Transcriptional enhancement of the mβglobin Gene by small RNAs

Abstract

Modern genomic regulation theories are evolving to include functional nonprotein coding RNA. There are several classes of these RNAs documented to alter gene expression. Researchers found evidence for transcription factories regulated by transcribed noncoding RNAs derived from promoter regions. The globin gene is regulated by a series of enhancers known as the Locus Control Region (LCR). Several sequenced noncoding small RNAs, derived from the LCR, were found to consistently align with specific sequences on globin gene promoters in mouse erythroleukemia (MEL) cells. We hypothesized that the small RNAs derived from the LCR have a significant upregulatory association with globin gene expression. In this study, we performed a quantitative reverse transcription PCR to determine the role of the small RNAs associated with the globin gene. qRT-PCR showed a link between the abundance of globin small RNAs and transcriptional upregulation of the globin gene in MEL cells induced to terminal differentiation. Plasmid DNA from E. coli, containing a GFP reporter gene and the globin gene sequence associated with the small RNAs, was transfected along with the small RNAs into MEL cells to investigate the regulatory role of small RNAs on the globin gene. However, there was poor uptake of the plasmid construct and flow cytometry detected a low percentage of cells expressing GFP resulting in inconclusive data. Future studies should reconstruct the plasmid and use a control plasmid that contains GFP without the globin gene sequence to identify potential issues with the reporter gene. Results can then be analyzed to determine if the small RNAs have significant effect on gene regulation. If there is an association, results can be extrapolated to examine other genes that have small RNAs repeatedly aligned with specific sequences.