Trypanosoma cruzi: RNA structure and post-transcriptional control of tubulin gene expression

Bartholomeu, Daniella Castanheira; Silva, Rosiane A.; Galvao, L. M. C.; El-Sayed, Najib M.; Donelson, John E.; Teixeira, Santuza Maria Ribeiro

doi:10.1016/s0014-4894(03)00034-1

Cited by 29 publications

(32 citation statements)

References 34 publications

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“…Before the availability of the whole genome, a combination of experimental approaches, such as parasite transfection with reporter genes and studies on a select group of T. cruzi genes, were used to identify various elements in the 3' UTRs that control mRNA abundance in response to changes in the parasite life cycle. That group includes members of the TS gene family (Weston et al 1999, Jäger et al 2008, Gentil et al 2009), amastins (Coughlin et al 2000, alpha and beta tubulins (Bartholomeu et al 2002, da Silva et al 2006, mucins (Di Noia et al 2000) and HSP . Table provides a list of genes that have fully or partially characterised regulatory sequences.…”

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“…T. cruzi tubulin genes are organised as a cluster of alternating copies of alpha and beta tubulins (Maingon et al 1988, Soares et al 1989). Although transcription rates of tubulin genes are similar in epimastigotes and amastigotes, the alpha and beta tubulin transcript levels are three to six-fold higher in epimastigotes compared to trypomastigotes and amastigotes (Gonzalez-Pino et al 1997, Bartholomeu et al 2002 due to the increased mRNA half-life in epimastigotes (Bartholomeu et al 2002). Alpha and beta tubulin mRNA levels and translation rates undergo a marked reduction during the differentiation of dividing epimastigotes into dividing metacyclic trypomastigotes (Rondinelli et al 1986).…”

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“…Alpha and beta tubulin mRNA levels and translation rates undergo a marked reduction during the differentiation of dividing epimastigotes into dividing metacyclic trypomastigotes (Rondinelli et al 1986). Transient transfection assays using the luciferase gene cloned upstream of the alpha tubulin 3' UTR demonstrated that elements in this region are responsible for the increased stability of alpha tubulin mRNA in epimastigotes (da Silva et al 2006); elements with similar functions may also be present in the 3' UTR of beta tubulin mRNA (Bartholomeu et al 2002). Higher levels of unpolymerised tubulins were found in amastigotes and trypomastigotes, which suggests that there is an autoregulatory mechanism to induce tubulin mRNA destabilisation in these two stages of the parasite life cycle (da Silva et al 2006).…”

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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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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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“…The power of current experimental methods allows one to pinpoint nucleotides in these regions and afterwards observe the effects on parameters such as transcription rates, gene expression and regulation, mRNA stability and half-life. Association of sequence motifs in 3'UTR of T. cruzi genes to defined cellular and molecular effects has gained sound experimental support (D'Orso & Frasch 2001, Bartholomeu et al 2002). Though both untranslated segments are expected to influence gene expression and function, most investigations points to 3'UTR playing a major role by the presence of cis-acting elements in T. cruzi genes (Nozak & Cross 1995, D'Orso et al 2003.…”

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“…This can be viewed as the result of structural differences, for the 3'UTR being on average 2-3 times longer than the 5'UTR increases the possibilities to influence mRNA processing (more sequence elements, protein binding sites, and secondary structures). Genes like glycoprotein 72 (gp72), glycoprotein 85 (gp85), glyceraldehyde 1-phosphate dehydrogenase (gapdh), amastin, β-tubulin, mucin, trans-sialidase and, FL-60 have elements (presumptively or accurately described) in their 3'UTR that in different ways affect gene expression in stage-specific process (Nozak & Cross 1995, Abuin et al 1999, Weston et al 1999, Coughlin et al 2000, Di Noia et al 2000, Bartholomeu et al 2002 The bottleneck created by the huge amount of nucleotide sequences offers new routes in the search of models and experimental strategies that translate the DNA code into biological phenomenon. Thus the DNA sequence to biological phenomenon translation is a process of searching for codes behind the code, i.e., the searching of a Meta code.…”

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The untranslated regions of genes from Trypanosoma cruzi: perspectives for functional characterization of strains and isolates

Brandão

2006

Mem. Inst. Oswaldo Cruz

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The sequencing of Trypanosoma cruzi genome has been completed and a great deal of information is now available. However, the organization of protozoa genomes is somewhat elusive and much effort must be applied to reveal all the information coded in the nucleotide sequences. Among the DNA segments that needs further investigation are the untranslated regions of genes. Many of the T. cruzi genes that were revealed by the genome sequencing lack information about the untranslated regions. In this paper, some features of these untranslated segments as well as their applications in T. cruzi populations are discussed.

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Expression of exogenous genes in Trypanosoma cruzi : improving vectors and electroporation protocols

DaRocha

Silva

Bartholomeu

et al. 2004

Parasitology Research

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104

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To improve transfection efficiency in Trypanosoma cruzi, we developed a new electroporation protocol and expression vectors which use luciferase and green and red fluorescent proteins as reporter genes. In transient transfections, the electroporation conditions reported here resulted in luciferase expression 100 times higher than the levels obtained with previously described protocols. To verify whether sequences containing different trans-splicing signals influence reporter gene expression, we compared DNA fragments corresponding to 5' untranslated plus intergenic (5' UTR plus Ig) regions from GAPDH, TcP2beta, alpha- and beta- tubulin and amastin genes. Vectors containing sequences derived from the first four genes presented similar efficiencies and resulted in luciferase expression in transiently transfected epimastigotes that was up to 10 times higher than that for a control vector. In contrast, the amastin 5' UTR plus Ig resulted in lower levels of reporter gene expression. We also constructed a vector containing an expression cassette designed to be targeted to the tubulin locus of the parasite.

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Trypanosoma cruzi: RNA structure and post-transcriptional control of tubulin gene expression

Cited by 29 publications

References 34 publications

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

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

The untranslated regions of genes from Trypanosoma cruzi: perspectives for functional characterization of strains and isolates

Expression of exogenous genes in Trypanosoma cruzi : improving vectors and electroporation protocols

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