scholarly journals Editosome RNase III domain interactions are essential for editing and differ between life cycle stages in Trypanosoma brucei

RNA ◽  
2019 ◽  
Vol 25 (9) ◽  
pp. 1150-1163 ◽  
Author(s):  
Suzanne M. McDermott ◽  
Jason Carnes ◽  
Kenneth Stuart
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Rnase Iii ◽  
Life Cycle Stages ◽  
Domain Interactions

scholarly journals Identification by Random Mutagenesis of Functional Domains in KREPB5 That Differentially Affect RNA Editing between Life Cycle Stages of Trypanosoma brucei

2015 ◽  
Vol 35 (23) ◽  
pp. 3945-3961 ◽  
Author(s):  
Suzanne M. McDermott ◽  
Jason Carnes ◽  
Kenneth Stuart
Keyword(s):  
Life Cycle ◽  
Amino Acid ◽  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Bloodstream Form ◽  
Single Amino Acid ◽  
Wild Type ◽  
Rnase Iii ◽  
Content Type ◽  
Life Cycle Stages

KREPB5 is an essential component of ∼20S editosomes inTrypanosoma bruceiwhich contains a degenerate, noncatalytic RNase III domain. To explore the function of this protein, we used a novel approach to make and screen numerous conditional nullT. bruceibloodstream form cell lines that express randomly mutagenized KREPB5 alleles. We identified nine single amino acid substitutions that could not complement the conditional loss of wild-type KREPB5. Seven of these were within the RNase III domain, and two were in the C-terminal region that has no homology to known motifs. Exclusive expression of these mutated KREPB5 alleles in the absence of wild-type allele expression resulted in growth inhibition, the loss of ∼20S editosomes, and inhibition of RNA editing in BF cells. Eight of these mutations were lethal in bloodstream form parasites but not in procyclic-form parasites, showing that multiple domains function in a life cycle-dependent manner. Amino acid changes at a substantial number of positions, including up to 7 per allele, allowed complementation and thus did not block KREPB5 function. Hence, the degenerate RNase III domain and a newly identified domain are critical for KREPB5 function and have differential effects between the life cycle stages ofT. bruceithat differentially edit mRNAs.

Diverse patterns of expression of the cytochrome c oxidase subunit I gene and unassigned reading frames 4 and 5 during the life cycle of Trypanosoma brucei

1985 ◽  
Vol 5 (11) ◽  
pp. 3041-3047
Author(s):  
D P Jasmer ◽  
J E Feagin ◽  
K Stuart
Keyword(s):  
Life Cycle ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Trypanosoma Brucei ◽  
Protein Coding ◽  
Oxidase Subunit ◽  
Life Cycle Stages ◽  
Multiple Processes ◽  
Mitochondrial Transcripts ◽  
Reading Frames

Transcription of a maxicircle segment from Trypanosoma brucei 164 that contains nucleotide (nt) sequences corresponding to cytochrome c oxidase subunit I (COI) and unassigned reading frames (URFs) 4 and 5 of other mitochondrial systems was investigated. Two major transcripts that differ in size by ca. 200 nt map to each of the COI and URF4 genes, while a single major transcript maps to URF5. In total RNA, the larger COI transcript is more abundant in procyclic forms (PFs) than in bloodstream forms (BFs), the smaller COI and both URF4 transcripts have similar abundances in both forms, and the single URF5 transcript is more abundant in BF than PF. These patterns of expression differ in poly(A)+ RNA as a result of a higher proportion of poly(A)+ mitochondrial transcripts in PFs than in BFs. In addition, small (300- to 500-nt) RNAs that are transcribed from C-rich sequences located between putative protein-coding genes also exhibit diverse patterns of expression between life cycle stages and differences in polyadenylation in PFs compared with BFs. These observations suggest that multiple processes regulate the differential expression of mitochondrial genes in T. brucei.

scholarly journals Intraflagellar transport during the assembly of flagella of different length in Trypanosoma brucei isolated from tsetse flies

2020 ◽  
Author(s):  
Eloïse Bertiaux ◽  
Adeline Mallet ◽  
Brice Rotureau ◽  
Philippe Bastin
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Live Cell Imaging ◽  
Intraflagellar Transport ◽  
Tsetse Fly ◽  
Tsetse Flies ◽  
Life Cycle Stages ◽  
Summary Statement ◽  
Cilia And Flagella ◽  
Multicellular Organisms

AbstractMulticellular organisms assemble cilia and flagella of precise lengths differing from one cell to another, yet little is known about the mechanisms governing these differences. Similarly, protists assemble flagella of different lengths according to the stage of their life cycle. This is the case of Trypanosoma brucei that assembles flagella of 3 to 30 µm during its development in the tsetse fly. It provides an opportunity to examine how cells naturally modulate organelle length. Flagella are constructed by addition of new blocks at their distal end via intraflagellar transport (IFT). Immunofluorescence assays, 3-D electron microscopy and live cell imaging revealed that IFT was present in all life cycle stages. IFT proteins are concentrated at the base, IFT trains are located along doublets 3-4 & 7-8 and travel bidirectionally in the flagellum. Quantitative analysis demonstrated that the total amount of IFT proteins correlates with the length of the flagellum. Surprisingly, the shortest flagellum exhibited a supplementary large amount of dynamic IFT material at its distal end. The contribution of IFT and other factors to the regulation of flagellum length is discussed.Summary statementThis work investigated the assembly of flagella of different length during the development of Trypanosoma brucei in the tsetse fly, revealing a direct correlation between the amount of intraflagellar transport proteins and flagellum length.

