How to find out the regulation of a gene (up/down) by the action of a miRNA & TF? Or if I put it across in the other way, given a TF/ miRNA, how to find whether the gene on which it acts gets up regulated or down regulated?
Also how to find the regulation of a TF on a particular miRNA (up regulation/ downregulation of miRNA)?
Is there any database/ tool to find these out?
Hi Sunitha,
miRNAs generally repress gene expression. As far as I know there are a bunch of articles linking miRNAs and gene activation, but often the mechanism of activation is indirect (e.g. miR-128 targeting nonsense-mediated decay machinery and hence increasing the abundance of mRNAs during neuronal differentiation in mammals). So whenever you query a database containing miRNA-target information (like www.mirbase.org), I guess you can fairly assume that miRNA will be downregulating its target gene.
I'm not a microRNA expert, so please correct me if you know of any miRNA upregulating the expression of its target gene.
On the other hand, what about checking if there is any knockdown experiment in GEO for your miRNA or TF of interest? You could perform Differential Expression analysis and identify which genes might be regulated by this miRNA or TF.
MiRNAs always will downregulate the mRNA target through a miRNA-RISC complex. The RISC complex will bind the miRNA and depending on the level of complementarity the mRNA , the mRNA will be either degraded (quite uncommon with miRNAs though it is the strategy used with siRNAs) or their translation will be inhibited (p.e. PolyA tail decapping)
The expression of miRNAs is independent of the expression of one specific gene, or the activation of their transcription factor. There are some cases in which there are miRNAs (p.e. miR-21), that have their own promotor, but it is not common.
I am sorry if I cannot be more clear but I don't completely understand what is your question.
@JP-B, miRNAs do not 'always' down-regulate.
@AP, here is one of the first papers that showed that they can up regulate too.
Vasudevan, S. et al. Switching from repression to activation: microRNAs can up- regulate translation. Science 318, 1931–1934 (2007)
Databases such as Targetscan, RNAhybrid and miranda can predict microRNA/mRNA targeting. miRTarBase will show you experimentally validated microRNA/mRNA interactions. And experimentally, 3' UTR luciferase reporter assays are often used along with other methods to validate this interaction. However, these are generally for miRNA mediated down-regulation of target mRNA. Although miRNA mediated activation of a mRNA target is possible, it likely suggests an indirect method of regulation.
For TF regulation of miRNA expression, you can use traditional promoter assays and examine primary transcript levels. However, this may or may not correlate with mature miRNA expression.
In silicon analysis using different databases like mirwalk, targetscan, miranda etc could inform you of the miRNA and its different predicted/validated target genes. Some others like the Ingenuity Analysis Pathway (IPA) gives you much more like their interactions and networks.
qPCR and luciferase assays are necessary to verify if a TF/gene is down regulated or upregulated. And to validate that, most researchers often use site-directed mutagenesis.
Traditionally, miRNAs repress translation. However, they can also active translation. There are several published journals as to that such as http://nar.oxfordjournals.org/content/early/2011/11/03/nar.gkr934.full; http://onlinelibrary.wiley.com/doi/10.1002/wrna.121/epdf
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