample_A_01.raw	Raw
	20241112_Sample_B_01.raw	Raw
	20241112_Sample_C_01.raw	Raw
	20241112_Sample_D_01.raw	Raw
	20241112_Sample_E_01.raw	Raw

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Characterization of two novel proteins involved in mitochondrial DNA anchoring in Trypanosoma brucei

Project description:Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Kinetoplastea. The parasite harbors a single mitochondrion with a singular mitochondrial genome that is known as the kinetoplast DNA (kDNA). The kDNA consists of a unique network of thousands of interlocked circular DNA molecules. To ensure proper inheritance of the kDNA to the daughter cells, the genome is physically linked to the basal body, the master organizer of the cell cycle in trypanosomes. The connection that spans, cytoplasm, mitochondrial membranes and the mitochondrial matrix is mediated by the Tripartite Attachment Complex (TAC). Using a combination of proteomics and RNAi we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and TbKAP68 as novel candidates of a complex that connects the TAC to the kDNA. Depletion of TbmtHMG44 or TbKAP68 each leads to a strong kDNA loss but not missegregation phenotype as previously defined for TAC components. We demonstrate that the proteins rely on both the TAC and the kDNA for stable localization to the interface between these two structures. In vitro experiments suggest a direct interaction between TbmtHMG44 and TbKAP68 and that recombinant TbKAP68 is a DNA binding protein. We thus propose that TbmtHMG44 and TbKAP68 are part of a distinct complex connecting the kDNA to the TAC.

2023-06-23 | PXD041551 | Pride

Characterization of the Novel Mitochondrial Genome Segregation Factor TAP110 in Trypanosoma brucei

Project description:Mitochondrial genome inheritance has been extensively studied in only a handful of model organisms, all belonging to the Opisthokonta eukaryotic supergroup. To understand mitochondrial evolution in more detail, it is important to include organisms of other supergroups such as Excavata (e.g. T. brucei) in our studies. To assure correct inheritance of the replicated kDNA to the two daughter cells in T. brucei , it is anchored to the flagellum that drives kDNA segregation during cell division. The structure connecting the kDNA to the basal body has been described as the tripartite attachment complex (TAC). Several components of that TAC structure and how they assemble has been described in the last few years. It remains elusive how the TAC, inside the mitochondrion, can connect to the kDNA. Here, we present data on the TAC associated protein 110 (TAP110). TAP110 is a 110 kDa protein that shows sequence similarities to a histone linker protein. With super-resolution microscopy, we show that TAP110 co-localizes with TAC102, a TAC component of the unilateral filaments in the mitochondrion. In contrast to other kDNA segregation factors characterized previously, TAP110 remains only partially associated with the flagellum after DNaseI and detergent treatment. Overexpression of TAP110 leads to a delay in the separation of the replicated kDNA networks and thus an increase in cells with replicated but non-segregated kDNA. The depletion of TAC102 leads to loss of TAP110, suggesting that TAP110 is more proximal to the kDNA than TAC102. Furthermore, we demonstrate that the TAC, but not the kDNA, is required for correct TAP110 localization. Blue native suggests that TAP110 might interact with other proteins to form a >669 kDa complex. Interestingly, TAP110 can only be solubilized in dyskinetoplastic cells suggesting a direct interaction with the kDNA.

2021-03-02 | PXD019665 | Pride

KRBP72 facilitates ATPase-dependent editing progression through a structural roadblock in mitochondrial A6 mRNA

Project description:Uridine insertion/deletion editing of mitochondrial mRNAs in kinetoplastids entails the coordinated action of three complexes. RNA Editing Catalytic Complexes (RECCs) catalyze the enzymatic reactions, while the RNA Editing Substrate Binding Complex (RESC) and RNA Editing Helicase 1 Complex coordinate interactions between RECCs, mRNAs, and hundreds of gRNAs that direct edited sequences. Additionally, numerous auxiliary factors are required for productive editing of specific mRNAs. Here, we elucidate the role of KRBP72, an editing auxiliary factor of the ABC ATPase family that exhibits RNA binding activity. In procyclic form T. brucei, KRBP72 knockdown leads to a pause in editing at the base of a predicted stem loop structure in ATP synthase subunit 6 (A6) mRNA. Enhanced cross-linking and affinity purification revealed KRBP72 binding sites both within and upstream of this stem loop. KRBP72 ATPase activity is essential for its A6 mRNA editing function; however, its RNA binding activity is dispensable. KRBP72 interacts with most RESC proteins in an RNA-dependent manner. By contrast, RESC12A associates with KRBP72 in an RNA-independent fashion, and RESC12A strongly promotes KRBP72’s interaction with RNA. Hence, KRBP72 ATPase activity facilitates the progression of editing through a challenging RNA secondary structure, highlighting this protein’s crucial role in A6 mRNA editing.

