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Key role of the 3' untranslated region in the cell cycle regulated expression of the Leishmania infantum histone H2A genes: minor synergistic effect of the 5' untranslated region.

Abanades DR, Ramírez L, Iborra S, Soteriadou K, González VM, Bonay P, Alonso C, Soto M - BMC Mol. Biol. (2009)

Bottom Line: Furthermore, it was determined that the addition of just the H2A 3' UTR to the CAT reporter gene is sufficient to achieve a similar pattern of post-transcriptional regulation indicating that this region contains the major regulatory sequences involved in the cell cycle dependent expression of the H2A genes.On the other hand, although CAT transcripts bearing the H2A 5' alone were translated both in the G1 and S phase, higher percentages of transcripts were detected on polyribosomes in the S phase correlating with an increase in the de novo synthesis of CAT.Thus, it can be concluded that this region also contributes, although to a minor extent than the 3' UTR, in the enhancement of translation in the S phase relative to the G1 phase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular, Universidad Autónoma de Madrid, CSIC-UAM, Nicolás Cabrera 1, 28049 Madrid, Spain. msoto@cbm.uam.es

ABSTRACT

Background: Histone synthesis in Leishmania is tightly coupled to DNA replication by a post-transcriptional mechanism operating at the level of translation.

Results: In this work we have analyzed the implication of the 5' and 3' untranslated regions (UTR) in the cell cycle regulated expression of the histone H2A in Leishmania infantum. For that purpose, L. infantum promastigotes were stably transfected with different plasmid constructs in which the CAT coding region used as a reporter was flanked by the 5' and 3' UTR regions of the different H2A genes. We report that in spite of their sequence differences, histone H2A 5' and 3' UTRs conferred a cell cycle dependent pattern of expression on the CAT reporter since de novo synthesis of CAT increased when parasites enter the S phase. Using one established L. infantum cell line we showed that CAT expression is controlled by the same regulatory events that control the endogenous histone gene expression. Thus, although we did not detect changes in the level of CAT mRNAs during cell cycle progression, a drastic change in the polysome profiles of CAT mRNAs was observed during the progression from G1 to S phase. In the S phase CAT mRNAs were on polyribosomal fractions, but in the G1 phase the association of CAT transcripts with ribosomes was impaired. Furthermore, it was determined that the addition of just the H2A 3' UTR to the CAT reporter gene is sufficient to achieve a similar pattern of post-transcriptional regulation indicating that this region contains the major regulatory sequences involved in the cell cycle dependent expression of the H2A genes. On the other hand, although CAT transcripts bearing the H2A 5' alone were translated both in the G1 and S phase, higher percentages of transcripts were detected on polyribosomes in the S phase correlating with an increase in the de novo synthesis of CAT. Thus, it can be concluded that this region also contributes, although to a minor extent than the 3' UTR, in the enhancement of translation in the S phase relative to the G1 phase.

Conclusion: Our findings indicate that both, the 5' and the 3' UTRs contain sequence elements that contribute to the cell cycle expression of L. infantum H2A. The 3' UTR region is essential for cell cycle dependent translation of the L. infantum H2A transcripts whereas the 5' UTR has a minor contribution in their S phase dependent translation.

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Effect of the H2A4 5' UTR on regulation of CAT expression. (A) Protein extracts from 35S-labelled L. infantum promastigotes transfected with pXCAT5'H2A and treated with 5 mM HU either for 12 h (lane 0) or at the indicated time period (in h) after removal of the drug, were used to immunoprecipitate CAT. After immunoprecipitation, protein samples were separated by SDS/PAGE and transferred on to a nitrocellulose membrane. Autoradiography (De novo panel) or Western blot with anti-CAT antibodies to reveal the total amount of CAT present in the samples (TP panel) is shown. (B) Densitometric analysis showing the ratio between the de novo synthesis of CAT and total amount of CAT (columns). The percentage of cells in the S phase is also included (black line). Northern blots prepared with RNA samples separated by sucrose gradients from promastigotes transfected with pXCAT5'H2A (C) or pXCATHSP83 (D) treated 12 with HU (lane 0) or 3 h after drug removal were sequentially hybridized with the CAT and the H4 probe. The autoradiographic exposure of the blots and the densitometric analysis is shown. Data correspond to one representative experiment of three independent assays with similar results.
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Figure 6: Effect of the H2A4 5' UTR on regulation of CAT expression. (A) Protein extracts from 35S-labelled L. infantum promastigotes transfected with pXCAT5'H2A and treated with 5 mM HU either for 12 h (lane 0) or at the indicated time period (in h) after removal of the drug, were used to immunoprecipitate CAT. After immunoprecipitation, protein samples were separated by SDS/PAGE and transferred on to a nitrocellulose membrane. Autoradiography (De novo panel) or Western blot with anti-CAT antibodies to reveal the total amount of CAT present in the samples (TP panel) is shown. (B) Densitometric analysis showing the ratio between the de novo synthesis of CAT and total amount of CAT (columns). The percentage of cells in the S phase is also included (black line). Northern blots prepared with RNA samples separated by sucrose gradients from promastigotes transfected with pXCAT5'H2A (C) or pXCATHSP83 (D) treated 12 with HU (lane 0) or 3 h after drug removal were sequentially hybridized with the CAT and the H4 probe. The autoradiographic exposure of the blots and the densitometric analysis is shown. Data correspond to one representative experiment of three independent assays with similar results.

