Fig. 1.

MCM3AP can be transcribed independently of GANP. (a) Domain organization of MCM3AP and GANP. GANP protein contains a domain that is identical to MCM3AP and transcribed from the same sequence. GANP also contains a region of homology to Sac3, a yeast mRNA export factor, and to nucleoporins, as well as a putative RNA recognition motif (RRM). (b) The promoter region of MCM3AP contains putative transcription factor binding sites for NF-IL6, AP-1, NF-κB, and p53. Diagram of the human GANP and MCM3AP genes. Exons 2–13 of MCM3AP are the same as exons 18–29 of GANP. MCM3AP exon 1 starts in GANP exon 17. A cluster of transcription factor binding sites was detected in GANP intron 16 using Genetyx version 10 software.  (c) 5′RACE identifies a transcription initiation site of MCM3AP within exon 17 of GANP. Two rounds of nested PCR were performed to identify the transcriptional start site of MCM3AP using pre-prepared RACE-ready cDNA from HeLa cells (Invitrogen). The first round was performed with primers homologous to the 5′RACE adapter sequence and to a region 50 bp downstream of the MCM3AP start codon. The second round was performed using the template from the first round with a different primer homologous to a region 70 bp upstream of the start codon. Products from the second round of a nested PCR reaction are shown. Transcription initiation site is indicated in green in (b). (d) MCM3AP promoter is cytokine responsive. Subconfluent HEK293 cells were treated or mock-treated with TNFα (Calbiochem) and IL6 (Calbiochem) and transfected with pGL3 (Promega) or pGL3-MCM3AP promoter and a constant amount of renilla luciferase control plasmid, pRL-TK (Promega) using Polyfect (Qiagen). Cells were harvested 40 h after transfection and assayed for luciferase activity (Promega). The firefly/renilla luciferase ratio was then calculated. Average results from three experiments are shown. (e) AP-1, NF-IL6, and NF-κB contribute to cytokine-mediated increase in promoter activity. Mutations of the AP-1 site (TGAGTAG to TGAGCCT), NF-IL6 site (TTTTGAAAT to TTTTGACCC), and each NF-κB site (GGGGTTTCAC to CCCGTTTCAC) were made using the QuikChange mutagenesis kit (Stratagene). The mutated promoter sequences were cloned into the pGL3 vector and sequenced for confirmation of the mutations. These plasmids were then transfected into HEK293 cells following cytokine treatment and assayed for luciferase activity as above.