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mRNA localization mechanisms in Trypanosoma cruzi.

Alves LR, Guerra-Slompo EP, de Oliveira AV, Malgarin JS, Goldenberg S, Dallagiovanna B - PLoS ONE (2013)

Bottom Line: Moreover, cruzipain mRNA was found inside reservosomes suggesting new unexpected functions for this vacuolar organelle.Our results strongly suggest that Trypanosoma cruzi have a core, basic mechanism of mRNA localization.This kind of controlled mRNA transport is ancient, dating back to early eukaryote evolution.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Regulação da Expressão Gênica, Instituto Carlos Chagas, Fiocruz-Paraná. Curitiba, Paraná, Brasil.

ABSTRACT
Asymmetric mRNA localization is a sophisticated tool for regulating and optimizing protein synthesis and maintaining cell polarity. Molecular mechanisms involved in the regulated localization of transcripts are widespread in higher eukaryotes and fungi, but not in protozoa. Trypanosomes are ancient eukaryotes that branched off early in eukaryote evolution. We hypothesized that these organisms would have basic mechanisms of mRNA localization. FISH assays with probes against transcripts coding for proteins with restricted distributions showed a discrete localization of the mRNAs in the cytoplasm. Moreover, cruzipain mRNA was found inside reservosomes suggesting new unexpected functions for this vacuolar organelle. Individual mRNAs were also mobilized to RNA granules in response to nutritional stress. The cytoplasmic distribution of these transcripts changed with cell differentiation, suggesting that localization mechanisms might be involved in the regulation of stage-specific protein expression. Transfection assays with reporter genes showed that, as in higher eukaryotes, 3'UTRs were responsible for guiding mRNAs to their final location. Our results strongly suggest that Trypanosoma cruzi have a core, basic mechanism of mRNA localization. This kind of controlled mRNA transport is ancient, dating back to early eukaryote evolution.

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Controls used for FISH validation.A) DNase I treatment before poly-T probe incubation. B) RNase A treatment before poly-T probe incubation. C) Cruzipain sense probes Cy-5 labeled in epimastigotes. D) β-tubulin sense probes Cy-3 labeled in epimastigotes. E) PFR2 sense probes Cy-3 labeled in epimastigotes. F) to J) Merged images, counterstaining with DAPI (blue) was used to identify nuclei (n), kinetoplast (k). Scale bars  = 10 µm.
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pone-0081375-g001: Controls used for FISH validation.A) DNase I treatment before poly-T probe incubation. B) RNase A treatment before poly-T probe incubation. C) Cruzipain sense probes Cy-5 labeled in epimastigotes. D) β-tubulin sense probes Cy-3 labeled in epimastigotes. E) PFR2 sense probes Cy-3 labeled in epimastigotes. F) to J) Merged images, counterstaining with DAPI (blue) was used to identify nuclei (n), kinetoplast (k). Scale bars  = 10 µm.

Mentions: We used FISH to investigate the subcellular distribution of transcripts for proteins with specific patterns of expression within cells. In all cases, sense probes were used and samples were initially treated with RNase and DNase as negative controls (Figure 1).


mRNA localization mechanisms in Trypanosoma cruzi.

Alves LR, Guerra-Slompo EP, de Oliveira AV, Malgarin JS, Goldenberg S, Dallagiovanna B - PLoS ONE (2013)

Controls used for FISH validation.A) DNase I treatment before poly-T probe incubation. B) RNase A treatment before poly-T probe incubation. C) Cruzipain sense probes Cy-5 labeled in epimastigotes. D) β-tubulin sense probes Cy-3 labeled in epimastigotes. E) PFR2 sense probes Cy-3 labeled in epimastigotes. F) to J) Merged images, counterstaining with DAPI (blue) was used to identify nuclei (n), kinetoplast (k). Scale bars  = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852752&req=5

pone-0081375-g001: Controls used for FISH validation.A) DNase I treatment before poly-T probe incubation. B) RNase A treatment before poly-T probe incubation. C) Cruzipain sense probes Cy-5 labeled in epimastigotes. D) β-tubulin sense probes Cy-3 labeled in epimastigotes. E) PFR2 sense probes Cy-3 labeled in epimastigotes. F) to J) Merged images, counterstaining with DAPI (blue) was used to identify nuclei (n), kinetoplast (k). Scale bars  = 10 µm.
Mentions: We used FISH to investigate the subcellular distribution of transcripts for proteins with specific patterns of expression within cells. In all cases, sense probes were used and samples were initially treated with RNase and DNase as negative controls (Figure 1).

Bottom Line: Moreover, cruzipain mRNA was found inside reservosomes suggesting new unexpected functions for this vacuolar organelle.Our results strongly suggest that Trypanosoma cruzi have a core, basic mechanism of mRNA localization.This kind of controlled mRNA transport is ancient, dating back to early eukaryote evolution.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Regulação da Expressão Gênica, Instituto Carlos Chagas, Fiocruz-Paraná. Curitiba, Paraná, Brasil.

ABSTRACT
Asymmetric mRNA localization is a sophisticated tool for regulating and optimizing protein synthesis and maintaining cell polarity. Molecular mechanisms involved in the regulated localization of transcripts are widespread in higher eukaryotes and fungi, but not in protozoa. Trypanosomes are ancient eukaryotes that branched off early in eukaryote evolution. We hypothesized that these organisms would have basic mechanisms of mRNA localization. FISH assays with probes against transcripts coding for proteins with restricted distributions showed a discrete localization of the mRNAs in the cytoplasm. Moreover, cruzipain mRNA was found inside reservosomes suggesting new unexpected functions for this vacuolar organelle. Individual mRNAs were also mobilized to RNA granules in response to nutritional stress. The cytoplasmic distribution of these transcripts changed with cell differentiation, suggesting that localization mechanisms might be involved in the regulation of stage-specific protein expression. Transfection assays with reporter genes showed that, as in higher eukaryotes, 3'UTRs were responsible for guiding mRNAs to their final location. Our results strongly suggest that Trypanosoma cruzi have a core, basic mechanism of mRNA localization. This kind of controlled mRNA transport is ancient, dating back to early eukaryote evolution.

Show MeSH