Heterodimers of the SnoN and Ski oncoproteins form preferentially over homodimers and are more potent transforming agents.

sno is a member of the ski oncogene family and shares ski 's ability to transform avian fibroblasts and induce muscle differentiation. Ski and SnoN are transcription factors that form both homodimers and heterodimers. They recognize a specific DNA binding site (GTCTAGAC) through which they repress transcription. Efficient homodimerization of Ski, mediated by a bipartite C-terminal domain consisting of five tandem repeats (TR) and a leucine zipper (LZ), correlates with efficient DNA binding and cellular transformation. The present study assesses the role of SnoN homodimerization and SnoN:Ski heterodimerization in the activities of these proteins. Unlike Ski, efficient homodimerization by SnoN is shown to require an upstream region of the protein in addition to the TR/LZ domain. Deletion of the TR/LZ from SnoN decreases its activity in transcriptional repression and cellular transformation. When co-expressed in vitro, c-Ski and SnoN preferentially form heterodimers. In vivo, they form heterodimers that bind the GTCTAGAC element. Tethered Ski:Sno hetero-dimers that lack TR/LZ domains are more active than either their monomeric counterparts, tethered Ski:Ski homodimers or full-length SnoN and c-Ski. This work demonstrates, for the first time, the differences between dimer formation by Ski and SnoN and underscores the importance of dimerization in their activity.

Kluyveromyces lactis SEF1 and its Saccharomyces cerevisiae homologue bypass the unknown essential function, but not the mitochondrial RNase P function, of the S. cerevisiae RPM2 gene.

RPM2 is a Saccharomyces cerevisiae nuclear gene required for normal cell growth yet the only known function of Rpm2p is as a protein subunit of yeast mitochondrial RNase P, an enzyme responsible for the 5' maturation of mitochondrial tRNAs. Since mitochondrial protein synthesis in S. cerevisiae is not essential for viability, RPM2 must provide another function in addition to its known role as a mitochondrial tRNA processing enzyme. During a search for RPM2 homologues from Kluyveromyces lactis, we recovered a K. lactis gene that compensates for the essential function but not the RNase P function of RPM2. We have named this gene SEF1 (Suppressor of the Essential Function), DNA sequence analysis of SEF1 reveals it contains a Zn(2)-Cys(6) binuclear cluster motif found in a growing number of yeast transcription factors. The SEF1 homologue of S. cerevisiae also compensates for the essential function of RPM2. The two proteins share 49% identity and 72% amino acid sequence similarity.

Mice lacking the ski proto-oncogene have defects in neurulation, craniofacial, patterning, and skeletal muscle development.

The c-ski proto-oncogene has been implicated in the control of cell growth and skeletal muscle differentiation. To determine its normal functions in vivo, we have disrupted the mouse c-ski gene. Our results show a novel role for ski in the morphogenesis of craniofacial structures and the central nervous system, and confirm its proposed function as a player in skeletal muscle development. Homozygous mutant mice show perinatal lethality resulting from exencephaly, a defect caused by failed closure of the cranial neural tube during neurulation. The timing of the neural tube defect in ski -/- embryos coincides with excessive apoptosis in the cranial neuroepithelium, as well as in the cranial mesenchyme. Homozygous ski mutants also exhibit a dramatic reduction in skeletal muscle mass, consistent with a defect in expansion of a myogenic precursor population. Nestin is an intermediate filament expressed in highly proliferative neuroepithelial stem cells and in myogenic precursors. Interestingly, we find decreased nestin expression in both the cranial neural tube and the somites of ski -/- embryos, compared with their normal littermates, but no reduction of nestin in the caudal neural tube. These results are consistent with a model in which ski activities are required for the successful expansion of a subset of precursors in the neuroepithelial or skeletal muscle lineages.

Enhanced expression of mouse c-ski accompanies terminal skeletal muscle differentiation in vivo and in vitro.

Overexpression of either v-ski, or the proto-oncogene, c-ski, in quail embryo fibroblasts induces the expression of myoD and myogenin, converting the cells to myoblasts capable of differentiating into skeletal myotubes. In transgenic mice, overexpression of ski also influences muscle development, but in this case it effects fully formed muscle, causing hypertrophy of fast skeletal muscle fibers. In attempts to determine whether endogenous mouse c-ski plays a role in either early muscle cell determination or late muscle cell differentiation, we analyzed mRNA expression during muscle development in mouse embryos and during in vitro terminal differentiation of skeletal myoblasts. To generate probes for these studies we cloned coding and 3' non-coding regions of mouse c-ski. In situ hybridization revealed low c-ski expression in somites, and only detected elevated levels of mRNA in skeletal muscle beginning at about 12.5 days of gestation. Northern analysis revealed a two-fold increase in c-ski mRNA during terminal differentiation of skeletal muscle cell lines in vitro. Our results suggest that c-ski plays a role in terminal differentiation of skeletal muscle cells not in the determination of cells to the myogenic lineage.

A carboxyl-terminal region of the ski oncoprotein mediates homodimerization as well as heterodimerization with the related protein SnoN.

Ski is a nuclear oncoprotein, and possibly a transcriptional factor, that has been shown to be involved in both transformation and myogenesis. In attempts to understand the molecular mechanisms underlying the function of Ski, the protein-protein interactions of Ski with itself and with its close relative, SnoN, were investigated. It was found that while both v-Ski and c-Ski bound themselves and each other as bacterial fusion proteins, only c-Ski formed homodimers that could be detected by covalent cross-linking of the native in vitro translated protein in solution. The results also showed that c-Ski formed heterodimers with SnoN. Deletion analysis showed that the carboxyl-terminal third of c-Ski, which is deleted in v-Ski, was required for stable dimer formation in solution. This region consists of two predicted structural motifs that constitute the c-Ski dimerization domain. The more amino-terminal motif is predicted to be mostly alpha helical and is comprised of five tandem repeats of 25 amino acids each and was required for c-Ski dimerization. The second motif is a predicted leucine zipper that was not required for dimerization but greatly increased the fraction of Ski protein detected as dimers. This minor c-Ski homodimerization domain appeared to be required for Ski-Sno heterodimer formation.