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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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Luciferase mRNA localization in T. cruzi with various UTRs.A) Luciferase probes labeled with Cy-5, showing the distribution of UTR-GAPDH as a control. B) Luciferase probes labeled with Cy-5, showing the distribution of UTR-β-tubulin. C) Luciferase probes labeled with Cy-5, showing the distribution of UTR-PFR2. D) to F) Merged images. G) Luciferase probes labeled with Cy-3, showing the distribution of UTR-Cruzipain. H) Cruzipain probes labeled with Cy-5, showing the distribution of cruzipain mRNA. I) Merged images. Counterstaining with DAPI (blue) was used to identify the nuclei (n) and kinetoplast (k), flagellum (f). Differential interference contrast images are shown for identification of the cellular body of the parasite and the flagellum. Scale bar  = 10 µm. White arrows indicate the position of the flagellum.
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pone-0081375-g007: Luciferase mRNA localization in T. cruzi with various UTRs.A) Luciferase probes labeled with Cy-5, showing the distribution of UTR-GAPDH as a control. B) Luciferase probes labeled with Cy-5, showing the distribution of UTR-β-tubulin. C) Luciferase probes labeled with Cy-5, showing the distribution of UTR-PFR2. D) to F) Merged images. G) Luciferase probes labeled with Cy-3, showing the distribution of UTR-Cruzipain. H) Cruzipain probes labeled with Cy-5, showing the distribution of cruzipain mRNA. I) Merged images. Counterstaining with DAPI (blue) was used to identify the nuclei (n) and kinetoplast (k), flagellum (f). Differential interference contrast images are shown for identification of the cellular body of the parasite and the flagellum. Scale bar  = 10 µm. White arrows indicate the position of the flagellum.

Mentions: In other eukaryotes, zipcode elements in the 3′-UTRs of the mRNA are recognized by specific proteins, which direct the mRNA to its subcellular localization. No orthologs of zipcode proteins or putative localization signals in transcripts have been described in trypanosomes. We investigated the possibility that similar elements guide mRNA localization in trypanosomes, by inserting the 3′-UTR containing the complete intergenic region of the β-tubulin, Cruzipain and PFR2 coding genes downstream from the firefly luciferase reporter gene in the pTcDUALuc vector. We transfected T. cruzi epimastigotes with these constructs and investigated the cytoplasmic localization of the luciferase mRNA by FISH. The intergenic region of the gapdh gene was used as a control. GAPDH transcripts had a diffuse cytoplasmic distribution in epimastigotes (Figure 7A and D). The pattern observed for the reporter transcript with the β-tubulin 3′-UTR was similar to that observed for the endogenous mRNA, although the perinuclear localization was less evident (Figure 7B and E). For constructs containing the PFR2 UTR, the distribution of the luciferase transcripts was predominantly in the posterior region of the cell and virtually indistinguishable from that of PFR2 transcripts in epimastigotes (Figure 7C and F). The results obtained with the cruzipain 3′UTR construct clearly demonstrate the localization of the luciferase transcripts in the reservosomes (Figure 7G), where they colocalize with the cruzipain mRNA (Figure 7H and I). These results suggest that the 3′UTRs of trypanosomes may contain localization elements similar to those present in other organisms.


mRNA localization mechanisms in Trypanosoma cruzi.

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

Luciferase mRNA localization in T. cruzi with various UTRs.A) Luciferase probes labeled with Cy-5, showing the distribution of UTR-GAPDH as a control. B) Luciferase probes labeled with Cy-5, showing the distribution of UTR-β-tubulin. C) Luciferase probes labeled with Cy-5, showing the distribution of UTR-PFR2. D) to F) Merged images. G) Luciferase probes labeled with Cy-3, showing the distribution of UTR-Cruzipain. H) Cruzipain probes labeled with Cy-5, showing the distribution of cruzipain mRNA. I) Merged images. Counterstaining with DAPI (blue) was used to identify the nuclei (n) and kinetoplast (k), flagellum (f). Differential interference contrast images are shown for identification of the cellular body of the parasite and the flagellum. Scale bar  = 10 µm. White arrows indicate the position of the flagellum.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0081375-g007: Luciferase mRNA localization in T. cruzi with various UTRs.A) Luciferase probes labeled with Cy-5, showing the distribution of UTR-GAPDH as a control. B) Luciferase probes labeled with Cy-5, showing the distribution of UTR-β-tubulin. C) Luciferase probes labeled with Cy-5, showing the distribution of UTR-PFR2. D) to F) Merged images. G) Luciferase probes labeled with Cy-3, showing the distribution of UTR-Cruzipain. H) Cruzipain probes labeled with Cy-5, showing the distribution of cruzipain mRNA. I) Merged images. Counterstaining with DAPI (blue) was used to identify the nuclei (n) and kinetoplast (k), flagellum (f). Differential interference contrast images are shown for identification of the cellular body of the parasite and the flagellum. Scale bar  = 10 µm. White arrows indicate the position of the flagellum.
Mentions: In other eukaryotes, zipcode elements in the 3′-UTRs of the mRNA are recognized by specific proteins, which direct the mRNA to its subcellular localization. No orthologs of zipcode proteins or putative localization signals in transcripts have been described in trypanosomes. We investigated the possibility that similar elements guide mRNA localization in trypanosomes, by inserting the 3′-UTR containing the complete intergenic region of the β-tubulin, Cruzipain and PFR2 coding genes downstream from the firefly luciferase reporter gene in the pTcDUALuc vector. We transfected T. cruzi epimastigotes with these constructs and investigated the cytoplasmic localization of the luciferase mRNA by FISH. The intergenic region of the gapdh gene was used as a control. GAPDH transcripts had a diffuse cytoplasmic distribution in epimastigotes (Figure 7A and D). The pattern observed for the reporter transcript with the β-tubulin 3′-UTR was similar to that observed for the endogenous mRNA, although the perinuclear localization was less evident (Figure 7B and E). For constructs containing the PFR2 UTR, the distribution of the luciferase transcripts was predominantly in the posterior region of the cell and virtually indistinguishable from that of PFR2 transcripts in epimastigotes (Figure 7C and F). The results obtained with the cruzipain 3′UTR construct clearly demonstrate the localization of the luciferase transcripts in the reservosomes (Figure 7G), where they colocalize with the cruzipain mRNA (Figure 7H and I). These results suggest that the 3′UTRs of trypanosomes may contain localization elements similar to those present in other organisms.

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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