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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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Cell cycle dependent expression of CAT is regulated at the translational level. (A) Northern-blot analysis of total RNA samples for promastigotes transfected with pXCAT5'III/3'I treated with 5 mM HU either for 12 h (lane 0) or at the indicated time (in h) after removal of the drug. The blot was sequentially probed with an oligonucleotide reverse and complementary to the 3' end of the CAT coding region and with a cDNA coding for L. infantum H4. Ethidium bromide staining of the corresponding gel is also shown (rRNA panel). (B) Graphic showing the ratio between the densitometric values of the blots hybridized with CAT probe (black columns) or H4 probe (white columns) and the rRNA bands revealed by ethidium bromide staining of the gel. Also the percentage of cells in the S phase of the cell cycle determined by flow cytometric analysis (black line) is shown. (C) Cytoplasmic extracts from L. infantum promastigotes transfected with pXCAT5'III/3'I and treated with 5 mM HU either for 12 h or 3 h after drug removal were separated on 15–40% sucrose linear gradients and RNA was purified and resolved in a 1% agarose-formaldehyde gel. The ethidium bromide staining of the gel is shown. Migration of 18 S, 24 S-α and 24 S-β are indicated. A graphic showing the densitometric values of the gel obtained 3 h after drug removal is included. The migration of the 80S particles has been marked in the gradients by an asterisk. (D) RNA from these gels were transferred on to nylon membranes and sequentially probed with the CAT and H4 probes. The autoradiographic exposure of the blot and the densitometric analysis is shown. Results are plotted as percentages of the total signal to allow direct comparison of the polysomal profiles. Data correspond to one representative experiment of three independent assays with similar results.
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Figure 4: Cell cycle dependent expression of CAT is regulated at the translational level. (A) Northern-blot analysis of total RNA samples for promastigotes transfected with pXCAT5'III/3'I treated with 5 mM HU either for 12 h (lane 0) or at the indicated time (in h) after removal of the drug. The blot was sequentially probed with an oligonucleotide reverse and complementary to the 3' end of the CAT coding region and with a cDNA coding for L. infantum H4. Ethidium bromide staining of the corresponding gel is also shown (rRNA panel). (B) Graphic showing the ratio between the densitometric values of the blots hybridized with CAT probe (black columns) or H4 probe (white columns) and the rRNA bands revealed by ethidium bromide staining of the gel. Also the percentage of cells in the S phase of the cell cycle determined by flow cytometric analysis (black line) is shown. (C) Cytoplasmic extracts from L. infantum promastigotes transfected with pXCAT5'III/3'I and treated with 5 mM HU either for 12 h or 3 h after drug removal were separated on 15–40% sucrose linear gradients and RNA was purified and resolved in a 1% agarose-formaldehyde gel. The ethidium bromide staining of the gel is shown. Migration of 18 S, 24 S-α and 24 S-β are indicated. A graphic showing the densitometric values of the gel obtained 3 h after drug removal is included. The migration of the 80S particles has been marked in the gradients by an asterisk. (D) RNA from these gels were transferred on to nylon membranes and sequentially probed with the CAT and H4 probes. The autoradiographic exposure of the blot and the densitometric analysis is shown. Results are plotted as percentages of the total signal to allow direct comparison of the polysomal profiles. Data correspond to one representative experiment of three independent assays with similar results.

Mentions: In order to analyze where the control of CAT gene expression is exerted, two different assays using promastigotes stably transfected with pXCAT5'III/3'I construct (containing the regulatory regions of gene H2A4) were performed. Firstly, a Northern blot of RNA isolated at various time points after HU release was hybridized with a CAT probe (Fig. 4A). Quantification of the obtained radioactive signals normalized to all ribosomal rRNA bands indicated that CAT mRNA levels remain constant throughout the cell cycle (Fig. 4B). Secondly, we analyzed the distribution of CAT mRNAs on polyribosomes during the progression from G1 and S phase. For that purpose the polysomal profile of the CAT transcripts in promastigotes at phase G1 (parasites treated for 12 h with 5 mM HU) and mid-S phase (3 h after HU release) was studied by sucrose gradient centrifugation of cytosolic extracts and Northern blotting. According to the rRNA species distribution on the gradients deduced from the ethidium bromide staining, fractions 1–5 should be considered to be free of functional ribosomes, since they either do not contain rRNAs or the rRNAs are not in equimolecular amounts. Equimolarity of the three larger 18 S, 24 S-α and 24 S-β rRNAs composing the L. infantum ribosome [29] was observed between aliquots 6–15 that correspond with the polyribosomal fractions (Fig. 4C). The distribution of the CAT mRNAs along the sucrose gradient fractions is shown in Fig 4D. During the G1 phase, CAT transcripts concentrate on the top of the gradient showing a peak out of the polysome fractions. On the contrary, when 50% of the cells were in the S phase CAT mRNA was detected in the bottom fractions. In particular, a secondary peak containing 57% of the CAT mRNAs was observed in the polyribosomal fractions. A similar pattern was obtained when blots were re-hybridized with a H4 probe although the percentage of H4 mRNAs in the polyribosome fractions was lower (41% of the H4 mRNAs) and the secondary peak was located in the central fractions of the sucrose gradient. The same results showed for H4 were obtained when a H2A coding region probe was tested in these blots (data not shown). Differences in the translational degree observed in the CAT mRNAs polysome profiles in the S phase relative to the endogenous H4 or H2A could be related to the influence of the CAT coding region on the secondary structure of the mRNAs since it has been recently described that the coding regions of different reporter genes may affect the translational efficiency in kinetoplastids [30]. Together, these results demonstrate that the cell-cycle related expression of CAT is controlled in the transfected parasites by the same regulatory events that control the expression of the histone genes.


