Abstract
BackgroundPurα 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.ResultsHere 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.ConclusionsResults 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.
Highlights
Pura 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
Three distinct regions for the initiation of human PURA transcription Because the Pura 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
The 5’ends of a second transcript size group are located approximately 1249 bp upstream of the translational start codon (Figure 1)
Summary
Pura 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 Pur protein family of sequence-specific singlestranded nucleic acid-binding proteins is extremely well conserved from bacteria through humans [1]. Human and mouse Pura differ by only 2 amino acids. Pura, has a primitive codon usage preference resembling that in bacteria. Pura was first identified due to its ability to bind a purine-rich sequence in an initiation simultaneously bound to the replication origin near the cMYC gene [9]. Pura has been associated with viral DNA replication. Pura binds the JC viral (JCV) origin of replication and at higher concentrations, inhibits JCV DNA replication [10]. At lower concentrations Pura cooperatively interacts with HIV-1 Tat and large T-antigen to enhance JCV DNA replication [11,12]
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