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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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Cruzipain mRNA within reservosomes and colocalization of TcRBP40 protein and cruzipain mRNA.A) Plane Z reconstruction from confocal images obtained with cruzipain probes labeled with Cy-5 in epimastigotes. B) Merged image counterstaining with DAPI (green); Differential interference contrast (DIC) 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. Colocalization of C) cruzipain mRNA labeled with Cy-5 and D) TcRBP40 protein. E) Merged image counterstaining with DAPI (blue) was used to identify the nuclei (n) and kinetoplast (k), flagellum (f). F) Western blot of protein extracts from the same fractions using antibodies against TcRBP40 and Histone H2AZ. G) RT-PCR of RNA obtained from the different cellular fractions of T. cruzi epimastigote form, S – soluble cytoplasm, P – pellet, R – reservosome enriched fraction. H) Quantitative PCR for Cruzipain, TcRPB15, TcRBP40 and L9 transcripts enrichment in the reservosome compared to the soluble cytoplasm fractions. The reference used was RNA Pol II and the error bars are indicated. *p-value <0.0035. I) Western blot of total (T), intact (I-R) or disrupted (D-R) reservosomal protein extracts using antibodies against Cruzipain, 40S ribosomal S7 and 60S ribosomal L26 proteins. J) Bioanalyzer's electropherograms of RNAs extracted from intact (I-R) and disrupted (D-R) reservosomal fractions. Peaks corresponding to rRNAs are shown in fraction I-R.
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pone-0081375-g003: Cruzipain mRNA within reservosomes and colocalization of TcRBP40 protein and cruzipain mRNA.A) Plane Z reconstruction from confocal images obtained with cruzipain probes labeled with Cy-5 in epimastigotes. B) Merged image counterstaining with DAPI (green); Differential interference contrast (DIC) 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. Colocalization of C) cruzipain mRNA labeled with Cy-5 and D) TcRBP40 protein. E) Merged image counterstaining with DAPI (blue) was used to identify the nuclei (n) and kinetoplast (k), flagellum (f). F) Western blot of protein extracts from the same fractions using antibodies against TcRBP40 and Histone H2AZ. G) RT-PCR of RNA obtained from the different cellular fractions of T. cruzi epimastigote form, S – soluble cytoplasm, P – pellet, R – reservosome enriched fraction. H) Quantitative PCR for Cruzipain, TcRPB15, TcRBP40 and L9 transcripts enrichment in the reservosome compared to the soluble cytoplasm fractions. The reference used was RNA Pol II and the error bars are indicated. *p-value <0.0035. I) Western blot of total (T), intact (I-R) or disrupted (D-R) reservosomal protein extracts using antibodies against Cruzipain, 40S ribosomal S7 and 60S ribosomal L26 proteins. J) Bioanalyzer's electropherograms of RNAs extracted from intact (I-R) and disrupted (D-R) reservosomal fractions. Peaks corresponding to rRNAs are shown in fraction I-R.

