Program Nr: 493

A conserved gene antisense to the proto-oncogene c-RAF encodes a multi-zinc-finger protein, MAKORIN2. T.A. Gray1, K. Azama2, K. Whitmore1, A. Min1, S. Abe2, R.D. Nicholls1. 1) Dept Genetics, Case Western Reserve Univ, Cleveland, OH; 2) Faculty of Agriculture, Ehime Univ., Matsuyama, Japan.
   High levels of c-RAF proto-oncogene expression occur in some human lung cancers, and recent studies have shown that over-expression of wildtype c-Raf in a mouse model leads to an increased incidence of lung tumorigenesis. The c-RAF proto-oncogene can be downregulated by antisense oligonucleotide therapy, culminating in a reduced growth rate for treated cells. Combined, the data suggest that regulation of c-RAF expression is an important factor in some cancers. Here, we describe a novel gene, MAKORIN RING zinc-finger 2 (MKRN2), that overlaps and is antisense to c-RAF. Northern blot analyses show that human and mouse MKRN2 and c-RAF are concurrently transcribed, suggesting that the mRNA products may form RNA duplexes. We have also identified MKRN2 orthologs in fish and birds, placing the gene duplication event that gave rise to this locus early in vertebrate evolution, at least 450 million years ago. All MAKORIN2 orthologs are well-conserved (56%-93% amino acid identity) and retain the hallmark RING and C3H zinc-finger composition of the MAKORIN protein family. While the function of MAKORIN2 is unknown, RING and C3H zinc-fingers suggest macromolecular and ribonucleoprotein roles, respectively, and poxviral derivatives homologous to the MAKORIN protein family circumvent apoptotic pathways. Taken together, our data identify a conserved vertebrate MKRN2 gene that overlaps the c-RAF locus in a tail-to-tail orientation. The conservation of this arrangement implies that it is evolutionarily advantageous, possibly due to co-regulation of the MKRN2 and c-RAF antisense mRNA and/or protein products. Present studies are examining a functional link between MKRN2 and c-RAF, and evaluating MKRN2 for disruption in human cancers.