A team out of the Chinese Academy of Sciences has reported both a new dual-fluorescent reporter system, and an insight on a suspected general phenomenon of functionally matched viral and cellular microRNA in viral-host interactions. You X, Zhang Z, Fan J, Cui Z, Zhang X-E (2012) Functionally Orthologous Viral and Cellular MicroRNAs Studied by a Novel Dual-Fluorescent Reporter System. PLoS ONE 7(4): e36157.
Now for context, the gist of what’s known about microRNAs (miRNA) is that they can be thought of as epigenetic post-transcriptional volume control. Modulating…fine tuning…dialling gene expression up or down. MicroRNAs are ~18-24 nucleotide molecules that are processed from long, highly evolutionarily conserved sequences in organisms and viruses. Such pre-miRNA sequences are located at protein gene introns, as individual genes, or as related polycistronic miRNA clusters. Cellular microRNAs participate in gene expression by acting on messenger RNAs through base pairing at mRNA 2-8 bp long “seed regions”. Due in part to microRNA not needing to be highly complementary to its mRNA targets, a single microRNA gene can have sprawling action among numerous mRNA molecules, containing its seed region. On the other hand, multiple microRNAs can also overlap activity, targeting the same mRNA. Overall, there is a gradation of affinity of microRNAs for target mRNAs.
The functional characterisation of microRNAs and microRNA biogenesis are both hot areas of Epigenetics research. Just this week, the first potential endogenous, intragenic, oncogenic microRNA, was reported in the journal Blood. The recent microRNA breast cancer predictive biomarker has also been big news. You probably already know that if you follow our @Epiexperts twitter stream!
Highly sensitive, luciferase reporter systems are the most popular way to study the functioning of microRNA sequences. Systems such as the Gaussia luciferase (GLuc) and Cypridina luciferase (Cluc) luciferase reporter systems are established for demonstrating decreased mRNA expression with increases in miRNA expression. See Lee, J.Y. et al. (2008) Development of a dual-luciferase reporter system for in vivo visualization of MicroRNA biogenesis and post-transcriptional regulation. Nucl. Med. 49, 285-294 for an example of bioluminescence reporting in real time, with in vivo cells and whole animal.
Xiangyu You et al. have cleverly engineered a single vector with the fluorescent protein mCherry indicating expression of an inserted mRNA sequence, a separate promoter for expression of an inserted pre-miRNA sequence, and another promoter for EGFP – an internal transfection control. The ratio of red to green fluorescence establishes microRNA activity levels in each transfected cell. More red meaning poorer microRNA activity, and less red demonstrating higher affinity and miRNA activity for that target mRNA. This plasmid could also potentially be used to study endogenous microRNA activity on an inserted mRNA gene, because microRNA target repression occurs in the 3′-UTR mCherry indicator.
Using this single plasmid system, they screened an SV40 miRNA, and an HIV miRNA. These miRNAs appeared to recognize identical seed regions as some human miRNAs found in miRBase, http://microrna.sanger.ac.uk/. The viral microRNAs indeed functioned to silence the predicted mRNA targets of the human cellular microRNAs. This data adds to the list of two other recently established herpesvirus microRNA orthologous, human microRNA pairs – generalising the precedent
Considering this pattern, you have to wonder how significant is the detriment to our genomes from viral microRNA…particularly with regard to cancer and immune system function.
You X, Zhang Z, Fan J, Cui Z, & Zhang XE (2012). Functionally Orthologous Viral and Cellular MicroRNAs Studied by a Novel Dual-Fluorescent Reporter System. PloS one, 7 (4) PMID: 22558366