A particular family of miRNA is unregulated in infected monocyte derived macrophages as validated by microarray as well as Taqman assay. However the gene targets are unregulated in the same system. How to explain this and how to look at the upstream regulators in the same signalling pathway the targets are a part of?
Hi ALok
Pretty interesting fact. I am not surprised about that.... Probably your relationships between miRNA and target in this particular system are biologically meaningless. They might be regulated by upstream effectors, and the power of the effector is higher than the negative regulatory effect exerted by the miRNA over the target.
More data could help to a deeper discussion.
Best
Paco
Hi ALok
Pretty interesting fact. I am not surprised about that.... Probably your relationships between miRNA and target in this particular system are biologically meaningless. They might be regulated by upstream effectors, and the power of the effector is higher than the negative regulatory effect exerted by the miRNA over the target.
More data could help to a deeper discussion.
Best
Paco
I agree with Francisco. Your background information is too vague to make a meaningful comment except complain about the vagueness of the question itself. Let me elaborate:
how do you define target genes, about how many target genes are we are talking here, how many are up-regulated how much.
How many genes change overall or how massive is the impact of infection on the transcriptome. Do you measure within a few hours of infection or "longterm" effects (48hours?). I would assume that immediate effects are run without much impact by microRNAs since the cells have better things to do then fine-tune gene expression. So, if you expect an inverse correlation between the microRNAs and their "target genes" (if you have defined them at all) then an " emergency" situation such as an infection, may not be the right place.
So, your expectations and ideas may have to be bend in order to explain the response of the cells. This looks like a "think twice" situation. But as stated, your background info is insufficient to make any suggestions about the biology of your observation.
With microRNAs you may have to leave the path of linear thinking and expect more complicated scenarios, lots of feedback regulation etc.
Dear Paco!!
I truly agree with that as the upstream master regulator is AKT while it is also being one of the target.
The downstream target being beclin-1 is high together with AKT. In case of AKT, the status of its post translational modification will be more important rather than its absolute level in this system.
There are several reports where a particular miRNA is not relevant to its previously validated target and hence both are unregulated.
Don't you think that someone must follow a kinetic of expression of the target for an extended time point, may be the higher level of miRNA is targeting at transcript level but the translated protein is much more stable and hence the opposite effect.
What I am trying to say is that the effect of higher level of this non-coding RNA is kind of a delayed response.
Alok
Hi Thomas!!
Sorry for the vagueness of the question itself.
Well the target in the screen is higher in expression till 36 h and then it declines. The level of miRNA is very high for more than 24 h and so.
I strongly believe that the effect of miRNA on target mRNA , protein and hence phenotype is a delayed response. I mean some one can just justify that well the miRNA was quite unregulated post infection and so was the target but as infection progressed there was a time point where the level of target reached plateau and then declined and hence there was the reversal of phenotype. It will be too immature to say that well the miRNA was unregulated at 12 h and so transcript should be low and hence protein. May be the relative stability of transcript and protein can make the situation different. Isn't it??
Thanks for insight>
Alok
Alok
Of course you always can think about an effect in longer times, that can affect the protein levels. However, if the transcriptional program is heavily activated, you will barely see anything at the protein level. I heard in a Tom Gingeras seminar a long time ago, that a single miRNA acting over a single site in a 3'-UTR is expected to reduce the protein levels in about 15-20%. If your transcription factor is active like hell, it will fight against the miRNA activity.
The problem with those things is when to stop... You can think in follow miRNA and target during an extended period and see what's happenning at the protein and RNA levels, but of course it depends on your patient.
Interesting... I am also dealing with AKT1 and miRNAs....
Tons of luck for you. Cheers
Paco
Its quite fascinating isn't it.
You have really helped me to have a different look on this problem.
:)
thanks again
warm regards
Alok
Hi Alok, do you mean up-regulated with "unregulated"? It still is hard to say what is going on. You could use a sensor for your microRNA and measure its activity in a time-course after infection. This will tell you how active your microRNA is on the reporter as a siRNA (if you use a perfect match as your sensor). Then you can compare this with your "target" gene kinetics and levels of microRNA.
It is also important to determine that your target genes are real and not just predicted. Then you can use a 3'UTR of this gene as your alternative sensor (when the microRNA functions as a microRNA). May sound overly complicated...
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