Abstract 4209: Hypusine pathway gene expression examined by single molecule RNA fluorescence in situ hybridization (smRNA-FISH)

Abstract

Abstract The fate of any given mRNA has remained difficult to experimentally follow and has left mechanisms of unexpected gene expression phenomena in the dark. Examples are differential stability of mRNA expression from different promoters, and inhibition of expression in trans from co-transfected plasmids encoding N1-spermidine/spermine, acetyltransferase and a second unrelated gene. Recent advances in single molecule fluorescence in situ hybridization (smRNA FISH) now bring such central aspects of cellular mRNA metabolism into view. Streamlining both the design and synthesis of probe sets of multiple tiled singly labeled 20-mer oligonucleotides with balanced GC content have made most mRNAs targetable. In contrast to traditional cell-disruptive RNA analyses - northern blotting, QPCR, and microarrays - that yield average and relative information on the content of the probed mRNA, smRNA FISH affords discrete and cell-specific copy numbers. That added spatial dimension can be applied to provide cell-to-cell variability and the actual range of mRNA numbers in a given cell within the field of focus, or to examine defined subsets of cells. The hypusine pathway genes (EIF5A1, EIF5A2, DHPS, DOHH, and SAT1) are essential with deep evolutionary conservation. The gene products are tightly linked to the growth of the cell with aberrant expression in cells that have lost proliferation control, and in several cancers. SAT1, encoding N1-spermidine/spermine, acetyl-transferase, in particular plays a pivotal function in regulating polyamine levels and in turn determining the cells’ proliferative state. Recently, vectors episomally expressing SAT1 were found to inhibit the expression from co-transfected plasmids. Inhibition only occurred with enzymatically active SAT1. To gain a better understanding of how the expression of the hypusine pathway genes may contribute to the transformed phenotype, the fate of these mRNAs and those of unrelated reporter genes was examined by smRNA FISH. Single cell expression data for the hypusine pathway genes is presented and compared with previous results from northern analyses. Data is also presented on the further use of the probe sets to examine changes in the numerical and spatial expression in cells after manipulation of the hypusine status. Lastly, data on the expression - mRNA and protein - in cells co-transfected with vectors for SAT1 and unrelated reporter genes is presented, distinguishing between post-transcriptional events that may cause the inhibition. In summary, we show smRNA FISH as a suitable tool to disseminate post-transcriptional events for genes relevant to polyamine homeostasis and regulated protein synthesis in normal and aberrant cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4209. doi:1538-7445.AM2012-4209