A novel role for RAX, the cellular activator of PKR, in synergistically stimulating SV40 large T antigen-dependent gene expression.
The double-stranded (ds) RNA-binding protein RAX was discovered as a stress-induced cellular activator of the dsRNA-dependent protein kinase (PKR), a key regulator of protein synthesis in response to viral infection and cellular stress. We now report a novel function of RAX, independent of PKR, to enhance SV40 promoter (origin)/enhancer-dependent gene expression. Several mammalian cell lines including COS-7, CV-1, and HeLa cells were tested. Results reveal that the SV40 large T antigen is required for RAX-mediated, synergistic enhancement of gene expression. RAX augments SV40 regulatory element-dependent DNA replication and transcription. The mechanism requires the SV40 enhancer, a viral transcriptional element that is necessary for efficient SV40 DNA replication in vivo. Mutational analysis reveals that the dsRNA-binding domains of RAX are required for the gene expression enhancing function. Thus, in addition to stimulating PKR activity, RAX can positively regulate both SV40 large T antigen-dependent DNA replication and transcription in a mechanism that may alter the interaction of the cellular factor(s) with the SV40 enhancer via the dsRNA-binding domains of RAX. This novel function of RAX may have implications for regulation of mammalian DNA replication and transcription because of the many similarities between the viral and cellular processes.
Yang
Mingli
M
University of Florida Shands Cancer Center and Department of Medicine, University of Florida, Gainesville, Florida 32610-0232, USA.
Ito
Takahiko
T
May
W Stratford
WS
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005136:Eye Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000502:physiology*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection; D019892:eIF-2 Kinase / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; D015852:Eukaryotic Initiation Factor-2; D005136:Eye Proteins; D018398:Homeodomain Proteins; C105441:Rax protein, mouse; D014157:Transcription Factors; D004247:DNA; D008715:Methionine; D008156:Luciferases; D019892:eIF-2 Kinase