how to test if a given microRNA regulates a gene of interest - microrna target (Sep/30/2009 )

Hi,
I am a newbie to this field so please forgive this simple question.
I have a gene of interest, X, which is predicted to have many
conserved target sites for microRNAs.
How do I go about finding out
1) which of the many microRNAs that target this gene are actually real and functional?
One way I can think of is to look at gene expression data and see
if the microRNA and the target show anticorrelation across many cell types.
Turns out that there are many microRNAs that are anticorrelated with this gene
(could be because I don't have that many expression data to rule out
spurious or chance anticorrelation from bonafide ones).
Another way is for me to clone the 3'UTR of the gene and also the
mutant 3'UTR with the 6bp pair deleted in a luciferase vector and
see if in a transfection system they are downregulated upon addision of
the microRNA.
But if many microRNAs are targetting this gene, I would have to do this
one by one. Is there an alternative?

What is the standard way of validating the action of a given microRNA on
a target or finding a list of microRNAs that act on this gene and their
relative effect?
I would be grateful if someone can also point me to relevant literature on
how to do this experimentally.

Sincere thanks
Lee

lesande on Sep 30 2009, 02:54 PM said:

You have alot to do.First u must clone 3'-UTR of your gene in a vector coding luciferae or metluciferae.You also must have the same vector with promoter(as positive control) and without promoter (as negative control or mock).also you must have something as internal standard like a vector coding for betagalactosidase but be careful this promoter should be different form luciferase one (otherwise there would be a competetion between promoters). Then you must try to transfect a cell line or somelines which express your gene or protein of interestand a cell line which does not express it.(the transfection step and the amount of DNA which must be transfected needs optimization).After 24 or 48 hour measure the luciferase and betagal activity.it will show you that is there any regulation or not.next step is mutagenizing the miRNA binding sites one by one to show that which miRNA is functional and it's cos of miRNA binding not other factors.after you find which miRNAs are functional you can transfect your cells inparallel with anti-miRNAs to block their activity also you can use anti dicer siRNAs to bloch miRNA maturation.
Then you can show regulation with qPCR and finally with westernblot.to make sure that the expression is blocked.

Here's a simpler approach.

Choi WY, Giraldez AJ, Schier AF. Target Protectors Reveal Dampening and Balancing of Nodal Agonist and Antagonist by miR-430. Science. 2007 Oct 12;318(5848):271-4. Epub 2007 Aug 30.

http://www.sciencemag.org/cgi/content/abstract/1147535

My opinion is that the luciferase assay is the best way to go initially, although later in vivo morpholino experiments would be nice after you've validated repression by luciferase. I think that it will be difficult to show that the morpholino is really specific for your mRNA. How could you show that any changes in protein "A" you see is not due to an indirect effect of repression of genuine target "B" being repressed, resulting in indirect repression of your gene of interest "A" (if that makes any sense - i.e. "B" is directly (or indirectly) regulating "A" and there is no direct miRNA-regulation of "A"). The morpholino might be specific for the miRNA binding site, but how many binding sites are there in the genome? Jon - please correct me if I'm wrong!

Hi miRNA man,

You are right, it takes careful controls to show specificity of a Morpholino-RNA interaction. The Morpholino oligos are more specific than most other antisense types (due to low per-base affinity and consequent long complementarity requirement), but off-target binding sometimes happens enough to cause misleading results. The problem of proving specificity for an oligo protecting an miRNA target site hasn't been thoroughly worked out as far as I know. The last paragraph of this discussion specifically addresses a new technique for controlling for the specificity of target protectors. Many of these techniques require careful control of oligo dose, so the solution concentrations of the Morpholinos should be confirmed by UV spectrometry prior to dosing.

