Development of a dual reporter system to identify regulatory cis-acting elements in untranslated regions of Trypanosoma cruzi mRNAs

Araújo, Patrícia Rosa de; Burle-Caldas, Gabriela A.; Silva-Pereira, Rosiane Aparecida da; Bartholomeu, Daniella Castanheira; DaRocha, Wanderson D.; Teixeira, Santuza Maria Ribeiro

doi:10.1016/j.parint.2011.01.006

Cited by 17 publications

(12 citation statements)

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“…The loci for one gene of each, the γ- and δ-subfamily of amastins [transcripts “a66;8439” and “a96;12664”, respectively (see Additional file 2: Figure S1a); hereafter referred to as γ- and δ-amastin], were selected for the site at which a cassette containing a drug resistance marker gene would be inserted because (i) these two amastin genes are highly expressed (Additional file 1: Table S1), (ii) amastin loci have been previously used to enhance expression of heterologous genes in other trypanosomatids [29, 30], and (iii) a knockdown of δ-amastin does not seem to affect proliferation of insect stages in other trypanosomatid species [31]. Amastins are surface glycoproteins of unknown function that can be grouped into four subfamilies (termed α to δ) and occur in some trypanosomatids as tandemly arrayed multicopy genes [32].…”

Section: Resultsmentioning

confidence: 99%

Development of a toolbox to dissect host-endosymbiont interactions and protein trafficking in the trypanosomatid Angomonas deanei

et al. 2016

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BackgroundBacterial endosymbionts are found across the eukaryotic kingdom and profoundly impacted eukaryote evolution. In many endosymbiotic associations with vertically inherited symbionts, highly complementary metabolic functions encoded by host and endosymbiont genomes indicate integration of metabolic processes between the partner organisms. While endosymbionts were initially expected to exchange only metabolites with their hosts, recent evidence has demonstrated that also host-encoded proteins can be targeted to the bacterial symbionts in various endosymbiotic systems. These proteins seem to participate in regulating symbiont growth and physiology. However, mechanisms required for protein targeting and the specific endosymbiont targets of these trafficked proteins are currently unexplored owing to a lack of molecular tools that enable functional studies of endosymbiotic systems.ResultsHere we show that the trypanosomatid Angomonas deanei, which harbors a β-proteobacterial endosymbiont, is readily amenable to genetic manipulation. Its rapid growth, availability of full genome and transcriptome sequences, ease of transfection, and high frequency of homologous recombination have allowed us to stably integrate transgenes into the A. deanei nuclear genome, efficiently generate null mutants, and elucidate protein localization by heterologous expression of a fluorescent protein fused to various putative targeting signals. Combining these novel tools with proteomic analysis was key for demonstrating the routing of a host-encoded protein to the endosymbiont, suggesting the existence of a specific endosymbiont-sorting machinery in A. deanei.ConclusionsAfter previous reports from plants, insects, and a cercozoan amoeba we found here that also in A. deanei, i.e. a member of a fourth eukaryotic supergroup, host-encoded proteins can be routed to the bacterial endosymbiont. This finding adds further evidence to our view that the targeting of host proteins is a general strategy of eukaryotes to gain control over and interact with a bacterial endosymbiont. The molecular resources reported here establish A. deanei as a time and cost efficient reference system that allows for a rigorous dissection of host-symbiont interactions that have been, and are still being shaped over evolutionary time. We expect this system to greatly enhance our understanding of the biology of endosymbiosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0820-z) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Development of a toolbox to dissect host-endosymbiont interactions and protein trafficking in the trypanosomatid Angomonas deanei

et al. 2016

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“…Using a luciferase reporter system, we have recently demonstrated that adding the 3' UTR from a tTS gene, which exhibits higher expression in trypomastigotes, resulted in increased luciferase activity in trypomastigotes without a corresponding increase in luciferase mRNA levels. These data indicate that this 3' UTR contains elements that mediate mRNA-specific translational control (Araújo et al 2011).…”

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confidence: 78%

“…Furthermore, treatment of epimastigotes with vinblastine, a drug that prevents microtubule assembly, reduced alpha and beta tubulin mRNAs levels, whereas the levels of GAPDH mRNA were unchanged (Urményi et al 1992 In an attempt to identify regulatory elements involved in modulating the half-lives of alpha and beta tubulin transcripts in response to changes in microtubule dynamics, epimastigotes were transiently transfected with vectors containing the luciferase gene associated with different regions of these mRNAs. These analyses clearly indicated the presence of elements in the 3' UTR of alpha tubulin transcripts that are responsible for the increased stability of this mRNA in epimastigotes (Araújo et al 2011). Moreover, these studies suggested the involvement of other sequences, possibly in the coding region, that acted as additional cis-acting elements to modulate tubulin mRNA stability in response to changes in microtubule dynamics (da Silva et al 2006).…”

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confidence: 96%

Regulatory elements involved in the post-transcriptional control of stage-specific gene expression in Trypanosoma cruzi: a review