Mentions: The posterior granular pattern of the cruzipain mRNA suggests the localization of the transcripts in or around reservosomes. Confocal microscopy observations suggested that the cruzipain mRNA was actually located inside the reservosomes (Figure 3A and B). To confirm if the granules observed for the cruzipain mRNA are indeed reservosomes, we performed immunolocalization assays with a TcRBP40 antibody and the cruzipain mRNA probe. TcRBP40 is a T. cruzi RNA-binding protein that is localized mainly in the reservosomes [26]. It is possible to observe that the cruzipain mRNA totally colocalizes with the TcRBP40 protein, confirming the reservosome localization for this mRNA (Figure 3 C, D and E). To further confirm the specific localization of cruzipain transcripts we purified reservosomes by cell fractionation as previously described [21], [27]. The identity of the reservosome fraction was shown by western blot of protein extracts using the antibody against TcRBP40. As a control, an antibody against histone H2AZ was used to quantify the possible contamination of this fraction with nuclear proteins or RNA (Figure 3F). RT-PCR analysis of RNA purified from the soluble cytosolic, an insoluble total (including nucleus) and the reservosome protein fractions showed the enrichment of cruzipain and TcRBP40 transcripts compared to control mRNAs, such as RNA pol II and kDNA associated protein (Figure 3G). The transcript levels of cruzipain, TcRBP40 and TcRBP15, a cytoplasmic RNA binding protein, in the reservosome fraction were then quantified by qPCR. For cruzipain, a 60-fold enrichment in the reservosome fraction was observed when compared to the cellular RNA (Figure 3H). This result shows the enrichment of the cruzipain transcript in this organelle. This result opened the possibility of translation inside this organelle. To study the presence of ribosomes we looked for the presence of ribosomal proteins and rRNA. Reservosomes were fractionated followed by membrane disruption to obtain the inside content of the organelle. Western blot assays showed that it was not possible to detect ribosomal proteins inside the vesicles (Figure 3I); however, ribosomal proteins were detected when the integrity of the reservosome was maintained. Cruzipain was detected in the total fraction as well as in the intact and disrupted organelle fractions (Figure 3I). As a complementary approach, the RNA fraction of the intact and the disrupted reservosome was extracted. The Bioanalyzer profile (Figure 3H) showed the presence of rRNA in the intact purified organelle. However, the analysis of the disrupted reservosome showed that rRNA was barely detected (Figure 3H). To further investigate if the cruzipain RNA in the reservosome is being stored or degraded, RNA was purified from the organelle followed by amplification using oligo-d(T) (Figure 3G). In T. cruzi, as in yeast, the main pathway of RNA degradation involves deadenylation of the poly-A tail, as no decapping enzyme has been described so far [28], [29]. The cruzipain amplification from the reservosome fraction indicates an intact poly-A tail, suggesting storage rather than degradation. The localization of cruzipain mRNA inside the reservosome is striking, nonetheless the biological role of this observation needs to be further investigated.


mRNA localization mechanisms in Trypanosoma cruzi.

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

Cruzipain mRNA within reservosomes and colocalization of TcRBP40 protein and cruzipain mRNA.A) Plane Z reconstruction from confocal images obtained with cruzipain probes labeled with Cy-5 in epimastigotes. B) Merged image counterstaining with DAPI (green); Differential interference contrast (DIC) 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. Colocalization of C) cruzipain mRNA labeled with Cy-5 and D) TcRBP40 protein. E) Merged image counterstaining with DAPI (blue) was used to identify the nuclei (n) and kinetoplast (k), flagellum (f). F) Western blot of protein extracts from the same fractions using antibodies against TcRBP40 and Histone H2AZ. G) RT-PCR of RNA obtained from the different cellular fractions of T. cruzi epimastigote form, S – soluble cytoplasm, P – pellet, R – reservosome enriched fraction. H) Quantitative PCR for Cruzipain, TcRPB15, TcRBP40 and L9 transcripts enrichment in the reservosome compared to the soluble cytoplasm fractions. The reference used was RNA Pol II and the error bars are indicated. *p-value <0.0035. I) Western blot of total (T), intact (I-R) or disrupted (D-R) reservosomal protein extracts using antibodies against Cruzipain, 40S ribosomal S7 and 60S ribosomal L26 proteins. J) Bioanalyzer's electropherograms of RNAs extracted from intact (I-R) and disrupted (D-R) reservosomal fractions. Peaks corresponding to rRNAs are shown in fraction I-R.
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Related In: Results  -  Collection

