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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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Analysis of the CAT cell cycle regulated expression in different constructs. (A) Ratio between the de novo CAT synthesis (normalised to the total amount of CAT) and number of cells in the S phase (3 h after HU removal) determined by three independent assays in cell lines transfected with pXCATHSP83, pXCAT5'III/3'I, pXCAT5'H2A and pXCAT3'H2A constructs. (B) Graphic showing the percentages of CAT mRNA on polysomes in the same cell lines. Data were obtained from the densitometric analysis of the blots performed with the RNAs separated on sucrose gradients from cell lines transfected with pXCATHSP83, pXCAT5'III/3'I, pXCAT5'H2A and pXCAT3'H2A constructs at G1 and S phase for three independent assays.
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Figure 7: Analysis of the CAT cell cycle regulated expression in different constructs. (A) Ratio between the de novo CAT synthesis (normalised to the total amount of CAT) and number of cells in the S phase (3 h after HU removal) determined by three independent assays in cell lines transfected with pXCATHSP83, pXCAT5'III/3'I, pXCAT5'H2A and pXCAT3'H2A constructs. (B) Graphic showing the percentages of CAT mRNA on polysomes in the same cell lines. Data were obtained from the densitometric analysis of the blots performed with the RNAs separated on sucrose gradients from cell lines transfected with pXCATHSP83, pXCAT5'III/3'I, pXCAT5'H2A and pXCAT3'H2A constructs at G1 and S phase for three independent assays.

Mentions: Taking into account the variations observed in the percentages of cells in the S-phase between experiments, three independent assays were performed with the cell lines transfected with pXCATHSP83, pXCAT5'III/3'I, pXCAT5'H2A and pXCAT3'H2A constructs. The ratio between the de novo synthesis of CAT (normalised to the total amount) and the amount of cells in the S phase in the different transfected cell lines was determined (Fig. 7A). An increase was observed in this ratio in both the pXCAT5'H2A and pXCAT3'H2A cell lines compared with pXCATHSP83 cell line. In addition, the ratio obtained with the parasites transfected with pXCAT3'H2A was higher than that observed for parasites transfected with pXCAT5'H2A. This ratio closely resembles the de novo synthesis ratio observed in the parasites transfected with the construct containing the two H2A regulatory regions (pXCAT5'III/3'I). In the same cell lines the percentage of CAT mRNA on polysomes was also analyzed (Fig. 7B). The distribution of the CAT transcripts on polysomes in parasites transfected with pXCAT3'H2A and pXCAT5'III/3'I constructs showed similar profiles, whereas a higher percentage of CAT transcripts was found on polysomes in the G1 phase in the pXCAT5'H2A transfected cell line. Altogether, these data indicate that although the 5' UTR and 3' UTR regions of the H2A gene are implicated in protein synthesis regulation, the 3' UTR plays the major role in the observed cell cycle dependent translation.


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)

Analysis of the CAT cell cycle regulated expression in different constructs. (A) Ratio between the de novo CAT synthesis (normalised to the total amount of CAT) and number of cells in the S phase (3 h after HU removal) determined by three independent assays in cell lines transfected with pXCATHSP83, pXCAT5'III/3'I, pXCAT5'H2A and pXCAT3'H2A constructs. (B) Graphic showing the percentages of CAT mRNA on polysomes in the same cell lines. Data were obtained from the densitometric analysis of the blots performed with the RNAs separated on sucrose gradients from cell lines transfected with pXCATHSP83, pXCAT5'III/3'I, pXCAT5'H2A and pXCAT3'H2A constructs at G1 and S phase for three independent assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691400&req=5

Figure 7: Analysis of the CAT cell cycle regulated expression in different constructs. (A) Ratio between the de novo CAT synthesis (normalised to the total amount of CAT) and number of cells in the S phase (3 h after HU removal) determined by three independent assays in cell lines transfected with pXCATHSP83, pXCAT5'III/3'I, pXCAT5'H2A and pXCAT3'H2A constructs. (B) Graphic showing the percentages of CAT mRNA on polysomes in the same cell lines. Data were obtained from the densitometric analysis of the blots performed with the RNAs separated on sucrose gradients from cell lines transfected with pXCATHSP83, pXCAT5'III/3'I, pXCAT5'H2A and pXCAT3'H2A constructs at G1 and S phase for three independent assays.
Mentions: Taking into account the variations observed in the percentages of cells in the S-phase between experiments, three independent assays were performed with the cell lines transfected with pXCATHSP83, pXCAT5'III/3'I, pXCAT5'H2A and pXCAT3'H2A constructs. The ratio between the de novo synthesis of CAT (normalised to the total amount) and the amount of cells in the S phase in the different transfected cell lines was determined (Fig. 7A). An increase was observed in this ratio in both the pXCAT5'H2A and pXCAT3'H2A cell lines compared with pXCATHSP83 cell line. In addition, the ratio obtained with the parasites transfected with pXCAT3'H2A was higher than that observed for parasites transfected with pXCAT5'H2A. This ratio closely resembles the de novo synthesis ratio observed in the parasites transfected with the construct containing the two H2A regulatory regions (pXCAT5'III/3'I). In the same cell lines the percentage of CAT mRNA on polysomes was also analyzed (Fig. 7B). The distribution of the CAT transcripts on polysomes in parasites transfected with pXCAT3'H2A and pXCAT5'III/3'I constructs showed similar profiles, whereas a higher percentage of CAT transcripts was found on polysomes in the G1 phase in the pXCAT5'H2A transfected cell line. Altogether, these data indicate that although the 5' UTR and 3' UTR regions of the H2A gene are implicated in protein synthesis regulation, the 3' UTR plays the major role in the observed cell cycle dependent translation.

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