Araújo

Teixeira

2011

Mem. Inst. Oswaldo Cruz

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Trypanosoma cruzi is an intracellular parasite that is transmitted by at least 40 different blood-sucking triatomine species to over 1,000 mammalian species (Brener et al. 2000). This parasite must adapt to enormous changes in its extracellular milieu, such as changes in environmental temperature as well as in the available nutrients inside each host. The parasite has a complex life cycle that is characterised by four stages; epimastigotes and metacyclic trypomastigotes are present in the insect vector, whereas intracellular amastigotes and bloodstream trypomastigotes are present in the mammalian host. Thus, this species must develop a broad set of molecular tools that allow it to multiply in the insect gut, to invade and multiply inside a large number of distinct mammalian cell types and to circumvent host immune defence systems. To meet such phenotypic plasticity, T. cruzi relies on unique mechanisms that can control the expression of its repertoire of about 12,000 genes. Because its genome is constitutively transcribed into long polycistronic primary transcripts, mRNAs for proteincoding genes must be processed through trans-splicing and polyadenylation reactions. The mRNAs must also interact with different protein factors in a complex posttranscriptional regulatory machinery that determines the levels of their protein product according to the cellular demands of the parasite in each stage of its life cycle.In the same issue that the T. cruzi genome was published (El-Sayed et al. 2005a), a study describing its proteome was also reported (Atwood et al. 2005). Proteins extracted from whole-cell and subcellular lysates of the four stages of T. cruzi were analysed by mass spectrometry (epimastigotes, metacyclic trypomastigotes, amastigotes and trypomastigotes), which identified 2,784 proteins belonging to the 1,168 protein groups in the annotated T. cruzi genome. Although about 30% of the identified proteins were found at all life-cycle stages, at least 248 proteins were only expressed at one stage, thereby demonstrating significant changes in the relative abundance of T. cruzi proteins throughout its life cycle. One of the main findings in these proteomic analyses was that the four parasite stages use distinct energy sources (Atwood et al. 2005); intracellular amastigotes upregulated proteins involved in lipid-dependent energy metabolism, such as enzymes of the citric acid cycle, whereas enzymes capable of catalysing the conversion of histidine to glutamate were more abundant in the insect epimastigote stage. Heat-shock proteins (HSP) and proteins involved in vesicular trafficking were also preferentially detected in amastigotes. Furthermore, enzymes involved in antioxidant defence were upregulated during the transformation of epimastigotes into invasive metacyclic trypomastigotes, whereas bloodstream trypomastigotes upregulated the surface expression of several large gene families that are known to be involved in interacting with the mammalian host (Atwood et al. 2005).In agreement with this proteomics data, glo...

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“…A variety of heterologous reporter systems (e.g., chloramphenicol acetyltransferase, β-galactosidase, β-glucuronidase, firefly (Fluc) and Renilla (Rluc) luciferases) have been employed to examine translational regulation and to identify cis -regulatory elements controlling mRNA abundance and translation in kinetoplastids (4,15–17). The studies presented herein describe the development of a Fluc/Rluc dual luciferase reporter system that allows post-transcriptional regulation to be readily assessed for multiple candidates derived from systems-level gene expression studies of kinetoplastid parasites.…”

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A dual luciferase system for analysis of post-transcriptional regulation of gene expression in Leishmania

Soysa

Carter

Yates

2014

Molecular and Biochemical Parasitology

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Gene expression in kinetoplastid parasites is regulated via post-transcriptional mechanisms that modulate mRNA turnover, translation rate, and/or post-translational protein stability. To facilitate the analysis of post-transcriptional regulation, a dual luciferase system was developed in which firefly and Renilla luciferase reporters genetically fused to compatible drug resistance genes are integrated in place of one allele of the gene of interest and of an internal control gene, respectively, in a manner that preserves the cognate pre-mRNA processing signals. The sensitivity and reproducibility of the assay coupled with the ability to rapidly assemble reporter integration constructs render the dual luciferase system suitable for analysis of multiple candidates derived from global expression analysis platforms. To demonstrate the utility of the system, regulation of three genes in response to purine starvation was examined in Leishmania donovani promastigotes. This dual luciferase system should be directly applicable to the analysis of post-transcriptional regulation in other kinetoplastids.

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Development of a dual reporter system to identify regulatory cis-acting elements in untranslated regions of Trypanosoma cruzi mRNAs

Cited by 17 publications

References 33 publications

Development of a toolbox to dissect host-endosymbiont interactions and protein trafficking in the trypanosomatid Angomonas deanei

Development of a toolbox to dissect host-endosymbiont interactions and protein trafficking in the trypanosomatid Angomonas deanei

Regulatory elements involved in the post-transcriptional control of stage-specific gene expression in Trypanosoma cruzi: a review

A dual luciferase system for analysis of post-transcriptional regulation of gene expression in Leishmania

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