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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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DNA constructs. (A) Schematic representation of the two Leishmania infantum H2A loci. The names of the genes are indicated below the maps. The locations of the different 5'(I-IV) and 3' UTR (I-III) are indicated on the diagram. Due to the sequence similarity 5'UTR-I and -II are represented as white boxes and 5'UTR-III and -IV are represented as black boxes. These sequences are in the EMBL databases under the accession numbers AJ419625 and AJ419627. (B) Generic representation of the pX63NEO recombinant vector. Position of the BamHI cut sites employed in the cloning are indicated as B. Positions of the 5'UTR + upstream regions (UPR) and 3' UTR + downstream regions (DNR) relative to CAT gene coding region are indicated. Transcriptional directions of the NEO and CAT genes are as indicated. (C) Schematic outlines of the constructs used in this work indicating the name of the clone and the type of 5'UTR+UPR and 3'UTR+DNR included around the CAT coding region. (D) Northern blots of RNA from promastigotes stably transfected with the different constructs and hybridized with radiollabeled oligonucleotide complementary to the 3' terminal region of CAT gene coding region. Ethidium bromide staining of the corresponding gel is also shown (rRNA panel).
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Figure 1: DNA constructs. (A) Schematic representation of the two Leishmania infantum H2A loci. The names of the genes are indicated below the maps. The locations of the different 5'(I-IV) and 3' UTR (I-III) are indicated on the diagram. Due to the sequence similarity 5'UTR-I and -II are represented as white boxes and 5'UTR-III and -IV are represented as black boxes. These sequences are in the EMBL databases under the accession numbers AJ419625 and AJ419627. (B) Generic representation of the pX63NEO recombinant vector. Position of the BamHI cut sites employed in the cloning are indicated as B. Positions of the 5'UTR + upstream regions (UPR) and 3' UTR + downstream regions (DNR) relative to CAT gene coding region are indicated. Transcriptional directions of the NEO and CAT genes are as indicated. (C) Schematic outlines of the constructs used in this work indicating the name of the clone and the type of 5'UTR+UPR and 3'UTR+DNR included around the CAT coding region. (D) Northern blots of RNA from promastigotes stably transfected with the different constructs and hybridized with radiollabeled oligonucleotide complementary to the 3' terminal region of CAT gene coding region. Ethidium bromide staining of the corresponding gel is also shown (rRNA panel).

Mentions: Two H2A loci are present in the L. infantum genome each one located in a different chromosome (Chr21 and Chr29 [24]) (Fig. 1A). The three gene copies located in each locus although highly conserved in the coding region present major sequence differences in the 5' UTRs and in the 3' UTRs. There are four types of 5' UTRs, named 5' UTR-I to -IV, with remarkable sequence conservation between the 5' UTR-I and 5' UTR-II and between the 5' UTR-III and 5' UTR-IV, respectively [25]. On the other hand, in the H2A loci there are three types of 3' UTR that are completely divergent in their nucleotide sequence (namely 3' UTR-I, -II and -III). An analysis of the relative steady-state levels of the different L. infantum H2A mRNAs revealed that transcripts containing the 3' UTR-I and 3' UTR-III account for the majority of the H2A transcripts in logarithmic-phase growing promastigotes. In addition, the 3' UTR-I containing transcripts are approximately 7-fold more abundant than those containing the 3' UTR-III. Very low steady-state levels were found for transcripts that contain the 3' UTR-II [25]. For that reason 3'UTR-II has been excluded in our analysis.


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)

DNA constructs. (A) Schematic representation of the two Leishmania infantum H2A loci. The names of the genes are indicated below the maps. The locations of the different 5'(I-IV) and 3' UTR (I-III) are indicated on the diagram. Due to the sequence similarity 5'UTR-I and -II are represented as white boxes and 5'UTR-III and -IV are represented as black boxes. These sequences are in the EMBL databases under the accession numbers AJ419625 and AJ419627. (B) Generic representation of the pX63NEO recombinant vector. Position of the BamHI cut sites employed in the cloning are indicated as B. Positions of the 5'UTR + upstream regions (UPR) and 3' UTR + downstream regions (DNR) relative to CAT gene coding region are indicated. Transcriptional directions of the NEO and CAT genes are as indicated. (C) Schematic outlines of the constructs used in this work indicating the name of the clone and the type of 5'UTR+UPR and 3'UTR+DNR included around the CAT coding region. (D) Northern blots of RNA from promastigotes stably transfected with the different constructs and hybridized with radiollabeled oligonucleotide complementary to the 3' terminal region of CAT gene coding region. Ethidium bromide staining of the corresponding gel is also shown (rRNA panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: DNA constructs. (A) Schematic representation of the two Leishmania infantum H2A loci. The names of the genes are indicated below the maps. The locations of the different 5'(I-IV) and 3' UTR (I-III) are indicated on the diagram. Due to the sequence similarity 5'UTR-I and -II are represented as white boxes and 5'UTR-III and -IV are represented as black boxes. These sequences are in the EMBL databases under the accession numbers AJ419625 and AJ419627. (B) Generic representation of the pX63NEO recombinant vector. Position of the BamHI cut sites employed in the cloning are indicated as B. Positions of the 5'UTR + upstream regions (UPR) and 3' UTR + downstream regions (DNR) relative to CAT gene coding region are indicated. Transcriptional directions of the NEO and CAT genes are as indicated. (C) Schematic outlines of the constructs used in this work indicating the name of the clone and the type of 5'UTR+UPR and 3'UTR+DNR included around the CAT coding region. (D) Northern blots of RNA from promastigotes stably transfected with the different constructs and hybridized with radiollabeled oligonucleotide complementary to the 3' terminal region of CAT gene coding region. Ethidium bromide staining of the corresponding gel is also shown (rRNA panel).
Mentions: Two H2A loci are present in the L. infantum genome each one located in a different chromosome (Chr21 and Chr29 [24]) (Fig. 1A). The three gene copies located in each locus although highly conserved in the coding region present major sequence differences in the 5' UTRs and in the 3' UTRs. There are four types of 5' UTRs, named 5' UTR-I to -IV, with remarkable sequence conservation between the 5' UTR-I and 5' UTR-II and between the 5' UTR-III and 5' UTR-IV, respectively [25]. On the other hand, in the H2A loci there are three types of 3' UTR that are completely divergent in their nucleotide sequence (namely 3' UTR-I, -II and -III). An analysis of the relative steady-state levels of the different L. infantum H2A mRNAs revealed that transcripts containing the 3' UTR-I and 3' UTR-III account for the majority of the H2A transcripts in logarithmic-phase growing promastigotes. In addition, the 3' UTR-I containing transcripts are approximately 7-fold more abundant than those containing the 3' UTR-III. Very low steady-state levels were found for transcripts that contain the 3' UTR-II [25]. For that reason 3'UTR-II has been excluded in our analysis.

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