Genomic characterization of the human mitochondrial tumor suppressor gene 1 (MTUS1): 5' cloning and preliminary analysis of the multiple gene promoters
© Zhou et al; licensee BioMed Central Ltd. 2009
Received: 18 February 2009
Accepted: 19 June 2009
Published: 19 June 2009
Mitochondrial tumor suppressor gene 1 (MTUS1) has been recently identified as a candidate tumor suppressor gene which resides in a genomic region (8p22) that shows frequent loss of heterozygosity (LOH) in several tumor types. It has been suggested that multiple gene promoters and alternative splicing lead to the expression of 5 known MTUS1 transcript variants.
Here, we characterized the 5' untranslated regions of the different transcript variants. We also cloned and functionally tested the alternatively utilized gene promoters that contribute to the production of different MTUS1 transcript variants.
Our results confirmed the early hypothesis that the transcript variants of MTUS1 gene are driven by multiple gene promoters.
The MTUS1 gene is located in a region (8p22) that shows frequent loss of heterozygosity (LOH) in several tumor types, including oral cancer . Alternative exon utilization leads to the production of 5 known transcript variants (designated as variant 1 to 5) . It has been suggested that the long form of transcript variants (variant 1, 2, and 3) are driven by a common gene promoter, while variant 4 and 5 are driven by 2 additional promoters . Variant 5 was the first transcript variant to be cloned independently in 2 laboratories, as a gene that is transiently upregulated during initiation of cell differentiation and quiescence . It represents an early component of the growth-inhibiting signaling cascade that interacts with angiotensin II AT2 receptor . Evidence supporting the tumor suppressor function of other MTUS1 variants comes from the study on Xenopus Icis gene, a homolog of MTUS1 variants 1 and 2, which regulates microtubule growth and spindle formation prior to anaphase . Here, we refined the genomic structure of the MTUS1 gene and functionally cloned the alternatively utilized gene promoters that control the production of these MTUS1 transcript variants. This will enhance our understanding on the regulation of this candidate tumor suppressor gene.
Materials and methods
To characterize the 5' untranslated regions (5'-UTR) of the MTUS1 transcript variants, 5'-RACE assays were carried out using human brain reference mRNA (Ambion Inc) and a FirstChoice RLM-RACE kit from Ambion, with primers specific for various transcript variants (Additional file 1). The RACE products were PCR amplified, gel purified and then sequenced. The sequence results have been submitted to the GenBank (accession numbers: FJ458439, FJ458440, FJ458441, FJ472826, and FJ472827 for exon -1a, -1b, -1c, 5, and 8, respectively).
The gene promoter prediction was carried out using MatInspector Professional http://genomatrix.de/cgi-bin/matinspector_prof/mat_fam.pl. The predicted transcription elements of the putative promoters were listed in Additional file 2. To assess the activities of potential gene promoters that control the productions of MTUS1 transcript variants, the following 4 fragments were PCR amplified using specific primers (Additional file 3) and Human Reference Genomic DNA (Promega): 1) a 2286 bp fragment (P1) located at the 5' flanking region of the MTUS1 gene; 2) a 773 bp fragment (P1') located at the 5' flanking region of exon 1; 3) a 529 bp fragment (P2) located at 5' flanking region of exon 5; and 4) a 733 bp fragment (P3) located at 5' flanking region of exon 8. The PCR products were then cloned into the KpnI/XhoI sites of pGL4.10 vector. After verification by DNA sequencing, the constructs were transiently transfected into cells using lipofectamine 2000 (Invitrogen). The pGL4.74 vector (Promega) was co-transfected as internal control for normalization of the transfection efficiency. After 48 hours, transfected cells were harvested with ice-cold phosphate-buffered saline, and dual luciferase assay were performed according to the manufacturer's protocol (Promega) using a Lumat LB 9507 Luminometer (Berthold Technologies).
Results and Discussion
MTUS1 gene promoter activity measured by Dual Luciferase assay1,2
In summary, our results refined the genomic structure of the MTUS1 gene and demonstrated the presence of multiple gene promoters. Characterization of this gene will now facilitate studies on the physiological and pathophysiological function and transcriptional regulation of the MTUS1.
This work was supported in part by NIH PHS grants DE014847, DE016569, CA114688, CA139596, and a grant from Prevent Cancer Foundation. We thank Ms. Katherine Long for excellent editorial assistance.
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