Mitochondrial Development during Life Cycle Differentiation of African Trypanosomes: Evidence for a Kinetoplast-dependent Differentiation Control Point

Timms, Mark; Deursen, Frederick J. van; Hendriks, Edward F.; Matthews, Keith R.

doi:10.1091/mbc.e02-05-0266

Cited by 39 publications

(43 citation statements)

References 44 publications

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“…Among seven genes associated with a nuclear defect (0N Ͼ 4.5%) were histone H3 (TFN1.218), cyclin 6 (TFN0.8), a (conserved hypothetical) protein with homology to the PRP38 pre-mRNA splicing factor (TFN1.212), and a probable arginine N-methyltransferase (TFN1.25). Topoisomerase II (TFN0.13) knockdown, as expected (35), and two additional conserved hypothetical proteins (TFN1.55 and TFN1.161) generated a kinetoplast defect (0K Ͼ 4.5%). BLAST analysis of the four conserved hypothetical genes associated with a block in cytokinesis (2N Ͼ 12%) revealed similarity to a histone methyltransferase (TFN1.…”

Section: Vol 5 2006 Systematic Analysis Of Gene Function In Trypanosupporting

confidence: 54%

Chromosome-Wide Analysis of Gene Function by RNA Interference in the African Trypanosome

et al. 2006

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Trypanosomatids of the order Kinetoplastida are major contributors to global disease and morbidity, and understanding their basic biology coupled with the development of new drug targets represents a critical need. Additionally, trypanosomes are among the more accessible divergent eukaryote experimental systems. The genome of Trypanosoma brucei contains 8,131 predicted open reading frames (ORFs), of which over half have no known homologues beyond the Kinetoplastida and a substantial number of others are poorly defined by in silico analysis. Thus, a major challenge following completion of the T. brucei genome sequence is to obtain functional data for all trypanosome ORFs. As T. brucei is more experimentally tractable than the related Trypanosoma cruzi and Leishmania spp. and shares >75% of their genes, functional analysis of T. brucei has the potential to inform a range of parasite biology. Here, we report methods for systematic mRNA ablation by RNA interference (RNAi) and for phenotypic analysis, together with online data dissemination. This represents the first systematic analysis of gene function in a parasitic organism. In total, 210 genes have been targeted in the bloodstream form parasite, representing an essentially complete phenotypic catalogue of chromosome I together with a validation set. Over 30% of the chromosome I genes generated a phenotype when targeted by RNAi; most commonly, this affected cell growth, viability, and/or cell cycle progression. RNAi against approximately 12% of ORFs was lethal, and an additional 11% had growth defects but retained short-term viability in culture. Although we found no evidence for clustering or a bias towards widely evolutionarily conserved genes within the essential ORF cohort, the putative chromosome I centromere is adjacent to a domain containing genes with no associated phenotype. Involvement of such a large proportion of genes in robust growth in vitro indicates that a high proportion of the expressed trypanosome genome is required for efficient propagation; many of these gene products represent potential drug targets.Protozoan parasites are responsible for a significant proportion of global morbidity, mortality, and economic hardship, and in most countries current control and treatment regimes are either failing or under serious threat (6). African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for tens to hundreds of thousands of human infections annually in areas of sub-Saharan Africa where the infection is endemic and is compounded by the impact of the disease on animal welfare and productivity. Without treatment trypanosomiasis is invariably fatal, and the need for new treatments is urgent. Mechanisms for combating pathogenic protozoa rely on development and exploitation of new drug targets and/or vaccine candidates as well as efforts at vector control, all of which require detailed understanding of the biology of the pathogen and its relationship with the host and vector. The presence of efficient antigenic variation in T. brucei make...

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Section: Vol 5 2006 Systematic Analysis Of Gene Function In Trypanosupporting

confidence: 54%

Chromosome-Wide Analysis of Gene Function by RNA Interference in the African Trypanosome

et al. 2006

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“…Finally, these analogs caused transformation to stumpy-like forms incapable of proliferation but not to fully committed procyclic forms, which appear to require a kinetoplastdependent event (35) for differentiation. Given their potency, it should, therefore, be possible to develop drugs based on these types of analogs as antitrypanosomal agents.…”

Section: Discussionmentioning

confidence: 99%

“…5B). After 48 h of treatment with 8-pCPT-2Ј-OMe-5Ј-AMP, NADH diaphorase activity, a marker of intermediate or stumpy forms (13,35,36), was detected (observed by the appearance of dark precipitates in the trypanosome body) (Fig. 5C).…”

Section: Brucei By 8-pcpt-2ј-o-me-5ј-amp (Squares) or 8-pcpt-2ј-o-me-mentioning

confidence: 99%

Hydrolysis products of cAMP analogs cause transformation ofTrypanosoma bruceifrom slender to stumpy-like forms