Mentions: Immunoprecipitation experiments with promastigotes transfected with the pXCAT5'H2A plasmid showed an increase in the metabolic labelling of CAT at 3 h after HU release (Fig. 6A). Densitometric analysis showed a 2-fold increase in the labelling of CAT when cells enter into the S phase (Fig. 6B). However, analysis of the polysomal profiles of cells transfected with pXCAT5'H2A revealed that CAT transcripts were actively translated at the G1 phase since they were associated with polyribosomal fractions (27% of the CAT mRNAs) at time 0 h after HU release (Fig. 6C). It was also observed that a higher percentage of transcripts were located in the polyribosomal fractions (44% of the CAT mRNAs) when cells progress into the S phase. In addition, the location of the reporter mRNAs in the bottom fractions of the sucrose gradient indicates a high translation rate for transcripts bearing the combination of the H2A 5' UTR and the CAT coding region in both cell cycle G1 and S phase. Subsequently, we analyzed the polysome profiles of the CAT transcripts in parasites transfected with the pXCATHSP83, a cell line that did not show cell cycle regulated expression of CAT. The results indicate that similar percentages of these transcripts were located into polysomes in the G1 (45% of the CAT mRNAs) and in the S phase (50%) (Fig. 6D). Contrary to CAT transcripts from the pXCAT5'H2A cell line, CAT transcripts from parasite transfected with PXCATHSP83 were in a similar active translational status along the cell cycle. Thus, it can be concluded that when the 5' UTR region of the H2A4 gene is located upstream of the reporter gene the translation level of CAT transcripts is increased in the S phase.


Key role of the 3' untranslated region in the cell cycle regulated expression of the Leishmania infantum histone H2A genes: minor synergistic effect of the 5' untranslated region.

Abanades DR, Ramírez L, Iborra S, Soteriadou K, González VM, Bonay P, Alonso C, Soto M - BMC Mol. Biol. (2009)

Effect of the H2A4 5' UTR on regulation of CAT expression. (A) Protein extracts from 35S-labelled L. infantum promastigotes transfected with pXCAT5'H2A and treated with 5 mM HU either for 12 h (lane 0) or at the indicated time period (in h) after removal of the drug, were used to immunoprecipitate CAT. After immunoprecipitation, protein samples were separated by SDS/PAGE and transferred on to a nitrocellulose membrane. Autoradiography (De novo panel) or Western blot with anti-CAT antibodies to reveal the total amount of CAT present in the samples (TP panel) is shown. (B) Densitometric analysis showing the ratio between the de novo synthesis of CAT and total amount of CAT (columns). The percentage of cells in the S phase is also included (black line). Northern blots prepared with RNA samples separated by sucrose gradients from promastigotes transfected with pXCAT5'H2A (C) or pXCATHSP83 (D) treated 12 with HU (lane 0) or 3 h after drug removal were sequentially hybridized with the CAT and the H4 probe. The autoradiographic exposure of the blots and the densitometric analysis is shown. Data correspond to one representative experiment of three independent assays with similar results.
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Related In: Results  -  Collection