For translation blocking or splice modifying Morpholinos, a common method of controlling for specificity is to use two distinct Morpholinos with non-overlapping target sites. If the oligos are used in separate experiments and produce the same change, this supports the hypothesis that the observed change is due to interactions with the targeted RNA and not due to unintended Morpholino-RNA interactions (since a random RNA binding to one oligo would be unlikely to have sufficient complementarity with the second oligo to cause the same change with each). Note that the two oligos will usually have somewhat different efficacies, and the minimum dose that causes the change will need to be independently determined. A second specificity test using the same pair of oligos involves coinjecting the pair of oligos and looking for dose synergy -- If a phenotype elicited by a single Morpholino can be reproduced by a coinjection where the sum of the concentrations of the two coinjected oligos is considerably less than the concentration of the original single oligo, this supports the hypothesis that the phenotype was specific.

Back to blocking miRNA activity with Morpholinos. First, when targeting the miRNA itself, one Morpholino is used which targets the guide strand (and usually overlaps the guide-Dicer site) and another oligo is used targeting the star strand (usually star-Dicer targeted). While the star-targeted oligo cannot bind to the miRNA on RISC (assuming the star strand doesn't load into RISC), the star-Dicer oligo can inhibit maturation of the miRNA (by protecting the Dicer site from cleavage). This means that the two oligos can be used as a specificity control pair; while the star oligo might produce a slightly weaker phenotype, the ability to mostly phenocopy the guide oligo lends support to the specificity hypothesis. However, this is complicated by the close sequence similarity within miRNA families; Morpholinos can generally still bind well enough to have some biological effect even if there is a mispair or two between the oligo and its target. To target one member of a family, I try to design oligos overlapping more loop sequence, since within a miRNA family the loop region is generally far less conserved than the guide or star sequences. The star-strand control was first proposed by Wigard Kloosterman: Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RH. Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development. PLoS Biol. 2007 Jul 24;5(8):e203

Next, targeting miRNA target sites. There are not very many reports of target-protecting with Morpholinos in the literature, so a consensus has not been reached about the techniques for specificity control. One possibility is to use two target-protecting oligos, each covering half of the miRNA target site and extending in different directions across the flanking sequence. First one or the other of these is used and the concentrations determined that produce a weak phenotype. When they are both used together, the phenotype should strengthen considerably, showing a dose-synergy effect IF the single-oligo phenotypes are caused by blocking the intended miRNA target site. A good confirmation is to drop the concentration of the coinjected oligos and determine whether they are still producing the phenotype of interest at a summed concentration below that required for a single oligo. This is not a well-vetted technique and I've not seen it published yet, but it is an approach that parallels the standard techniques used for other classes of Morpholino targets.

miRNA man,

On rereading our original post I see that I did not directly address the situation you posed. While the two-oligo strategy I outlined helps to show that a particular effect is due to modulation of the expression of a particular mRNA, it does not prove the pathway for that modulation. It is still possible that the oligo pair is modulating expression of a different mRNA which encodes a protein (r.g. a transcription factor) itself modulating the expression of the mRNA of interest. However, the probability is very low that the different mRNA bears sufficient homology with the miRNA response element and its surroundings (50 bases for two oligos) that the two oligos would pass a specificity test by modulating this different RNA.

You present an interesting problem. The luciferase assay is useful in that it shows that the oligos are interacting with the putative miRNA response element (MRE), but is that sufficient proof that the expression of the gene of interest is primarily modulated through binding its MRE and not the more circuitous pathway as you proposed? What experiment would rigorously demonstrate that there is no other pathway involved in vivo?

Hi Jon,

Your double morpholino strategy is a very good one and I agree that it would be unlikely for another 3' UTR to have similar 50 bp of sequence. What I gathered from the paper you cited was that they used a single morpholino to block the MRE in the 3' UTR. In this case it was convincing because they came at the problem from several other directions (for example the in vivo GFP reporters and Dicer knockout) and came to the same conclusion. Without the other corroborating data their conclusion wouldn't be as strong. Of course the same principle applies to the luciferase approach since it usually has drawbacks, such as being an overexpression system in many cases.

The most rigorous experiment that I can think of would be to use HITS-CLIP or other similar precipitation/sequencing technologies that are being developed. Hopefully these techniques will become the norm in the near future, given time and reduced costs.

Hi miRNA man,

I agree, HITS-CLIP looks like a great strategy to directly show interactions. I think it will be hard to find a more convincing technique.