Regulation of Purα gene transcription: Evidence for autoregulation of Purα promoter

Abstract

The single-stranded DNA and RNA binding protein, Puralpha, has recently received special attention as this protein, by associating with the specific nucleotide sequence (GGN repeats) and/or several important cellular and viral proteins regulates crucial biological events such as transcription, replication, and cell proliferation. In this study, we focused on the promoter activity of the Puralpha upstream DNA sequence and demonstrated that the sequence spanning 6,000 nucleotides upstream of the Puralpha transcription start site has promoter activity in various cell types. Results from promoter deletion studies revealed that this region encompasses various regulatory motifs which differentially participate in the promoter activity of Puralpha in various cells. The transcription start site of Puralpha is surrounded by the GA/GC-rich sequence which exhibits the ability to interact with Puralpha, suggesting a role for autoregulation of Puralpha transcription. Results from co-transfection studies revealed that ectopic expression of Puralpha reduced transcriptional activity of the Puralpha promoter and the region located between amino acid residues, 1-85 of Puralpha is important for the observed autoregulatory event. The regulatory protein of the human neurotropic virus, JCV, T-antigen, which interacts with Puralpha, decreased transcriptional activity of the Puralpha promoter. Co-expression of JCV T-antigen and Puralpha had no significant effect on the suppression of Puralpha gene transcription by either protein. The importance of this finding in light of earlier results showing down regulation of Puralpha during JCV infection of glial cells is discussed.

Regulation of the Pur-alpha promoter by E2F-1

“…The plasmids pCDNA‐E‐RNA and pCDNA‐P‐RNA have been described previously [Darbinian et al, 2004]. Luciferase reporter plasmids containing the mouse pPurα and promoter deletion mutants have been described previously [Muralidharan et al, 2001] except for pPurα (δ5′UTR). This was made by PCR amplifying the portion of the promoter without the 5′UTR from pPurα (1090 bp), cloning into pCR‐TII (Invitrogen) and subcloning the 1.1‐Kb δ5′UTR promoter fragment into the Sma I site of pGL3 (Promega, Madison, WI).…”

“…Binding to this motif has been demonstrated in RNA gel shift assays and ectopic expression of RNA containing this motif inhibits E2F activity [Darbinian et al, 2004]. This motif is found near the 5′ end of the Purα primary transcript in close juxtaposition to the site where Purα has been shown to bind, which is located close to the transcription start site and mediates Purα autoregulation of its own promoter [Muralidharan et al, 2001].…”

“…This study was prompted by two observations. Firstly, Purα binds to a GC/GA‐rich sequence that is located close to the transcription start site within its own promoter and inhibits gene expression, that is, Purα is autoregulated [Muralidharan et al, 2001]. Secondly, ectopic expression of E2F‐1 in glial cells increased the level of Purα detected by Western blot [Darbinian et al, 1999].…”

Posttranscriptional Self-Regulation by the Lyme Disease Bacterium's BpuR DNA/RNA-Binding Protein

“…Noting that BpuR can bind double-stranded DNA (19), we could not disregard that possibility for BpuR. Moreover, eukaryotic PUR-domain proteins may govern their own transcription through direct interactions with noncoding DNA (35). To that end, EMSAs were performed with recombinant BpuR protein and two dsDNA probes that span the bpuR 5= noncoding region.…”

“…BpuR shares significant structural and sequence identity with eukaryotic PUR-domain proteins, which are critical pre-and posttranscriptional regulatory factors (30)(31)(32)(33)(34)(35)(36). BpuR is a transcriptional regulatory factor in the Lyme disease spirochete Borrelia burgdorferi and its own production is controlled by the bacterium (19).…”