Identification of the gene immediately downstream of the murine INK4a/ARF locus

The tumor suppressor gene ARF is formed by three exons, namely exons 1 beta, 2 and 3. Here, we show that embryo fibroblasts from mice genetically deficient in exons 2 and 3 (Delta 2,3) express a transcript formed by exon 1 beta followed by the 3'-terminal exon of the gene immediately downstream of the INK4a/ARF locus, which we have called NTp16 (Next-To-p16). The chimeric ARF-NTp16 transcript is not detectable in wild-type fibroblasts but its expression level in Delta 2,3 fibroblasts is 30% compared to the level of the normal ARF transcript in wild-type cells. Expression of the ARF-NTp16 transcript in Delta 2,3 cells is subject to normal regulatory features, such as upregulation by the accumulation of cell doublings, and by the presence of oncogenic Ras or E1a. The chimeric ARF-NTp16 transcript has the potential to encode a 17kDa peptide; however, this peptide is not accumulated in cells at detectable levels, probably reflecting poor codon usage or protein instability. We conclude that Delta 2,3 cells do not retain ARF functionality, at least to a significant extent. Interestingly, the expression pattern of the full-length NTp16 gene is altered in several tissues by the presence of the Delta 2,3 mutation. Finally, these data identify the gene immediately downstream of the INK4a/ARF locus, a region that has been previously proposed to contain another tumor suppressor different from the INK4a/ARF genes.