2025-01-29 | GSE272305 | GEO

Pentatricopeptide repeat poly(A) binding protein from mitochondria of trypanosomes

Project description:In Trypanosoma brucei, most mitochondrial mRNAs undergo U-insertion/deletion editing, and 3′ adenylation and uridylation. The internal sequence changes and terminal extensions are coordinated: Pre-editing addition of the short (A) tail protects the edited transcript against 3′-5′ degradation, while post-editing A/U-tailing renders mRNA competent for ribosome recruitment. Participation of a poly(A) binding protein (PABP) in coupling of editing and 3′ modification processes has been inferred, but its identity and mechanism of action remained elusive. We report identification of KPAF4, a pentatricopeptide repeat-containing PABP which sequesters the A-tail and impedes exonucleolytic degradation. Conversely, KPAF4 inhibits uridylation of A-tailed transcripts and, therefore, premature A/U-tailing of partially-edited mRNAs. This quality check point prevents translation of incompletely edited mRNAs. Our findings also implicate the RNA editing substrate binding complex (RESC) in mediating the interaction between the 5′-end bound pyrophosphohydrolase MERS1 and 3′-end associated KPAF4 to enable mRNA circularization. This event is critical for transcript stability during the editing process.

2019-04-02 | PXD012008 | Pride

A-T to G-C base editing efficiency at targeted gene sites in HEK293T cell line

Project description:A-T to G-C base editing efficiency at targeted gene sites in HEK293T cells using the dCas12f-ABE design or the Cas12f-ABE design. Found that the total A-T to G-C conversion efficiency of Circular gRNAs exhibited about two-fold increase compared with U6 gRNAs. We further analyzed the pattern for A-T to G-C conversion on the target site, and observed that the most efficient base editing occurred in a narrow window A3 (3bp downstream of the PAM) similar to U6 gRNAs. In summary, Circular gRNAs with dCas12f-ABE design could enhance A-T to G-C base editing efficiency in a narrow window.

2024-03-11 | GSE260967 | GEO

A post-transcriptional respiratome regulon in trypanosomes

Project description:Post-transcriptional regulons coordinate the expression of groups of genes in eukaryotic cells, yet relatively few have been characterised. Parasitic trypanosomatids are particularly good models for studies on such mechanisms because they exhibit almost exclusive polycistronic, and unregulated, transcription. Here, we identify the Trypanosoma brucei ZC3H39/40 RNA-binding proteins as regulators of the respiratome; the mitochondrial electron transport chain (complexes I-IV) and the FoF1-ATP synthase (complex V). A high-throughput RNAi screen initially implicated both ZC3H proteins in variant surface glycoprotein (VSG) gene silencing. This link was confirmed and both proteins were shown to form a cytoplasmic ZC3H39/40 complex. Transcriptome and mRNA-interactome analyses indicated that the impact on VSG silencing was indirect, while the ZC3H39/40 complex specifically bound and stabilised transcripts encoding respiratome-complexes. Quantitative proteomic analyses revealed specific positive control of respiratome protein abundance. Our findings establish a link between the mitochondrial respiratome and VSG gene silencing. They also reveal a major respiratome regulon controlled by the conserved trypanosomatid ZC3H39/40 RNA-binding proteins.

2019-11-13 | PXD012450 | Pride

Pooled CRISPR screening with single-cell transcriptome read-out

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ILF2 and ILF3 shield transcripts from RNA editing to enable human cell fate transitions

Project description:Chromatin regulation and RNA processing are both fundamental for establishing cell identity, yet how they interact to direct cell fate remains unclear. To uncover factors that coordinate this interplay, we screened dual DNA- and RNA-binding proteins (DRBPs) and identified ILF2 and ILF3 as critical regulators of human cell fate. We show that ILF2 and ILF3 control human, but not mouse, gastrulation and maintain the self-renewal of adult progenitor cells across all three germ layers. Mechanistically, ILF2 and ILF3 interact with and inhibit the RNA-editing enzyme ADAR, which catalyzes adenosine-to-inosine conversion in primate-specific repetitive elements. Acute degradation of the ILF2-ILF3 complex triggers widespread RNA misediting and missplicing of transcripts encoding key cell fate regulators. These misedited transcripts are degraded, destabilizing the proteome and rewiring the epigenetic landscape of stem and progenitor cells. Our findings reveal a human-specific mechanism linking RNA processing to chromatin regulation and identify an evolutionary safeguard that prevents aberrant RNA editing and retrotransposon activation, enabling human cell fate transitions.

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Project description:In E. coli, editing efficiency (EE) with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA DNA double-strand break (DSB)-induced cell death. We found that EE with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, non-targeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise EE. These effects on EE were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs and differences in rates of dissociation of Cas9:gRNA complexes from target sites account for the observed variations in EE between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.

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