Effect of actinomycin D onTrypanosoma cruzi

Cruz, M. Queiroz da; Bräscher, H. M.; Vargens, J. R.; Oliveira-Lima, A.J.

doi:10.1007/bf02041263

Cited by 5 publications

(2 citation statements)

References 6 publications

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“…To determine whether the RNA Pol II localization was dependent on transcription, immunofluorescence assays using the anti-CTD antibodies were performed in cells previously treated with actinomycin D, which has been shown to effectively block all transcription events in T. cruzi (18,39). As seen in Fig.…”

Section: Resultsmentioning

confidence: 99%

Actively Transcribing RNA Polymerase II Concentrates on Spliced Leader Genes in the Nucleus of Trypanosoma cruzi

Dossin

Schenkman

2005

Eukaryot Cell

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RNA polymerase II of trypanosomes, early diverging eukaryotes, transcribes long polycistronic messages, which are not capped but are processed by trans-splicing and polyadenylation to form mature mRNAs. The same RNA polymerase II also transcribes the genes coding for the spliced leader RNA, which are capped, exported to the cytoplasm, processed, and reimported into the nucleus before they are used as splicing donors to form mRNAs from pre-mRNA polycistronic transcripts. As pre-mRNA and spliced leader transcription events appear to be uncoupled, we studied how the RNA polymerase II is distributed in the nucleus of Trypanosoma cruzi. Using specific antibodies to the T. cruzi RNA polymerase II unique carboxy-terminal domain, we demonstrated that large amounts of the enzyme are found concentrated in a domain close to the parasite nucleolus and containing the spliced leader genes. The remaining RNA polymerase II is diffusely distributed in the nucleoplasm. The spliced leader-associated RNA polymerase II localization is dependent on the cell transcriptional state. It disperses when transcription is blocked by ␣-amanitin and actinomycin D. Tubulin genes are excluded from this domain, suggesting that it may exclusively be the transcriptional site of spliced leader genes. Trypomastigote forms of the parasite, which have reduced spliced leader transcription, show less RNA polymerase II labeling, and the spliced leader genes are more dispersed in the nucleoplasm. These results provide strong evidences that transcription of spliced leader RNAs occurs in a particular domain in the T. cruzi nucleus.Trypanosomes belong to a group of early diverging flagellated protozoa that causes several parasitic diseases, including Chagas' disease, leishmaniosis, and African trypanosomiasis. As with most eukaryotes, they also have three RNA polymerases characterized by a variable sensitivity to ␣-amanitin (15, 48). In trypanosomatids, the ␣-amanitin-sensitive RNA polymerase II (RNA Pol II), which transcribes most of the protein coding genes, catalyzes the transcription of long polycistronic messages (25, 47). These long polycistronic messages are then processed in a trans-splicing reaction by addition of a 30-to 40-nucleotide sequence derived from the spliced leader (SL) RNA at the 5Ј end of each cistron, followed by addition of a poly(A) tail at the 3Ј end (3, 42). The cap is added at the 5Ј end of the SL RNA during its transcription, and then it is transferred with the 30 to 40 nucleotides to the mature mRNA in the trans-splicing reaction (21, 29, 37). Transcription is constitutive for almost all genes characterized to date and varies in overall rates according to the parasite developmental stages (18). Thus, most regulation of gene expression in these organisms seems to occur posttranscriptionally either by the stability of the processed mRNAs or by translational controls as discussed in several reviews (8,23,44,49). Recently, a bidirectional promoter was identified in strand-switch regions of chromosomes 1 (31) and 3 (30) of Leishmania ...

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Section: Resultsmentioning

confidence: 99%

Actively Transcribing RNA Polymerase II Concentrates on Spliced Leader Genes in the Nucleus of Trypanosoma cruzi

Dossin

Schenkman

2005

Eukaryot Cell

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“…Treatment with actinomycin D, which blocks transcription in T. cruzi (52), was used to study the stability of amastin, TS, and …”

Section: Resultsmentioning

confidence: 99%

Expression of trans-Sialidase and 85-kDa Glycoprotein Genes in Trypanosoma cruzi Is Differentially Regulated at the Post-transcriptional Level by Labile Protein Factors

Abuin¹,

Freitas‐Junior²,

Colli³

et al. 1999

Journal of Biological Chemistry

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To adapt to different environments, Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, expresses a different set of proteins during development. To begin to understand the mechanism that controls this differential gene expression, we have analyzed the levels of amastin and trans-sialidase mRNAs and the mRNAs encoding members of the 85-kDa glycoprotein gene family, which are differentially expressed in the T. cruzi stages found in the mammalian host. Amastin mRNA is expressed predominantly in intracellular and proliferative amastigotes. trans-Sialidase mRNAs are found mostly in forms undergoing transformation from amastigotes to trypomastigotes inside infected cells, whereas mRNAs encoding the 85-kDa glycoproteins appear only in the infective trypomastigotes released from the cells. The genes coding for these mRNA species are constitutively transcribed in all stages of T. cruzi cells, suggesting that expression is controlled post-transcriptionally during differentiation. Inhibition of transcription by actinomycin D revealed that each mRNA species has a relatively long half-life in stages where it accumulates. In the case of the transsialidase and 85-kDa glycoprotein genes, mRNA accumulation was induced by treatment with the protein synthesis inhibitor cycloheximide at the stages that preceded the normal accumulation. Therefore, mRNA stabilization may account for mRNA accumulation. mRNA degradation could be promoted by proteins with high turnover, or stabilization could be promoted by forming a complex with the translational machinery at defined times in development. Identification of the factors that induce mRNA degradation or stabilization is essential to the understanding of control of gene expression in these organisms.Gene expression in Trypanosoma cruzi as well as in other trypanosomes is largely controlled at the post-transcriptional level (1-6), although there are a few exceptions where promoters and transcriptional activation have been described (7). Well defined RNA polymerase II initiation sites have not been found, and most genes are transcribed as part of polycistronic units and are processed by trans-splicing (8, 9). During processing, capped spliced leader sequence is added to the 5Ј-end, and a polyadenosine tail is added to the 3Ј-end of mRNA (10), probably ensuring mRNA stability and transport to the cytoplasm (11). In particular, correct splicing and polyadenylation, as well as the existence of specific 3Ј-untranslated portions of the transcribed genes, have been shown to promote either mRNA stability (12) or an increase in the translational efficiency (13). However, the nature of controlling factors and how environmental modifications induce differential expression remain obscure.T. cruzi provides an attractive model to study how modifications in the environment induce differential gene expression. In the insect host, the epimastigote form of the parasite proliferates in the gut. In the posterior gut, where the nutrients become scarce, the parasite transforms into metacyclic ...

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The challenge of Chagas' disease chemotherapy: An update of drugs assayed against Trypanosoma cruzi

Castro

1993

Acta Tropica

130

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Effect of actinomycin D onTrypanosoma cruzi

Cited by 5 publications

References 6 publications

Actively Transcribing RNA Polymerase II Concentrates on Spliced Leader Genes in the Nucleus of Trypanosoma cruzi

Actively Transcribing RNA Polymerase II Concentrates on Spliced Leader Genes in the Nucleus of Trypanosoma cruzi

Expression of trans-Sialidase and 85-kDa Glycoprotein Genes in Trypanosoma cruzi Is Differentially Regulated at the Post-transcriptional Level by Labile Protein Factors

The challenge of Chagas' disease chemotherapy: An update of drugs assayed against Trypanosoma cruzi

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