RNAit: an automated web-based tool for the selection of RNAi targets in Trypanosoma brucei

Redmond, Seth; Vadivelu, Jamuna; Field, Mark C.

doi:10.1016/s0166-6851(03)00045-8

Cited by 229 publications

(201 citation statements)

References 9 publications

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“…Selection of the gene sequences for RNAi was done with RNAit, a prediction algorithm designed to prevent potential cross-talk and hence off-target effects (34). Cloning of the gene fragments into the tetracycline-inducible vector, pALC14 (a kind gift of André Schneider, University of Bern), was performed as described previously (35), using two separate PCR products obtained with primers Tb3020-F (5Ј-GCCCAAGCTTGGATCCCGCTG-CAATCAACACACTCT-3Ј) and Tb3020-R (5Ј-GCTCT-AGACTCGAGTGGGAACACCTGTGAAACAA-3Ј) (spanning nucleotides 842-1181 of Tb11.02.3020), resulting in plasmid pAG3020.…”

Section: Methodsmentioning

confidence: 99%

myo-Inositol Uptake Is Essential for Bulk Inositol Phospholipid but Not Glycosylphosphatidylinositol Synthesis in Trypanosoma brucei

González-Salgado

Steinmann

Greganova

et al. 2012

Journal of Biological Chemistry

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Background: Intracellular myo-inositol homeostasis involves both de novo synthesis and uptake of myo-inositol from the environment. Results: Down-regulation of the myo-inositol transporter in Trypanosoma brucei causes depletion of bulk inositol lipids, but not glycosylphosphatidylinositols, and leads to parasite death. Conclusion: De novo synthesis of myo-inositol is not sufficient to ensure bulk inositol lipid production. Significance: myo-Inositol metabolism in T. brucei is compartmentalized.

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Section: Methodsmentioning

confidence: 99%

myo-Inositol Uptake Is Essential for Bulk Inositol Phospholipid but Not Glycosylphosphatidylinositol Synthesis in Trypanosoma brucei

González-Salgado

Steinmann

Greganova

et al. 2012

Journal of Biological Chemistry

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“…In situ C-terminal tagging of MRB8620 and MRB3010 was facilitated by previously described vectors . To generate RNAi knockdowns, PCR primers were designed using the RNAit tool (Redmond et al 2003) for amplifying an appropriate region of the MRB8620 ORF from T. brucei genomic DNA, for cloning into the p2T7-177 vector (Wickstead et al 2002) allowing inducible RNAi. The linearized constructs were electroporated into the PS 29-13 strain by an established protocol .…”

Section: Generation Of Cell Lines and Their Manipulationmentioning

confidence: 99%

Integrity of the core mitochondrial RNA-binding complex 1 is vital for trypanosome RNA editing

Huang

Faktorová

Křížová

et al. 2015

RNA

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Trypanosoma brucei is the causative agent of the human and veterinarian diseases African sleeping sickness and nagana. A majority of its mitochondrial-encoded transcripts undergo RNA editing, an essential process of post-transcriptional uridine insertion and deletion to produce translatable mRNA. Besides the well-characterized RNA editing core complex, the mitochondrial RNA-binding 1 (MRB1) complex is one of the key players. It comprises a core complex of about six proteins, guide RNA-associated proteins (GAPs) 1/2, which form a heterotetramer that binds and stabilizes gRNAs, plus MRB5390, MRB3010, and MRB11870, which play roles in initial stages of RNA editing, presumably guided by the first gRNA:mRNA duplex in the case of the latter two proteins. To better understand all functions of the MRB1 complex, we performed a functional analysis of the MRB8620 core subunit, the only one not characterized so far. Here we show that MRB8620 plays a role in RNA editing in both procyclic and bloodstream stages of T. brucei, which reside in the tsetse fly vector and mammalian circulatory system, respectively. While RNAi silencing of MRB8620 does not affect procyclic T. brucei fitness when grown in glucose-containing media, it is somewhat compromised in cells grown in the absence of this carbon source. MRB8620 is crucial for integrity of the MRB1 core, such as its association with GAP1/2, which presumably acts to deliver gRNAs to this complex. In contrast, GAP1/2 is not required for the fabrication of the MRB1 core. Disruption of the MRB1 core assembly is followed by the accumulation of mRNAs associated with GAP1/2.

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“…The 1674 bp gene was identified with a temporary systematic ID of Tb10.70.3220 as of February 26, 2005. The T. brucei SPT2 735 bp RNAi fragment was verified for specificity by the RNA-it program (Redmond et al, 2003), generated from 29-13 strain genomic DNA by polymerase chain reaction amplification using primers 5Ј-AAGGCAGTAACTGAAGCCGA-3Ј and 5Ј-CAAGCTATCGCTGACCACAA-3Ј, and ligated into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA). The RNAi fragment was directionally subcloned using XhoI and HindIII into the RNAi vector pZJM (Wang et al, 2000), which supports expression of double-stranded (ds) RNA from Tet-inducible T7 promoters.…”

Section: Dna Constructsmentioning

confidence: 99%

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

Fridberg

Olson

Nakayasu

et al. 2008

Journal of Cell Science

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Sphingolipids and their metabolites have been thought crucial for cell growth and cell cycle progression, membrane and protein trafficking, signal transduction, and formation of lipid rafts; however, recent studies in trypanosomes point to the dispensability of sphingolipids in some of these processes. In this study, we explore the requirements for de novo sphingolipid biosynthesis in the insect life cycle stage of the African trypanosome Trypanosoma brucei by inhibiting the enzyme serine palmitoyltransferase (SPT2) by using RNA interference or treatment with a potent SPT2 inhibitor myriocin. Mass spectrometry revealed that upon SPT2 inhibition, the parasites contained substantially reduced levels of inositolphosphorylceramide. Although phosphatidylcholine and cholesterol levels were increased to compensate for this loss, the cells were ultimately not viable. The most striking result of sphingolipid reduction in procyclic T. brucei was aberrant cytokinesis, characterized by incomplete cleavage-furrow formation, delayed kinetoplast segregation and emergence of cells with abnormal DNA content. Organelle replication continued despite sphingolipid depletion, indicating that sphingolipids act as second messengers regulating cellular proliferation and completion of cytokinesis. Distention of the mitochondrial membrane, formation of multilamellar structures within the mitochondrion and near the nucleus, accumulation of lipid bodies and, less commonly, disruption of the Golgi complex were observed after prolonged sphingolipid depletion. These findings suggest that some aspects of vesicular trafficking may be compromised. However, flagellar membrane targeting and the association of the flagellar membrane protein calflagin with detergent-resistant membranes were not affected, indicating that the vesicular trafficking defects were mild. Our studies indicate that sphingolipid biosynthesis is vital for cell cycle progression and cell survival, but not essential for the normal trafficking of flagellar membrane-associated proteins or lipid raft formation in procyclic T. brucei.

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RNAit: an automated web-based tool for the selection of RNAi targets in Trypanosoma brucei

Cited by 229 publications

References 9 publications

myo-Inositol Uptake Is Essential for Bulk Inositol Phospholipid but Not Glycosylphosphatidylinositol Synthesis in Trypanosoma brucei

myo-Inositol Uptake Is Essential for Bulk Inositol Phospholipid but Not Glycosylphosphatidylinositol Synthesis in Trypanosoma brucei

Integrity of the core mitochondrial RNA-binding complex 1 is vital for trypanosome RNA editing

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

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