Abstract 1998: Alternative polyadenylation of eukaryotic initiation factor 5A-2 (eIF5A-2) mRNAs examined by quadruplex single molecule RNA fluorescence in situ hybridization (smRNA-FISH)

Abstract

Abstract Normal expression of the minor oncogene EIF5A2 in the hypusine pathway is restricted to brain and testis. However aberrant expression has been noted in multiple cancers, including ovarian gastric, colorectal and pancreatic, and artificial overexpression causes tumor growth in naked mice. Unlike cDNAs from the homologous gene EIF5A1, which are readily translated into protein upon transient transfection, EIF5A2-mRNAs only associate with monosomes without production of eIF5A-2 protein. Here we present data from multiple EIF5A2-positive cell lines on the steady state levels of the four different EIF5A2-mRNAs that arise through differential polyadenylation. While considering qPCR and RNA-Seq, we chose to apply Stellaris single molecule RNA fluorescence in situ hybridization with four unique probe sets, each reporting on the dependent A, B, C, or D segments. The probe sets were designed with multiple tiled singly labeled 18-22-mer oligonucleotides with balanced GC content, and hence similar hybridization characteristics, which in turn enabled the simultaneous quadruplex smRNA FISH. Stellaris RNA FISH leaves the cell intact, and also does not require RNA isolation or nucleic acid amplification that may introduce an otherwise hidden experimental bias. The single molecule spots revealed by widefield microscopy and subsequent image analysis co-localized with a high degree of confidence. Taking advantage of the added spatial dimension that RNA FISH affords, we also determined the localization of each mRNA variant and recorded the cell-to-cell variability and the actual range of mRNA copy numbers per cell. Our data is consistent with previous gene expression analysis data, but also provide actual counts of each mRNA variant. The results presented here have direct applications for the expression of the minor oncogene EIF5A2, and the regulated protein synthesis of its mRNAs, as well as of other mRNAs in polyamine and hypusine pathways. Further, the techniques and probes developed here can be directly transferred to the analysis of other mRNA variants that may arise through alternative transcription start site use, alternative splicing or alternative 3’ end processing in normal and aberrant cells. Citation Format: Hans E. Johansson, Raymund H. Yin, Swati Mandal, Myung-Hee Park. Alternative polyadenylation of eukaryotic initiation factor 5A-2 (eIF5A-2) mRNAs examined by quadruplex single molecule RNA fluorescence in situ hybridization (smRNA-FISH). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1998.