If we observe that a miRNA, say hsa-mir-233, targets a mRNA, say XXX, in a given tissue in disease condition, can we say that always hsa-mir-233 targets mRNA XXX regardless of type of tissue and disease condition?
$\begingroup$ miRNA's can be tissue-specific, and their mRNA target can also be tissue-specific. I'd be careful about generalizing. $\endgroup$– CKMDec 9, 2015 at 21:14
1$\begingroup$ by tissue-specific, you mean miRNA may target mRNA in tissue A, but not in tissue B? $\endgroup$– user4704857Dec 9, 2015 at 21:16
$\begingroup$ To avoid confusion, yes, the miRNA can be targeted to it's specific mRNA if it ends up in another tissue. To me there are some confounding factors that may alter it's net effect in different tissues or disease states. $\endgroup$– CKMDec 9, 2015 at 21:22
First you have to define what you really mean by miRNA-X targets mRNA-Y. If you mean direct targeting then there are assays to verify it.
To verify if the miRNA can potentially target the mRNA independently, what is routinely done is a reporter downregulation assay (usually luciferase). You clone the 3'UTR of your target mRNA downstream of the reporter gene and check the activity of luciferase in the presence and absence of miRNA. You do that by co-transfecting the miRNA and the luciferase construct, collecting the cell lysate after some time (typically 12/24/36h post transfection) and measuring luciferase activity using bioluminescence counter. This just establishes the molecular possibility of targeting but does not really say if the same actually happens in a given tissue.
Reporter assays like this can be modified for in-vivo conditions too. Express the reporter-3'UTR construct in your organism (or specific tissue). Use another reporter with a mutated target site in the 3'UTR which abolishes miRNA targeting and compare the activities of the two. In zebrafish, people also use modified-backbone oligonucleotides (morpholinos) to mask the target site such that the miRNA can no longer target the mRNA. This assay is called target-protection assay.
There are other assays which do not involve reporter genes but actually capture miRNA and mRNA bound to the Ago-complex. This is done by protein-RNA crosslinking followed by immunoprecipitation of the protein (Ago) which is then followed by sequencing of the bound RNA. See this post for more details. These assays will give you tissue specific information but they only tell you if a miRNA and mRNA are bound together. They do not tell whether the mRNA activity is downregulated or not.
Targeting of the mRNA by miRNA, in-vivo (which has already been established by ectopic reporter assays) depends on many factors, the foremost of which is the relative concentrations of these molecules and the competitors. To produce an effect, the miRNA has to be in sufficient concentration for each of its target (if the miRNA has many targets then the per-target availability will go down).
So, if a miRNA-X actually targets i.e. downregulates a mRNA-Y in a specific condition, it need not do it in another condition. However, we would still say that mRNA-Y is a valid target of miRNA-X if it is so in at least one condition and better if it has been validated using reporter assays (ectopic).
mRNA targeting mechanism is highly specific and depends on various factors. the target mRNA and miRNA complex is highly conserved even from an evolutionary point of view.There are many in-vitro studies which shows the importance of miRNA concentration.Now under any kind of disease state the normal functioning of cells is hindered and this directly effects the level of transcription. the rate can either increase or decrease.
mRNA target region for miRNA in the 3'UTR region is highly specific, and its dependent on base pair complementarity between mRNA and miRNA, due to which the miRNA-mRNA duplex remains exclusive. there are multiple miRNAs which have been found for one particular mRNA, if u go through the database TargetHumanScan database(http://www.targetscan.org/) u will find many miRNAs for one particular mRNA, and vice-versa.
The concern here is not weather any mRNA can be regulated by the particular miRNA, but, whether its positive regulation or negative regulation which is needed by the cell at that particular diseased state.
$\begingroup$ miRNA hardly ever cause positive regulation. They are primarily repressors. $\endgroup$– WYSIWYGDec 11, 2015 at 20:33
As well as the above, it's worth noting that the usage of 3' UTRs and polyadenylation sites can vary from tissue to tissue, so that an mRNA might have the cognate miRNA binding site in one tissue, but not another.