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Regulation of PURA gene transcription by three promoters generating distinctly spliced 5-prime leaders: a novel means of fine control over tissue specificity and viral signals.

Wortman MJ, Hanson LK, Martínez-Sobrido L, Campbell AE, Nance JA, García-Sastre A, Johnson EM - BMC Mol. Biol. (2010)

Bottom Line: Chromatin immunoprecipitation reveals that IRF-3 protein binds hPURA promoter sequences at TSS II in vivo.The viral infection alters the degree of splicing of the 5'-UTR introns of TSS II transcripts.Results provide evidence for a novel mechanism of transcriptional control by multiple promoters used differently in various tissues and cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, 700 W, Olney Road, Norfolk, VA 23507, USA. wortmamj@evms.edu

ABSTRACT

Background: Purα is an evolutionarily conserved cellular protein participating in processes of DNA replication, transcription, and RNA transport; all involving binding to nucleic acids and altering conformation and physical positioning. The distinct but related roles of Purα suggest a need for expression regulated differently depending on intracellular and external signals.

Results: Here we report that human PURA (hPURA) transcription is regulated from three distinct and widely-separated transcription start sites (TSS). Each of these TSS is strongly homologous to a similar site in mouse chromosomal DNA. Transcripts from TSS I and II are characterized by the presence of large and overlapping 5'-UTR introns terminated at the same splice receptor site. Transfection of lung carcinoma cells with wild-type or mutated hPURA 5' upstream sequences identifies different regulatory elements. TSS III, located within 80 bp of the translational start codon, is upregulated by E2F1, CAAT and NF-Y binding elements. Transcription at TSS II is downregulated through the presence of adjacent consensus binding elements for interferon regulatory factors (IRFs). Chromatin immunoprecipitation reveals that IRF-3 protein binds hPURA promoter sequences at TSS II in vivo. By co-transfecting hPURA reporter plasmids with expression plasmids for IRF proteins we demonstrate that several IRFs, including IRF-3, down-regulate PURA transcription. Infection of NIH 3T3 cells with mouse cytomegalovirus results in a rapid decrease in levels of mPURA mRNA and Purα protein. The viral infection alters the degree of splicing of the 5'-UTR introns of TSS II transcripts.

Conclusions: Results provide evidence for a novel mechanism of transcriptional control by multiple promoters used differently in various tissues and cells. Viral infection alters not only the use of PURA promoters but also the generation of different non-coding RNAs from 5'-UTRs of the resulting transcripts.

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Comparison of human (A) and mouse (B) PURA transcriptional start sites and 5' untranslated regions. Three distinct transcriptional start sites (TSSs) initiate transcription of human PURA. Human and mouse EST databases were utilized to determine TSSs of PURA. Numbering is from the A in the translationally- initiating ATG. A. TSS I - III are shown for humans. B. Two TSSs as determined from ESTs are shown for mouse. The one most distant from ATG has previously been identified [28]. The new mouse TSS is very homologous to human TSS III, which is verified in this report. Human transcripts starting at sites I and II are characterized by introns of dissimilar lengths, as indicated in A. The genomic locations of human and mouse PURA are given.
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Figure 1: Comparison of human (A) and mouse (B) PURA transcriptional start sites and 5' untranslated regions. Three distinct transcriptional start sites (TSSs) initiate transcription of human PURA. Human and mouse EST databases were utilized to determine TSSs of PURA. Numbering is from the A in the translationally- initiating ATG. A. TSS I - III are shown for humans. B. Two TSSs as determined from ESTs are shown for mouse. The one most distant from ATG has previously been identified [28]. The new mouse TSS is very homologous to human TSS III, which is verified in this report. Human transcripts starting at sites I and II are characterized by introns of dissimilar lengths, as indicated in A. The genomic locations of human and mouse PURA are given.

Mentions: Because the Purα protein sequence is so highly conserved [1], we sought to analyze hPURA transcriptional regulation for clues as to how the regulation of PURA expression can adapt to important cellular processes. As hPURA has no TATA-like sequence, there is no single obvious site for the initiation of transcription. To locate established PURA transcription start sites (TSSs) we queried both mouse and human EST databases using the BLAST algorithm [26]. Because Purα is very widely distributed in human tissues, the large database of Purα ESTs provides multiple high-quality sequences. Intriguingly, the PURA transcripts could be grouped into three distinct lengths, each of which contains multiple closely-spaced 5'ends (Figure 1, Table 1). The discrete nature of these length groups indicates that each corresponds to a separate transcriptional start site (TSS). The 5' ends of the transcripts in TSS I, located most distant from the translational start codon, correspond to the only thus-far-demonstrated mouse TSS [27,28]. The sequence surrounding this TSS is strongly conserved in mouse and humans (Additional file 1, Figure S1) and is, therefore, likely to be a human TSS. We confirmed the utilization of TSS I in humans using hPURA promoter specific primers 0 and 14 (Figure 2A) in rt-PCR analysis of human lung RNA. These primers, whose 5' ends are located 6133 and 115 bp respectively, upstream of the translational start codon, yield an 80 bp rt-PCR product (Figure 2B). Sequencing this product confirmed the removal of a 5,939 base intron (Figure 2C). The Group I transcripts are, therefore, characterized by the removal of a large intron of 5,481 bp in mouse and 5,939 bp in human. In both species this intron is completely within the 5'-UTR. It is not extensively analyzed here, as it has already been analyzed in the mouse [28].


