Silencing and Overexpression of the Ubiquitin-Conjugating Enzyme E2B in Murine Erythroleukemia Cells

The ubiquitin-conjugating enzyme E2B functions in the ubiquitination of proteins within eukaryotes. The addition of ubiquitin facilitates further modifications or protein degradation. Previous qPCR analysis in Friend virus anemia cells (FVA) showed a significant increase in E2B mRNA level during terminal erythroid differentiation (TED). The goal of the current work was to analyze the expression of E2B on the mRNA and protein levels while also modifying the gene's expression to determine if it is necessary for TED. It was found that the basal E2B mRNA expression did not change in the murine erythroleukemia (MEL) cell model of TED. However, in the more relevant FVA cell model, a significant increase of E2B mRNA was observed. The increase in mRNA found only in FVA cells points to a function in the later stages of TED. A cell line was also generated to attempt overexpression of E2B in MEL cells, but this failed to produce significant results. Lentiviruses were used to generate transduced MEL cells with silenced E2B expression via RNAi to determine if the basal level of E2B expression contributed to TED. Subsequent qPCR analysis found that the RNAi silencing was insignificant. The conclusion from our studies of E2B expression in both cell models is that E2B likely functions in the later stages of TED during enucleation or reticulocyte maturation, since these stages are not reached by MEL cells. Further studies are needed to optimize the overexpression of E2B to determine if this has any effect on the MEL cell differentiation phenotype.