An in vitro system from Plasmodium falciparum active in endogenous mRNA translation

Ferreras, Ana Celia; Triana, Ledia; Correia, Heriberto; Sanchez, Erlinda; Herrera, Flor

doi:10.1590/s0074-02762000000200017

Cited by 7 publications

(11 citation statements)

References 26 publications

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“…The effects of these compounds were examined in a cell-free system from P. falciparum that we developed very recently (Ferreras et al 2000). This system is able to translate endogenous mRNA giving about 25-fold stimulation over the activity at time zero which indicates that the parasite cell-free system contains polyribosomes, tRNA, aminoacyltRNA synthetases and factors that allow the system to complete the steps of elongation and termination of the protein synthesis mechanism.…”

Section: Resultsmentioning

confidence: 99%

“…The erythrocytes were isolated and the parasites released from them as described by Ferreras et al (2000).…”

Section: Methodsmentioning

confidence: 99%

“…Preparation of the parasite cell-free system -This was performed as described by Ferreras et al (2000).…”

Section: Methodsmentioning

confidence: 99%

“…Protein synthesis -Cell-free protein synthesis was carried out in a standard reaction mixture (Ferreras et al 2000) containing the following components in a volume of 50 µl: 20 mM Hepes/KOH pH 7.4; 100 mM KOAc; 5 mM Mg(OAc) 2 ; 2 mM DTT; 10% glycerol; 0.5 mM ATP; 0.1 mM GTP; 20 mM creatine phosphate; 10 µg of creatine phosphokinase; 40 µM (each) of 19 aminoacids (except methionine) and 1.7 µM L-[ 3 H]methionine (77x 10 3 cpm/ pmol), 140 µg of protein of S-30 fraction and variable amounts of artemisinin, chloroquine or primaquine as indicated in the legend to the Fig. 2.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we developed a P. falciparum cell-free system that contains polyribosomes, tRNA, aminoacyl-tRNA synthetases and factors that allow the system to complete the steps of elongation and termination of the protein synthesis mechanism (Ferreras et al 2000). Using this system we could determine that the translation of endogenous mRNA is not the target for artemisinin, primaquine and chloroquine which were highly effective inhibiting cultures of P. falciparum.…”

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confidence: 99%

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Effect of Antimalarial Drugs on Plasmodia Cell-Free Protein Synthesis

Ferreras

Triana

Sanchez

et al. 2002

Mem. Inst. Oswaldo Cruz

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Section: Resultsmentioning

confidence: 99%

“…The erythrocytes were isolated and the parasites released from them as described by Ferreras et al (2000).…”

Section: Methodsmentioning

confidence: 99%

“…Preparation of the parasite cell-free system -This was performed as described by Ferreras et al (2000).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Effect of Antimalarial Drugs on Plasmodia Cell-Free Protein Synthesis

Ferreras

Triana

Sanchez

et al. 2002

Mem. Inst. Oswaldo Cruz

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Polysome profiling of the malaria parasite Plasmodium falciparum

Lacsina

LaMonte

Nicchitta

et al. 2011

Molecular and Biochemical Parasitology

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In the malaria parasite Plasmodium falciparum, global studies of translational regulation have been hampered by the inability to isolate malaria polysomes. We describe here a novel method for polysome profiling in Plasmodium falciparum, a powerful approach which allows both a global view of translation and the measurement of ribosomal loading and density for specific mRNAs. Simultaneous lysis of infected erythrocytes and parasites releases stable, intact malaria polysomes, which are then purified by centrifugation through a sucrose cushion. The polysomes are resuspended, separated by velocity sedimentation and then fractionated, yielding a characteristic polysome profile reflecting the global level of translational activity in the parasite. RNA isolated from specific fractions can be used to determine the density of ribosomes loaded onto a particular transcript of interest, and is free of host ribosome contamination. Thus, our approach opens translational regulation in malaria to genome-wide analysis.

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Dynamical features of thePlasmodium falciparumribosome during translation

Sun

Blomqvist

et al. 2015

Nucleic Acids Res

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Plasmodium falciparum, the mosquito-transmitted Apicomplexan parasite, causes the most severe form of human malaria. In the asexual blood-stage, the parasite resides within erythrocytes where it proliferates, multiplies and finally spreads to new erythrocytes. Development of drugs targeting the ribosome, the site of protein synthesis, requires specific knowledge of its structure and work cycle, and, critically, the ways they differ from those in the human host. Here, we present five cryo-electron microscopy (cryo-EM) reconstructions of ribosomes purified from P. falciparum blood-stage schizonts at sub-nanometer resolution. Atomic models were built from these density maps by flexible fitting. Significantly, our study has taken advantage of new capabilities of cryo-EM, in visualizing several structures co-existing in the sample at once, at a resolution sufficient for building atomic models. We have discovered structural and dynamic features that differentiate the ribosomes of P. falciparum from those of mammalian system. Prompted by the absence of RACK1 on the ribosome in our and an earlier study we confirmed that RACK1 does not specifically co-purify with the 80S fraction in schizonts. More extensive studies, using cryo-EM methodology, of translation in the parasite will provide structural knowledge that may lead to development of novel anti-malarials.

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An in vitro system from Plasmodium falciparum active in endogenous mRNA translation

Abstract: An in vitro translation system has been prepared from Plasmodium falciparum by saponin lysis of infected-erythrocytes to free parasites which were homogeneized with glass beads, centrifuged to obtain a S-30 fraction followed by

Cited by 7 publications

References 26 publications

Effect of Antimalarial Drugs on Plasmodia Cell-Free Protein Synthesis

Effect of Antimalarial Drugs on Plasmodia Cell-Free Protein Synthesis

Polysome profiling of the malaria parasite Plasmodium falciparum

Dynamical features of thePlasmodium falciparumribosome during translation

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