Thio-phosphate is incorporated into nucleic acids of all types of cells and this modification leads to the stabilization and accumulation of mRNA in vivo. The present findings demonstrate a global shift in distribution of proteins on 2D gels from E. coli and Human HEK 293 cells grown in the presence of thio-phosphate. Computer scanning of 2D gels for E. coli detected 815 spots and of these spots, 28% showed significant changes. 125 spots were increased an average of 5.5 fold while a total of 104 spots were decreased an average of 4.9 fold. In general those spots with an initial mass of .038% or less were enhanced while those with an initial mass of .176% were reduced. In HEK 293 cells, 1772 spots were identified of which 17.45% showed significant changes. The average fold increase was 4.9 and the average fold decrease was 3.2. The average initial mass of those spots increased was .014% and of those decreased was .049%. The results show a clear shift in the distribution of proteins. In fact in HEK 293 cells the fraction of total mass increased in those proteins that were enhanced was 5.28% and nearly equal to the 5.7% fraction of total mass decreased for those proteins that were reduced.
For E. coli, it was possible to identity two protein spots by comparing the MW and pI values with computed MW and pIs in the TagIdent database, based on their characteristically large MWs. Additional spots were identified using a database of 715 proteins known to be detected in 2D gels. For 14 of these candidate genes in E. coli it was possible to cross-reference them for mRNA half-life using a database of 2679 known half-lives. The results revealed an increase in the expression of proteins for those genes whose mRNA half-life was less than the half-life of total mRNA (6.8 min). Conversely, those whose mRNA half-life was longer than average were reduced.
Identifying human proteins from 2D spot data is harder because of more extensive post-translational modifications. However, using TagIdent two neural specific proteins were detected because of their distinctive size, namely, nestin and neurabin. Several additional proteins were identified using Shaw’s database of genes expressed in 293 cells and the compute MW/PI function of SIB Ex Pasy. Shaw’s database uses DNA microarray data to report mRNA levels in these cells. The most abundant of these mRNAs, GSA, appeared inhibited using thio-phosphate while the rest, expressed at 1/10 or less of GSA levels appeared enhanced, such as alpha-internexin, another type VI intermediate filiament. Other ubiquitous proteins identified with the TMIG-2D Page database or SIB Ex Pasy also showed a correlation between mRNA half-life and enhancement versus inhibition.
Overall the findings are consistent with a model wherein as cellular mRNA accumulates in the cell owing to nuclease resistance imparted by a modified backbone, competition occurs for a limited number of ribosomes. Those proteins derived from mRNAs less stable than the cellular average preferentially accumulate in the presence of the analogue allowing them compete more effectively for protein synthesis. This results in a global shift in the distribution of proteins in the cell favoring those proteins that are naturally less abundant owing to being derived from less stable mRNAs at the expense of more abundant proteins derived from naturally more stable mRNAs.