Genome-scale RNAi screens for high-throughput phenotyping in bloodstream-form African trypanosomes

Glover, Lucy; Alsford, Sam; Baker, Nicola; Turner, Daniel J.; Sánchez-Flores, Alejandro; Hutchinson, Sebastian; Hertz‐Fowler, Christiane; Berriman, Matthew; Horn, David

doi:10.1038/nprot.2015.005

Cited by 52 publications

(84 citation statements)

References 48 publications

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“…The number of different protein variants that can be assayed in a single screen is limited only by the allele library size and the transfection efficiency of the mutant allele library (53). Strategies to increase transfection efficiency (54) would allow the generation of larger mutant libraries that could also be screened in an alternative highthroughput fashion, using deep sequencing, analogous to that used to screen previously described genome-wide RNA interference libraries in T. brucei (55,56). The methodology can also be adapted to probe more specific questions.…”

Section: Discussionmentioning

confidence: 99%

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

McDermott

Carnes

Stuart

2015

Molecular and Cellular Biology

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KREPB5 is an essential component of ϳ20S editosomes in Trypanosoma brucei which 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 null T. brucei bloodstream 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 ϳ20S 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 of T. brucei that differentially edit mRNAs.T he kinetoplastid parasite Trypanosoma brucei is the etiologic agent of human African trypanosomiasis, which is transmitted by the tsetse fly and is a health threat to millions of people in sub-Saharan Africa. T. brucei is a deeply divergent eukaryote, the study of which advanced the understanding of many fundamental biological processes and eukaryotic evolution. Indeed, a number of these processes, such as trans splicing, polycistronic transcription, antigenic variation, glycosylphosphatidylinositol anchoring, and mitochondrial RNA editing, were first described in trypanosomes and provided novel paradigms for eukaryotic biology (1-11). Kinetoplastids are named for their distinctive mitochondrial DNA network, known as the kinetoplast DNA (kDNA), within their single mitochondrion. The kDNA in T. brucei is comprised of ϳ50 identical maxicircles and thousands of heterogeneous minicircles (12, 13). The ϳ22-kb maxicircles encode two rRNAs and mRNAs for 18 mitochondrial proteins, 12 of which undergo posttranscriptional RNA editing to generate translatable open reading frames (ORFs). RNA editing involves the precise insertion and deletion of uridylylates (Us) at hundreds and tens of editing sites (ESs), respectively. The minicircles encode numerous diverse ϳ60-nucleotide guide RNAs (gRNAs) which specify edited sequences (14-16). Editing progresses generally, but not precisely, 3= to 5= with respect to the mRNA, each gRNA specifies the editing of numerous ESs, and multiple gRNAs are required for complete editing of most transcripts. T. brucei undergoes stage-specific adaptations in the bloodstream of the mammalian host and in the tsetse fly, particularly in mitochondrial function (17,18), and a number of maxicircle transcripts are ...

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Section: Discussionmentioning

confidence: 99%

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

McDermott

Carnes

Stuart

2015

Molecular and Cellular Biology

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“…Determinants of ISM resistance were identified using an RNAi library screen as previously described (15). Briefly, the RNAi screen was carried out with 4.5 nM ISM.…”

Section: Methodsmentioning

confidence: 99%

“…Reads were mapped to the T. brucei 927 reference genome (v6, tritrypdb.org) with Bowtie 2 (54) using the following parameters: very-sensitive-local-phred33. Alignment files were manipulated with SAMtools (55) and a custom script (15), and data were further assessed using the Artemis genome browser (56).…”