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Show All Figures
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pone-0081375-g003: Cruzipain mRNA within reservosomes and colocalization of TcRBP40 protein and cruzipain mRNA.A) Plane Z reconstruction from confocal images obtained with cruzipain probes labeled with Cy-5 in epimastigotes. B) Merged image counterstaining with DAPI (green); Differential interference contrast (DIC) 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. Colocalization of C) cruzipain mRNA labeled with Cy-5 and D) TcRBP40 protein. E) Merged image counterstaining with DAPI (blue) was used to identify the nuclei (n) and kinetoplast (k), flagellum (f). F) Western blot of protein extracts from the same fractions using antibodies against TcRBP40 and Histone H2AZ. G) RT-PCR of RNA obtained from the different cellular fractions of T. cruzi epimastigote form, S – soluble cytoplasm, P – pellet, R – reservosome enriched fraction. H) Quantitative PCR for Cruzipain, TcRPB15, TcRBP40 and L9 transcripts enrichment in the reservosome compared to the soluble cytoplasm fractions. The reference used was RNA Pol II and the error bars are indicated. *p-value <0.0035. I) Western blot of total (T), intact (I-R) or disrupted (D-R) reservosomal protein extracts using antibodies against Cruzipain, 40S ribosomal S7 and 60S ribosomal L26 proteins. J) Bioanalyzer's electropherograms of RNAs extracted from intact (I-R) and disrupted (D-R) reservosomal fractions. Peaks corresponding to rRNAs are shown in fraction I-R.
Mentions: The posterior granular pattern of the cruzipain mRNA suggests the localization of the transcripts in or around reservosomes. Confocal microscopy observations suggested that the cruzipain mRNA was actually located inside the reservosomes (Figure 3A and B). To confirm if the granules observed for the cruzipain mRNA are indeed reservosomes, we performed immunolocalization assays with a TcRBP40 antibody and the cruzipain mRNA probe. TcRBP40 is a T. cruzi RNA-binding protein that is localized mainly in the reservosomes [26]. It is possible to observe that the cruzipain mRNA totally colocalizes with the TcRBP40 protein, confirming the reservosome localization for this mRNA (Figure 3 C, D and E). To further confirm the specific localization of cruzipain transcripts we purified reservosomes by cell fractionation as previously described [21], [27]. The identity of the reservosome fraction was shown by western blot of protein extracts using the antibody against TcRBP40. As a control, an antibody against histone H2AZ was used to quantify the possible contamination of this fraction with nuclear proteins or RNA (Figure 3F). RT-PCR analysis of RNA purified from the soluble cytosolic, an insoluble total (including nucleus) and the reservosome protein fractions showed the enrichment of cruzipain and TcRBP40 transcripts compared to control mRNAs, such as RNA pol II and kDNA associated protein (Figure 3G). The transcript levels of cruzipain, TcRBP40 and TcRBP15, a cytoplasmic RNA binding protein, in the reservosome fraction were then quantified by qPCR. For cruzipain, a 60-fold enrichment in the reservosome fraction was observed when compared to the cellular RNA (Figure 3H). This result shows the enrichment of the cruzipain transcript in this organelle. This result opened the possibility of translation inside this organelle. To study the presence of ribosomes we looked for the presence of ribosomal proteins and rRNA. Reservosomes were fractionated followed by membrane disruption to obtain the inside content of the organelle. Western blot assays showed that it was not possible to detect ribosomal proteins inside the vesicles (Figure 3I); however, ribosomal proteins were detected when the integrity of the reservosome was maintained. Cruzipain was detected in the total fraction as well as in the intact and disrupted organelle fractions (Figure 3I). As a complementary approach, the RNA fraction of the intact and the disrupted reservosome was extracted. The Bioanalyzer profile (Figure 3H) showed the presence of rRNA in the intact purified organelle. However, the analysis of the disrupted reservosome showed that rRNA was barely detected (Figure 3H). To further investigate if the cruzipain RNA in the reservosome is being stored or degraded, RNA was purified from the organelle followed by amplification using oligo-d(T) (Figure 3G). In T. cruzi, as in yeast, the main pathway of RNA degradation involves deadenylation of the poly-A tail, as no decapping enzyme has been described so far [28], [29]. The cruzipain amplification from the reservosome fraction indicates an intact poly-A tail, suggesting storage rather than degradation. The localization of cruzipain mRNA inside the reservosome is striking, nonetheless the biological role of this observation needs to be further investigated.

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