Regulation of PURA gene transcription by three promoters generating distinctly spliced 5-prime leaders: a novel means of fine control over tissue specificity and viral signals.

Wortman MJ, Hanson LK, Martínez-Sobrido L, Campbell AE, Nance JA, García-Sastre A, Johnson EM - BMC Mol. Biol. (2010)

Comparison of human (A) and mouse (B) PURA transcriptional start sites and 5' untranslated regions. Three distinct transcriptional start sites (TSSs) initiate transcription of human PURA. Human and mouse EST databases were utilized to determine TSSs of PURA. Numbering is from the A in the translationally- initiating ATG. A. TSS I - III are shown for humans. B. Two TSSs as determined from ESTs are shown for mouse. The one most distant from ATG has previously been identified [28]. The new mouse TSS is very homologous to human TSS III, which is verified in this report. Human transcripts starting at sites I and II are characterized by introns of dissimilar lengths, as indicated in A. The genomic locations of human and mouse PURA are given.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992531&req=5

Figure 1: Comparison of human (A) and mouse (B) PURA transcriptional start sites and 5' untranslated regions. Three distinct transcriptional start sites (TSSs) initiate transcription of human PURA. Human and mouse EST databases were utilized to determine TSSs of PURA. Numbering is from the A in the translationally- initiating ATG. A. TSS I - III are shown for humans. B. Two TSSs as determined from ESTs are shown for mouse. The one most distant from ATG has previously been identified [28]. The new mouse TSS is very homologous to human TSS III, which is verified in this report. Human transcripts starting at sites I and II are characterized by introns of dissimilar lengths, as indicated in A. The genomic locations of human and mouse PURA are given.
Mentions: Because the Purα protein sequence is so highly conserved [1], we sought to analyze hPURA transcriptional regulation for clues as to how the regulation of PURA expression can adapt to important cellular processes. As hPURA has no TATA-like sequence, there is no single obvious site for the initiation of transcription. To locate established PURA transcription start sites (TSSs) we queried both mouse and human EST databases using the BLAST algorithm [26]. Because Purα is very widely distributed in human tissues, the large database of Purα ESTs provides multiple high-quality sequences. Intriguingly, the PURA transcripts could be grouped into three distinct lengths, each of which contains multiple closely-spaced 5'ends (Figure 1, Table 1). The discrete nature of these length groups indicates that each corresponds to a separate transcriptional start site (TSS). The 5' ends of the transcripts in TSS I, located most distant from the translational start codon, correspond to the only thus-far-demonstrated mouse TSS [27,28]. The sequence surrounding this TSS is strongly conserved in mouse and humans (Additional file 1, Figure S1) and is, therefore, likely to be a human TSS. We confirmed the utilization of TSS I in humans using hPURA promoter specific primers 0 and 14 (Figure 2A) in rt-PCR analysis of human lung RNA. These primers, whose 5' ends are located 6133 and 115 bp respectively, upstream of the translational start codon, yield an 80 bp rt-PCR product (Figure 2B). Sequencing this product confirmed the removal of a 5,939 base intron (Figure 2C). The Group I transcripts are, therefore, characterized by the removal of a large intron of 5,481 bp in mouse and 5,939 bp in human. In both species this intron is completely within the 5'-UTR. It is not extensively analyzed here, as it has already been analyzed in the mouse [28].

Bottom Line: Chromatin immunoprecipitation reveals that IRF-3 protein binds hPURA promoter sequences at TSS II in vivo.The viral infection alters the degree of splicing of the 5'-UTR introns of TSS II transcripts.Results provide evidence for a novel mechanism of transcriptional control by multiple promoters used differently in various tissues and cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, 700 W, Olney Road, Norfolk, VA 23507, USA. wortmamj@evms.edu

ABSTRACT

Background: Purα is an evolutionarily conserved cellular protein participating in processes of DNA replication, transcription, and RNA transport; all involving binding to nucleic acids and altering conformation and physical positioning. The distinct but related roles of Purα suggest a need for expression regulated differently depending on intracellular and external signals.

Results: Here we report that human PURA (hPURA) transcription is regulated from three distinct and widely-separated transcription start sites (TSS). Each of these TSS is strongly homologous to a similar site in mouse chromosomal DNA. Transcripts from TSS I and II are characterized by the presence of large and overlapping 5'-UTR introns terminated at the same splice receptor site. Transfection of lung carcinoma cells with wild-type or mutated hPURA 5' upstream sequences identifies different regulatory elements. TSS III, located within 80 bp of the translational start codon, is upregulated by E2F1, CAAT and NF-Y binding elements. Transcription at TSS II is downregulated through the presence of adjacent consensus binding elements for interferon regulatory factors (IRFs). Chromatin immunoprecipitation reveals that IRF-3 protein binds hPURA promoter sequences at TSS II in vivo. By co-transfecting hPURA reporter plasmids with expression plasmids for IRF proteins we demonstrate that several IRFs, including IRF-3, down-regulate PURA transcription. Infection of NIH 3T3 cells with mouse cytomegalovirus results in a rapid decrease in levels of mPURA mRNA and Purα protein. The viral infection alters the degree of splicing of the 5'-UTR introns of TSS II transcripts.

Conclusions: Results provide evidence for a novel mechanism of transcriptional control by multiple promoters used differently in various tissues and cells. Viral infection alters not only the use of PURA promoters but also the generation of different non-coding RNAs from 5'-UTRs of the resulting transcripts.

Show MeSH
Related in: MedlinePlus