MCM3AP is transcribed from a promoter within an intron of the overlapping gene for GANP

Abstract

MCM3 acetylase (MCM3AP) and germinal-centre associated nuclear protein (GANP) are transcribed from the same locus and are therefore confused in databases because the MCM3 acetylase DNA sequence is contained entirely within the much larger GANP sequence and the entire MCM3AP sequence is identical to the carboxy terminus of GANP. Thus, the MCM3AP and GANP genes are read in the same reading frame and MCM3AP is an N-terminally truncated region of GANP. However, we show here that MCM3AP and GANP are different proteins, occupying different locations in the cell and transcribed from different promoters. Intriguingly, a promoter for MCM3AP lies within an intron of GANP. This report is an interesting example in nature of two separate gene products from the same locus that perform two entirely different functions in the cell. Therefore, to avoid further confusion, they should now be referred to as separate but overlapping genes.

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.

Fig. 2

MCM3AP and GANP have different localisations in the cell. (a) MCM3AP can shuttle between the cytoplasm and nucleus. 293 T cells were transfected with EYFP-MCM3AP plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Twenty-four hours after transfection, cells were treated with leptomycin B for 90 min and analysed by immunofluorescence as described previously. (b) MCM3AP localises to the nucleus and cytoplasm, whereas GANP localises to the nuclear envelope. MCM3AP and GANP stable cell lines were generated as follows. Full-length GANP or MCM3AP sequence was amplified by PCR, adding KpnI and XhoI restriction enzyme ends. The KpnI site was designed to create a Kozak sequence around the start codon in both cases. The PCR fragment was ligated into pUB6/V5-His A (Invitrogen) in the correct reading frame to add C-terminal V5 and poly-His tags. Then, KpnI and PmeI were used to excise the protein and tags to ligate into pcDNA5/FRT/To (Invitrogen), which had been mutated to remove an upstream PmeI site. This construct was co-transfected with pOG44 into Flp-In TREx 293 cells according to the manufacturer's protocol (Invitrogen). This after antibiotic selection produces a cell line that can be induced to express GANP or MCM3AP by the addition of 0.1 mg/ml doxycycline. GANP and MCM3AP localisations were determined by immunofluorescence with anti-V5 antibody (Invitrogen). (c) Expression of GANP-V5 and MCM3AP-V5 were validated by Western blot using anti-V5 antibody.

Fig. 3

GANP is cleaved during apoptosis into a fragment that still contains the MCM3AP sequence. (a) HEK293 cells were treated with TNFa and analysed by Western blotting with antibodies against GANP, PARP (Calbiochem), and tubulin (Abcam). The cleavage product is ∼30 kDa larger than MCM3AP and indicated by a red arrow. Dashed arrow indicates the predicted position of MCM3AP. (b) Recombinant caspase-8 cleaves GANP into a  110-kDa fragment. Structure-bound fraction from HEK293 cells was incubated with recombinant caspase-8 (Calbiochem) for 1 h at 30 °C and analysed by Western blotting with an anti-GANP antibody. (c) Mapping of caspase-8 cleavage site. A fragment of GANP spanning residues 689–1315 was expressed and purified in E. coli, and 100 μg was incubated with recombinant caspase-8 in an in vitro cleavage reaction under identical experimental conditions to the in vivo reaction above. Samples were analysed by SDS-PAGE and Coomassie staining, and N-terminal sequencing was carried out on the bands by the University of Cambridge Department of Biochemistry to determine the identity of the cleavage site.