Laxman¹,

Riechers²,

Sadı́lek

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

110

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African sleeping sickness is a disease caused by Trypanosoma brucei. T. brucei proliferate rapidly in the mammalian bloodstream as long, slender forms, but at higher population densities they transform into nondividing, short, stumpy forms. This is thought to be a mechanism adopted by T. brucei to establish a stable hostparasite relationship and to allow a transition into the insect stage of its life cycle. Earlier studies have suggested a role for cAMP in mediating this transformation. In this study, using membranepermeable nucleotide analogs, we show that it is not the cAMP analogs themselves but rather the hydrolyzed products of membrane-permeable cAMP analogs that prevent proliferation of T. brucei. The metabolic products are more potent than the cAMP analogs, and hydrolysis-resistant cAMP analogs are not antiproliferative. We further show that the antiproliferative effect of these membrane-permeable adenosine analogs is caused by transformation into forms resembling short, stumpy bloodstream forms. These data suggest that the slender-to-stumpy transformation of T. brucei may not be mediated directly by cAMP and also raise the possibility of using such adenosine analogs as antitrypanosomal drugs.adenosine ͉ EPACs ͉ phosphodiesterases ͉ trypanosomes

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“…Indeed, BF cytochrome c oxidase subunit 6 null mutants are viable (60). One possibility is that BFs require the COII protein either for complex IV function or for an unknown function.…”

Section: Discussionmentioning

confidence: 99%

RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

Carnes

Trotter

Peltan

et al. 2008

Molecular and Cellular Biology

103

150

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Trypanosoma brucei has three distinct ϳ20S editosomes that catalyze RNA editing by the insertion and deletion of uridylates. Editosomes with the KREN1 or KREN2 RNase III type endonucleases specifically cleave deletion and insertion editing site substrates, respectively. We report here that editosomes with KREPB2, which also has an RNase III motif, specifically cleave cytochrome oxidase II (COII) pre-mRNA insertion editing site substrates in vitro. Conditional repression and mutation studies also show that KREPB2 is an editing endonuclease specifically required for COII mRNA editing in vivo. Furthermore, KREPB2 expression is essential for the growth and survival of bloodstream forms. Thus, editing in T. brucei requires at least three compositionally and functionally distinct ϳ20S editosomes, two of which distinguish between different insertion editing sites. This unexpected finding reveals an additional level of complexity in the RNA editing process and suggests a mechanism for how the selection of sites for editing in vivo is controlled.RNA editing recodes most of the mitochondrial mRNAs in trypanosomatids by the insertion and deletion of uridine nucleotides (U's) using information provided by guide RNAs (gRNAs) (58). RNA editing is developmentally regulated and is required for parasites that cycle between insect vector and mammalian host (54). Proteins that perform catalytic steps of RNA editing have been identified: endonucleases KREN1 or KREN2 cleave at deletion or insertion sites, respectively; terminal uridylyl transferase (TUTase) KRET2 adds U's at insertion sites; U specific exoribonuclease (exoUase) KREX1 removes U's at deletion sites; and ligases KREL1 or KREL2 rejoin mRNA fragments after U addition or removal (5,13,25,30,34,55,61). These catalytic steps are coordinated by the multiprotein editosomes that sediment at ϳ20S on glycerol gradients (9, 43). KREPB2 was identified as an editosome component by mass spectrometry, and found to contain an RNase III motif harboring amino acids that are critical for catalysis, a U1 Zn 2ϩ finger, and double-stranded RNA binding. It has sequence similarity to KREN1 and KREN2, which also contain these three motifs (23,41,64). These data strongly suggest an RNA editing endonuclease role for KREPB2.KREN1 and KREN2 were shown to be RNA editing endonucleases using both RNA interference (RNAi) and conditional knockout cell lines (5, 61). Both KREN1 and KREN2 are essential for the normal growth of PF and BF cells, and repression of their expression by either method led to dramatic growth defects or death, respectively. Such repression of KREN1 eliminated in vitro cleavage by ϳ20S editosomes of a deletion site in a synthetic substrate modeled on ATPase subunit 6 (A6) pre-mRNA but did not alter cleavage of an insertion site in a substrate derived from the same mRNA. These studies and other data have shown that KREN1 is an editing endonuclease with a preference for sites from which U's are deleted (29). Similarly, repression of KREN2 eliminated in vitro cleavage at insertion ed...

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Mitochondrial Development during Life Cycle Differentiation of African Trypanosomes: Evidence for a Kinetoplast-dependent Differentiation Control Point

Cited by 39 publications

References 44 publications

Chromosome-Wide Analysis of Gene Function by RNA Interference in the African Trypanosome

Chromosome-Wide Analysis of Gene Function by RNA Interference in the African Trypanosome

Hydrolysis products of cAMP analogs cause transformation ofTrypanosoma bruceifrom slender to stumpy-like forms

RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

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