Today’s blog is from guest blogger Ken Doyle of Loquent, LLC. Here, Ken reviews a 2014 paper highlighting specific considerations for using reporter assays to study miRNA-mediated gene regulation.
The accelerated pace of research into noncoding RNAs has revealed multiple regulatory roles for microRNAs (miRNAs). These diminutive noncoding RNA species—typically 20-24 nucleotides in length—are now known to mediate a broad range of biological functions in plants and animals. In humans, miRNAs have been implicated in various aspects of development, differentiation, and metabolism. They are known to regulate an assortment of genes involved in processes from neuronal development to stem cell division. Dysregulation of miRNA expression is associated with many disease states, including neurodegenerative disorders, cardiovascular disease, and cancer.
Typically, miRNAs act as post-transcriptional repressors of gene expression, either by targeted degradation of messenger RNA (mRNA) or by interfering with mRNA translation. Most miRNAs exert these effects by binding to specific sequences called microRNA response elements (MREs). These sequences are found most often within the 3´-untranslated regions (3´-UTRs) of animal genes, while they may occur within coding sequences in plant genes.
Studies of the regulatory roles played by miRNAs often involve cell-based assays that use a reporter gene system, such as luciferase or green fluorescent protein. In a standard assay, the reporter gene is cloned upstream of the 3´-UTR sequence being studied; this construct is then cotransfected with the miRNA into cells in culture. A study by Campos-Melo et al., published in September 2014, examined this experimental approach for miRNAs from spinal cord tissues, using firefly luciferase as the reporter gene and Renilla luciferase as a transfection control.
Normalization against the effect of the miRNA and cellular factors on the luciferase coding sequence is essential to obtain the specific impact of the miRNA on the 3’UTR (untranslated region) target. (Campos-Melo et al, 2014.)
Controls for Effects of miRNA on Reporter Gene Activity
The authors note that many miRNA studies do not include rigorous normalization of the expression data, other than including a transfection control and a “minus miRNA” negative control. Therefore, such analyses could conceivably miss the indirect effects of miRNAs on MREs within the reporter gene transcript itself.
To address these potential errors in measuring reporter gene expression, the authors report a miRNA-specific protocol for normalization of luciferase expression. The study was designed to examine miRNA-mediated regulation of the neurofilament (NEFL) mRNA 3´-UTR. The control plasmid (pmirGLO) construct included the firefly and Renilla luciferase coding sequences, while the experimental construct (pmirGLO-NEFL-3´UTR) used the same vector with the NEFL mRNA 3´-UTR cloned downstream from the firefly luciferase coding sequence.
The miRNAs studied in this experiment included miR-507 with four predicted MREs and miR-518e* with one predicted MRE within the NEFL 3´-UTR. Further, miR-507 had two predicted MREs within the firefly luciferase coding region. These miRNAs and a negative control (miR-let-7a) were transfected independently, together with pmirGLO or pmirGLO-NEFL-3´UTR, into HEK293T cells. Luciferase activity was measured as the mean of six replicates for each experimental condition.
In the initial analysis, the authors normalized each sample’s firefly luciferase activity with the respective Renilla luciferase activity, to derive the mean luciferase activity for each experiment. Based on this analysis, they observed that both miR-507 and miR-518e* downregulated luciferase activity from pmirGLO-3´UTR; interestingly, the negative control miRNA (miR-let-7a) also appeared to downregulate luciferase activity.
A Two-Step Normalization Process
For the control plasmid pmirGLO, the authors also observed that all three miRNAs downregulated luciferase activity, even though this plasmid did not contain the NEFL 3´-UTR. Therefore, they performed a second step of normalization in which they calculated the effect of the three miRNAs on firefly luciferase expression with or without the NEFL mRNA 3´-UTR sequence. This two-step normalization procedure yielded the specific effect of the miRNA under study on the NEFL 3´-UTR, independent of the effect of the miRNA on the luciferase coding region itself.
Without this two-step normalization procedure and following the standard practice of normalizing luciferase activity only to a transfection control, the authors would have erroneously concluded that miR-518e* and miR-let-7a could both downregulate the expression of NEFL mRNA. In fact, after eliminating the effects of the miRNAs studied on the luciferase reporter coding sequence, their results showed that miR-507 downregulated NEFL mRNA expression, miR-518e* upregulated it, and the control miR-let-7a had no statistically significant effect. Based on these results and previous studies, the authors observe that although miRNAs are typically known for downregulation of gene expression, they may also play upregulatory roles for some transcripts.
Here’s the Paper
Campos-Melo D. et al. (2014) Comprehensive Luciferase-Based Reporter Gene Assay Reveals Previously Masked Up-Regulatory Effects of miRNAs. Int. J. Mol. Sci. 15, 15592–15602.
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