Section: Methodsmentioning

confidence: 99%

Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes

Baker

Hamilton

Wilkes

et al. 2015

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

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Kinetoplastid parasites cause lethal diseases in humans and animals. The kinetoplast itself contains the mitochondrial genome, comprising a huge, complex DNA network that is also an important drug target. Isometamidium, for example, is a key veterinary drug that accumulates in the kinetoplast in African trypanosomes. Kinetoplast independence and isometamidium resistance are observed where certain mutations in the F 1 -γ-subunit of the two-sector F 1 F o -ATP synthase allow for F o -independent generation of a mitochondrial membrane potential. To further explore kinetoplast biology and drug resistance, we screened a genome-scale RNA interference library in African trypanosomes for isometamidium resistance mechanisms. Our screen identified 14 V-ATPase subunits and all 4 adaptin-3 subunits, implicating acidic compartment defects in resistance; V-ATPase acidifies lysosomes and related organelles, whereas adaptin-3 is responsible for trafficking among these organelles. Independent strains with depleted V-ATPase or adaptin-3 subunits were isometamidium resistant, and chemical inhibition of the V-ATPase phenocopied this effect. While drug accumulation in the kinetoplast continued after V-ATPase subunit depletion, acriflavine-induced kinetoplast loss was specifically tolerated in these cells and in cells depleted for adaptin-3 or endoplasmic reticulum membrane complex subunits, also identified in our screen. Consistent with kinetoplast dispensability, V-ATPase defective cells were oligomycin resistant, suggesting ATP synthase uncoupling and bypass of the normal F o -A6-subunit requirement; this subunit is the only kinetoplast-encoded product ultimately required for viability in bloodstream-form trypanosomes. Thus, we describe 30 genes and 3 protein complexes associated with kinetoplast-dependent growth. Mutations affecting these genes could explain natural cases of dyskinetoplasty and multidrug resistance. Our results also reveal potentially conserved communication between the compartmentalized two-sector rotary ATPases.brucei | mitochondrion | nagana | petite | samorin

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“…Whole-genome RNAi libraries are now allowing the development of functional screens to identify essential components involved in basic biochemical processes in African trypanosomes [18]. A whole-genome RNAi screen designed to select for derepression of a silent T. brucei telomere has allowed the identification of the first ESB-specific marker, a protein called VEX1 [19].…”

Section: How Is Vsg Expression Controlled In a Monoallelic Fashion?mentioning

confidence: 99%

How to create coats for all seasons: elucidating antigenic variation in African trypanosomes

Ooi

Rudenko

2017

Emerging Topics in Life Sciences

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Extracellular parasites of the mammalian bloodstream face considerable challenges including incessant assault by the immune system. African trypanosomes are consummate survivors in this inclement environment and are renowned for their supremely sophisticated strategy of antigenic variation of their protective surface coat during the course of chronic infections. Recent developments are making us realize how complex this antigenic machinery is and are allowing us to tackle previously intractable problems. However, many of the simplest (and arguably the most important) questions still remain unanswered! How is the extraordinary heterogeneity of variant surface glycoprotein variants during a chronic infection generated?The bloodstream form of Trypanosoma brucei is coated with a dense covering of ∼10 7 variant surface glycoprotein (VSG) molecules, which extend as antigenically variable rods connected to the cell surface via glycosylphosphatidylinositol (GPI) anchors [1]. This GPI attachment allows VSGs to move freely over the cell surface [2]. Tight packing of these VSGs produces a protective barrier shielding invariant surface proteins from recognition by host antibodies and preventing trypanosome lysis by the alternative pathway of the complement system [3]. Extraordinarily high rates of recycling of surface VSG allow selective removal of host cell molecules including antibodies and complement from the trypanosome surface, thereby functioning as a 'coat-cleaning ' machine [4]. This prolongs trypanosome survival in rising antibody titres and facilitates escape from macrophages [5].Each trypanosome has a vast repertoire of VSG genes [6], differing in number and content between different strains, presumably as a consequence of diversifying forces. For example, the 'VSGnome' of T. brucei 427 contains more than 2500 different VSG genes, of which more than 80% are not immediately functional [7]. Switching the active VSG can entail transcriptional switches between the ∼15 telomeric VSG expression site (ES) transcription units (Figure 1). More importantly, DNA rearrangements and predominantly gene conversions can copy new VSGs (or segments of VSGs) into the active ES, thereby resulting in an antigenic switch [8] (Figure 1). This extraordinary ability to construct novel patchwork, or mosaic, VSGs is key for trypanosome survival [9]. The continuous recombination of various permutations and combinations of sections of VSG genes (or pseudogenes) allows the trypanosome to construct an almost infinite number of antigenically distinct VSG coat types.A given wave of trypanosome parasitaemia is normally predominantly composed of a major VSG variant, although it has long been known that minor VSG variants are also present within each wave. However, there is possibly phenomenal heterogeneity within these infection waves, as next-generation RNA sequencing of RNA transcripts has documented an extraordinary variety of minor VSG variants [10]. These experiments argue that a considerable percentage of the available VSG repertoire is disp...

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Genome-scale RNAi screens for high-throughput phenotyping in bloodstream-form African trypanosomes

Cited by 52 publications

References 48 publications

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

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

Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes

How to create coats for all seasons: elucidating antigenic variation in African trypanosomes

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