scholarly journals Microtubule polyglutamylation is important for regulating cytoskeletal architecture and motility in Trypanosoma brucei

2020 ◽  
Vol 133 (18) ◽  
pp. jcs248047 ◽  
Author(s):  
Jana Jentzsch ◽  
Adal Sabri ◽  
Konstantin Speckner ◽  
Gertrud Lallinger-Kube ◽  
Matthias Weiss ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Dynamic Behaviour ◽  
Cell Movement ◽  
Post Translational Modifications ◽  
Life Cycle Stages ◽  
Associated Proteins ◽  
Flagellar Axoneme

ABSTRACTThe shape of kinetoplastids, such as Trypanosoma brucei, is precisely defined during the stages of the life cycle and governed by a stable subpellicular microtubule cytoskeleton. During the cell cycle and transitions between life cycle stages, this stability has to transiently give way to a dynamic behaviour to enable cell division and morphological rearrangements. How these opposing requirements of the cytoskeleton are regulated is poorly understood. Two possible levels of regulation are activities of cytoskeleton-associated proteins and microtubule post-translational modifications (PTMs). Here, we investigate the functions of two putative tubulin polyglutamylases in T. brucei, TTLL6A and TTLL12B. Depletion of both proteins leads to a reduction in tubulin polyglutamylation in situ and is associated with disintegration of the posterior cell pole, loss of the microtubule plus-end-binding protein EB1 and alterations of microtubule dynamics. We also observe a reduced polyglutamylation of the flagellar axoneme. Quantitative motility analysis reveals that the PTM imbalance correlates with a transition from directional to diffusive cell movement. These data show that microtubule polyglutamylation has an important role in regulating cytoskeletal architecture and motility in the parasite T. brucei.This article has an associated First Person interview with the first author of the paper.

scholarly journals Developmental regulation of edited CYb and COIII mitochondrial mRNAs is achieved by distinct mechanisms in Trypanosoma brucei

2020 ◽  
Vol 48 (15) ◽  
pp. 8704-8723
Author(s):  
Joseph T Smith Jr. ◽  
Eva Doleželová ◽  
Brianna Tylec ◽  
Jonathan E Bard ◽  
Runpu Chen ◽  
...  
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
High Throughput Sequencing ◽  
Environmental Changes ◽  
Developmental Regulation ◽  
Complex Life Cycle ◽  
Mrna Levels ◽  
Developmentally Regulated ◽  
Life Cycle Stages

Abstract Trypanosoma brucei is a parasitic protozoan that undergoes a complex life cycle involving insect and mammalian hosts that present dramatically different nutritional environments. Mitochondrial metabolism and gene expression are highly regulated to accommodate these environmental changes, including regulation of mRNAs that require extensive uridine insertion/deletion (U-indel) editing for their maturation. Here, we use high throughput sequencing and a method for promoting life cycle changes in vitro to assess the mechanisms and timing of developmentally regulated edited mRNA expression. We show that edited CYb mRNA is downregulated in mammalian bloodstream forms (BSF) at the level of editing initiation and/or edited mRNA stability. In contrast, edited COIII mRNAs are depleted in BSF by inhibition of editing progression. We identify cell line-specific differences in the mechanisms abrogating COIII mRNA editing, including the possible utilization of terminator gRNAs that preclude the 3′ to 5′ progression of editing. By examining the developmental timing of altered mitochondrial mRNA levels, we also reveal transcript-specific developmental checkpoints in epimastigote (EMF), metacyclic (MCF), and BSF. These studies represent the first analysis of the mechanisms governing edited mRNA levels during T. brucei development and the first to interrogate U-indel editing in EMF and MCF life cycle stages.

Developmental aspects of uridine addition within mitochondrial transcripts of Trypanosoma brucei

1988 ◽  
Vol 8 (3) ◽  
pp. 1259-1265
Author(s):  
J E Feagin ◽  
K Stuart
Keyword(s):  
Life Cycle ◽  
Cytochrome Oxidase ◽  
Trypanosoma Brucei ◽  
Cytochrome B ◽  
Respiratory System ◽  
Life Cycle Stage ◽  
Stage Specificity ◽  
Life Cycle Stages ◽  
Mitochondrial Transcripts ◽  
Cytochrome Oxidase Ii

The mitochondrial respiratory system is absent in slender bloodstream forms of Trypanosoma brucei, incomplete in stumpy bloodstream forms, and complete in procyclic (insect) forms. The steady-state abundance of transcripts of some mitochondrially encoded components of the respiratory system correlates with its differential expression in different life cycle stages. Recently, it was reported that uridines which are not encoded in the genome are added to cytochrome b and cytochrome oxidase II transcripts. We now report that the (U)+ transcripts of both genes are found in procyclic forms and to some degree in stumpy forms but are absent in slender forms. The uridine additions to cytochrome oxidase II correct a frameshift in the gene and presumably allow production of a full-length protein, whereas those added to cytochrome b create an in-frame AUG which extends the N terminus of the predicted protein by 20 amino acids. The stage specificity of uridine additions to these transcripts thus reflects the life cycle stage during which the protein products would be used. Transcripts of MURF2, a gene of unknown function, have additional uridines in both slender and procyclic forms which create two in-frame AUGs. MURF2 transcripts additionally differ from the DNA sequence in ways which cannot be explained by uridine addition alone, implying that other processes alter these transcripts.

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