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Show All Figures
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Figure 6: Effect of the H2A4 5' UTR on regulation of CAT expression. (A) Protein extracts from 35S-labelled L. infantum promastigotes transfected with pXCAT5'H2A and treated with 5 mM HU either for 12 h (lane 0) or at the indicated time period (in h) after removal of the drug, were used to immunoprecipitate CAT. After immunoprecipitation, protein samples were separated by SDS/PAGE and transferred on to a nitrocellulose membrane. Autoradiography (De novo panel) or Western blot with anti-CAT antibodies to reveal the total amount of CAT present in the samples (TP panel) is shown. (B) Densitometric analysis showing the ratio between the de novo synthesis of CAT and total amount of CAT (columns). The percentage of cells in the S phase is also included (black line). Northern blots prepared with RNA samples separated by sucrose gradients from promastigotes transfected with pXCAT5'H2A (C) or pXCATHSP83 (D) treated 12 with HU (lane 0) or 3 h after drug removal were sequentially hybridized with the CAT and the H4 probe. The autoradiographic exposure of the blots and the densitometric analysis is shown. Data correspond to one representative experiment of three independent assays with similar results.
Mentions: Immunoprecipitation experiments with promastigotes transfected with the pXCAT5'H2A plasmid showed an increase in the metabolic labelling of CAT at 3 h after HU release (Fig. 6A). Densitometric analysis showed a 2-fold increase in the labelling of CAT when cells enter into the S phase (Fig. 6B). However, analysis of the polysomal profiles of cells transfected with pXCAT5'H2A revealed that CAT transcripts were actively translated at the G1 phase since they were associated with polyribosomal fractions (27% of the CAT mRNAs) at time 0 h after HU release (Fig. 6C). It was also observed that a higher percentage of transcripts were located in the polyribosomal fractions (44% of the CAT mRNAs) when cells progress into the S phase. In addition, the location of the reporter mRNAs in the bottom fractions of the sucrose gradient indicates a high translation rate for transcripts bearing the combination of the H2A 5' UTR and the CAT coding region in both cell cycle G1 and S phase. Subsequently, we analyzed the polysome profiles of the CAT transcripts in parasites transfected with the pXCATHSP83, a cell line that did not show cell cycle regulated expression of CAT. The results indicate that similar percentages of these transcripts were located into polysomes in the G1 (45% of the CAT mRNAs) and in the S phase (50%) (Fig. 6D). Contrary to CAT transcripts from the pXCAT5'H2A cell line, CAT transcripts from parasite transfected with PXCATHSP83 were in a similar active translational status along the cell cycle. Thus, it can be concluded that when the 5' UTR region of the H2A4 gene is located upstream of the reporter gene the translation level of CAT transcripts is increased in the S phase.

Bottom Line: Furthermore, it was determined that the addition of just the H2A 3' UTR to the CAT reporter gene is sufficient to achieve a similar pattern of post-transcriptional regulation indicating that this region contains the major regulatory sequences involved in the cell cycle dependent expression of the H2A genes.On the other hand, although CAT transcripts bearing the H2A 5' alone were translated both in the G1 and S phase, higher percentages of transcripts were detected on polyribosomes in the S phase correlating with an increase in the de novo synthesis of CAT.Thus, it can be concluded that this region also contributes, although to a minor extent than the 3' UTR, in the enhancement of translation in the S phase relative to the G1 phase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular, Universidad Autónoma de Madrid, CSIC-UAM, Nicolás Cabrera 1, 28049 Madrid, Spain. msoto@cbm.uam.es

ABSTRACT

Background: Histone synthesis in Leishmania is tightly coupled to DNA replication by a post-transcriptional mechanism operating at the level of translation.

Results: In this work we have analyzed the implication of the 5' and 3' untranslated regions (UTR) in the cell cycle regulated expression of the histone H2A in Leishmania infantum. For that purpose, L. infantum promastigotes were stably transfected with different plasmid constructs in which the CAT coding region used as a reporter was flanked by the 5' and 3' UTR regions of the different H2A genes. We report that in spite of their sequence differences, histone H2A 5' and 3' UTRs conferred a cell cycle dependent pattern of expression on the CAT reporter since de novo synthesis of CAT increased when parasites enter the S phase. Using one established L. infantum cell line we showed that CAT expression is controlled by the same regulatory events that control the endogenous histone gene expression. Thus, although we did not detect changes in the level of CAT mRNAs during cell cycle progression, a drastic change in the polysome profiles of CAT mRNAs was observed during the progression from G1 to S phase. In the S phase CAT mRNAs were on polyribosomal fractions, but in the G1 phase the association of CAT transcripts with ribosomes was impaired. Furthermore, it was determined that the addition of just the H2A 3' UTR to the CAT reporter gene is sufficient to achieve a similar pattern of post-transcriptional regulation indicating that this region contains the major regulatory sequences involved in the cell cycle dependent expression of the H2A genes. On the other hand, although CAT transcripts bearing the H2A 5' alone were translated both in the G1 and S phase, higher percentages of transcripts were detected on polyribosomes in the S phase correlating with an increase in the de novo synthesis of CAT. Thus, it can be concluded that this region also contributes, although to a minor extent than the 3' UTR, in the enhancement of translation in the S phase relative to the G1 phase.

Conclusion: Our findings indicate that both, the 5' and the 3' UTRs contain sequence elements that contribute to the cell cycle expression of L. infantum H2A. The 3' UTR region is essential for cell cycle dependent translation of the L. infantum H2A transcripts whereas the 5' UTR has a minor contribution in their S phase dependent translation.

Show MeSH
Related in: MedlinePlus