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)

Cell cycle dependent expression of CAT is regulated at the translational level. (A) Northern-blot analysis of total RNA samples for promastigotes transfected with pXCAT5'III/3'I treated with 5 mM HU either for 12 h (lane 0) or at the indicated time (in h) after removal of the drug. The blot was sequentially probed with an oligonucleotide reverse and complementary to the 3' end of the CAT coding region and with a cDNA coding for L. infantum H4. Ethidium bromide staining of the corresponding gel is also shown (rRNA panel). (B) Graphic showing the ratio between the densitometric values of the blots hybridized with CAT probe (black columns) or H4 probe (white columns) and the rRNA bands revealed by ethidium bromide staining of the gel. Also the percentage of cells in the S phase of the cell cycle determined by flow cytometric analysis (black line) is shown. (C) Cytoplasmic extracts from L. infantum promastigotes transfected with pXCAT5'III/3'I and treated with 5 mM HU either for 12 h or 3 h after drug removal were separated on 15–40% sucrose linear gradients and RNA was purified and resolved in a 1% agarose-formaldehyde gel. The ethidium bromide staining of the gel is shown. Migration of 18 S, 24 S-α and 24 S-β are indicated. A graphic showing the densitometric values of the gel obtained 3 h after drug removal is included. The migration of the 80S particles has been marked in the gradients by an asterisk. (D) RNA from these gels were transferred on to nylon membranes and sequentially probed with the CAT and H4 probes. The autoradiographic exposure of the blot and the densitometric analysis is shown. Results are plotted as percentages of the total signal to allow direct comparison of the polysomal profiles. Data correspond to one representative experiment of three independent assays with similar results.
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Figure 4: Cell cycle dependent expression of CAT is regulated at the translational level. (A) Northern-blot analysis of total RNA samples for promastigotes transfected with pXCAT5'III/3'I treated with 5 mM HU either for 12 h (lane 0) or at the indicated time (in h) after removal of the drug. The blot was sequentially probed with an oligonucleotide reverse and complementary to the 3' end of the CAT coding region and with a cDNA coding for L. infantum H4. Ethidium bromide staining of the corresponding gel is also shown (rRNA panel). (B) Graphic showing the ratio between the densitometric values of the blots hybridized with CAT probe (black columns) or H4 probe (white columns) and the rRNA bands revealed by ethidium bromide staining of the gel. Also the percentage of cells in the S phase of the cell cycle determined by flow cytometric analysis (black line) is shown. (C) Cytoplasmic extracts from L. infantum promastigotes transfected with pXCAT5'III/3'I and treated with 5 mM HU either for 12 h or 3 h after drug removal were separated on 15–40% sucrose linear gradients and RNA was purified and resolved in a 1% agarose-formaldehyde gel. The ethidium bromide staining of the gel is shown. Migration of 18 S, 24 S-α and 24 S-β are indicated. A graphic showing the densitometric values of the gel obtained 3 h after drug removal is included. The migration of the 80S particles has been marked in the gradients by an asterisk. (D) RNA from these gels were transferred on to nylon membranes and sequentially probed with the CAT and H4 probes. The autoradiographic exposure of the blot and the densitometric analysis is shown. Results are plotted as percentages of the total signal to allow direct comparison of the polysomal profiles. Data correspond to one representative experiment of three independent assays with similar results.
Mentions: In order to analyze where the control of CAT gene expression is exerted, two different assays using promastigotes stably transfected with pXCAT5'III/3'I construct (containing the regulatory regions of gene H2A4) were performed. Firstly, a Northern blot of RNA isolated at various time points after HU release was hybridized with a CAT probe (Fig. 4A). Quantification of the obtained radioactive signals normalized to all ribosomal rRNA bands indicated that CAT mRNA levels remain constant throughout the cell cycle (Fig. 4B). Secondly, we analyzed the distribution of CAT mRNAs on polyribosomes during the progression from G1 and S phase. For that purpose the polysomal profile of the CAT transcripts in promastigotes at phase G1 (parasites treated for 12 h with 5 mM HU) and mid-S phase (3 h after HU release) was studied by sucrose gradient centrifugation of cytosolic extracts and Northern blotting. According to the rRNA species distribution on the gradients deduced from the ethidium bromide staining, fractions 1–5 should be considered to be free of functional ribosomes, since they either do not contain rRNAs or the rRNAs are not in equimolecular amounts. Equimolarity of the three larger 18 S, 24 S-α and 24 S-β rRNAs composing the L. infantum ribosome [29] was observed between aliquots 6–15 that correspond with the polyribosomal fractions (Fig. 4C). The distribution of the CAT mRNAs along the sucrose gradient fractions is shown in Fig 4D. During the G1 phase, CAT transcripts concentrate on the top of the gradient showing a peak out of the polysome fractions. On the contrary, when 50% of the cells were in the S phase CAT mRNA was detected in the bottom fractions. In particular, a secondary peak containing 57% of the CAT mRNAs was observed in the polyribosomal fractions. A similar pattern was obtained when blots were re-hybridized with a H4 probe although the percentage of H4 mRNAs in the polyribosome fractions was lower (41% of the H4 mRNAs) and the secondary peak was located in the central fractions of the sucrose gradient. The same results showed for H4 were obtained when a H2A coding region probe was tested in these blots (data not shown). Differences in the translational degree observed in the CAT mRNAs polysome profiles in the S phase relative to the endogenous H4 or H2A could be related to the influence of the CAT coding region on the secondary structure of the mRNAs since it has been recently described that the coding regions of different reporter genes may affect the translational efficiency in kinetoplastids [30]. Together, these results demonstrate that the cell-cycle related expression of CAT is controlled in the transfected parasites by the same regulatory events that control the expression of the histone genes.

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