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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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Ratio between the de novo CAT synthesis and number of cells in the S phase of the cell cycle. Ratio between the de novo CAT synthesis (normalised to the total amount of CAT) and the number of cells in the S phase (3 h after HU removal) determined by three independent assays. The assays were performed for the transfectants bearing both the 5' and 3' UTR H2A gene regulatory regions as well as for the transfectant with the HSP83 gene UTRs
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Figure 3: Ratio between the de novo CAT synthesis and number of cells in the S phase of the cell cycle. Ratio between the de novo CAT synthesis (normalised to the total amount of CAT) and the number of cells in the S phase (3 h after HU removal) determined by three independent assays. The assays were performed for the transfectants bearing both the 5' and 3' UTR H2A gene regulatory regions as well as for the transfectant with the HSP83 gene UTRs

Mentions: Fig. 2 summarizes the studies on the effect of the flanking sequences of H2A genes on the cell cycle dependent expression of the CAT gene. For that purpose the different transfected promastigote cell lines were treated for 12 h with hydroxyurea (HU) and after drug removal cells were collected at different time periods and stained with propidium iodide to determine the percentage of cells in the different cell-cycle phases. HU removal resulted in a semi-synchronous entry into the cell cycle in L. infantum promastigotes, in line with previous reports [11]. At the same time the rate of the de novo synthesis of CAT was determined by immunoprecipitation with an anti-CAT polyclonal antibody on protein extracts labelled with [35S]methionine. The autoradiograph exposure of the blot showed that 35S-labelling of CAT reached a maximum at 3 h and then decreased to initial levels 9 h after HU removal for all the cell lines containing the regulatory regions of the different H2A genes (Fig. 2A–D). Densitometric analysis of the results showed a 3- to 4-fold increase at the 3 h point, correlating to the maximum percentage of cells in the S phase determined by flow cytometry. On the other hand, the amount of radioactivity bound to the immunoprecipitated CAT in the cell line transfected with pXCATHSP83 plasmid did not show the cell cycle specific expression profile observed for the other constructs (Fig. 2E). Since in the context of these experiments variations were observed in the percentage of cells in the S phase (3 h after HU removal) three independent experiments were performed with all the cell lines showed in Fig. 2, and the ratio between the de novo synthesis (normalised to the total amount of CAT) and cells in the S phase (3 h after HU removal) was determined. A significant increase was observed in this ratio when comparing the transfectants bearing the different H2A gene regulatory regions to the transfectant with the HSP83 gene UTRs (Fig. 3). In Fig. 3 it is also shown that transfectants bearing the 3' UTR-III, independently of the 5' UTR, have a moderately higher ratio which is indicative of a higher translational potential.


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)

Ratio between the de novo CAT synthesis and number of cells in the S phase of the cell cycle. Ratio between the de novo CAT synthesis (normalised to the total amount of CAT) and the number of cells in the S phase (3 h after HU removal) determined by three independent assays. The assays were performed for the transfectants bearing both the 5' and 3' UTR H2A gene regulatory regions as well as for the transfectant with the HSP83 gene UTRs
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Ratio between the de novo CAT synthesis and number of cells in the S phase of the cell cycle. Ratio between the de novo CAT synthesis (normalised to the total amount of CAT) and the number of cells in the S phase (3 h after HU removal) determined by three independent assays. The assays were performed for the transfectants bearing both the 5' and 3' UTR H2A gene regulatory regions as well as for the transfectant with the HSP83 gene UTRs
Mentions: Fig. 2 summarizes the studies on the effect of the flanking sequences of H2A genes on the cell cycle dependent expression of the CAT gene. For that purpose the different transfected promastigote cell lines were treated for 12 h with hydroxyurea (HU) and after drug removal cells were collected at different time periods and stained with propidium iodide to determine the percentage of cells in the different cell-cycle phases. HU removal resulted in a semi-synchronous entry into the cell cycle in L. infantum promastigotes, in line with previous reports [11]. At the same time the rate of the de novo synthesis of CAT was determined by immunoprecipitation with an anti-CAT polyclonal antibody on protein extracts labelled with [35S]methionine. The autoradiograph exposure of the blot showed that 35S-labelling of CAT reached a maximum at 3 h and then decreased to initial levels 9 h after HU removal for all the cell lines containing the regulatory regions of the different H2A genes (Fig. 2A–D). Densitometric analysis of the results showed a 3- to 4-fold increase at the 3 h point, correlating to the maximum percentage of cells in the S phase determined by flow cytometry. On the other hand, the amount of radioactivity bound to the immunoprecipitated CAT in the cell line transfected with pXCATHSP83 plasmid did not show the cell cycle specific expression profile observed for the other constructs (Fig. 2E). Since in the context of these experiments variations were observed in the percentage of cells in the S phase (3 h after HU removal) three independent experiments were performed with all the cell lines showed in Fig. 2, and the ratio between the de novo synthesis (normalised to the total amount of CAT) and cells in the S phase (3 h after HU removal) was determined. A significant increase was observed in this ratio when comparing the transfectants bearing the different H2A gene regulatory regions to the transfectant with the HSP83 gene UTRs (Fig. 3). In Fig. 3 it is also shown that transfectants bearing the 3' UTR-III, independently of the 5' UTR, have a moderately higher ratio which is indicative of a higher translational potential.

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