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.