A Novel Differentiation Factor?

Saturday, February 13, 2016
Elizabeth Frayne, University of Phoenix, Tempe, AZ
Background: The present study using mammalian cells extends previous findings in bacteria and yeast suggesting that thiophosphate is incorporated into mRNA and leads to the stabilization and accumulation of mRNA in the cell.  As there are a limited number of ribosomes in the cell, the resultant increase in total mRNA leads to greater competition for translation. Under these conditions there is a preferential accumulation of proteins derived from mRNAs with half-lives less than the cellular mRNA average, at the expense of those proteins derived from mRNAs with half-lives greater than the cellular average. Furthermore, in HEK293 cells a neuronal identity is enhanced. Methods: 2D dimensional polyacrylamide gel analysis was performed on cultured HEK 293 cells treated with or without thio-phosphate. Images of 2D gels were analyzed with SameSpots software (Non-Linear Dynamics).The thio-phosphate used for these studies was chemically synthesized and purified to biological grade. Results: Significant changes were observed in the cultured human cells. Out of 1772 spots detected via computer scanning, 17.4% showed a significant change with a fold increase of > 1.7 and p value < 0.05 or a fold increase of ≥ 3.0. A plot of the % initial mass versus fold increase showed a statistically significant correlation of -0.13, p< .05. The average fold increase was 4.9 and the average fold decrease was 3.2. The maximum increase was 59 fold and the maximum decrease was 25 fold. The average initial mass of those increased was .014% and of those decrease was .049%.  The results showed a clear shift in the distribution of proteins. Using TagIdent two elevated neural specific proteins were identified because of their distinctive size, namely, nestin (type VI intermediate filament) and neurabin (actin binding protein). Several additional proteins were identified using Shaw’s database of genes expressed in 293 cells that are specifically expressed in neuronal cells and the compute MW/pI function of SIB Ex Pasy. The database uses DNA microarray data to report mRNA levels in these cells. The most abundant of these mRNAs, GSA (X56009), appeared inhibited using thio-phosphate whereas the rest, expressed at 1/10 or less of GSA levels appeared enhanced, such as alpha-internexin, another type IV intermediate filament. Conclusions: The results support the possibility of the phosphate analogue as a promoter of the differentiated state.  HEK 293 cells are not kidney cells in origin and the current findings support a neural identity, based on the identification of some characteristic neural proteins important for axonal outgrowth. Neurabin is a tissue specific actin filament binding protein involved in elongation of the growth cone. Alpha internexin can form neuronal intermediate filaments as a homopolymer or as a heteropolymer with nestin (also elevated). Further studies are needed to explore the connection between thio-phosphate as a mRNA stabilization tool and it use in promoting cellular differentiation.