promoter analysis of miRNA's - how to look for regulatory elements in the promoters (Aug/05/2008 )
Hi,
I have a few miRNAs that I am interested in studying. One of the aspects I want to look at is what type of regulatory elements are present in the promoters of those specific miRNA's.
What I did once before for 'messengerRNA's is go to ensembl and export a 1kB region upstream of the location of the gene. I am not sure if thats the right way to do it! Does anyone have any comments?
If it is an acceptable approach, then, the problem with the miRNA's becomes that some miRNA's are transcibed as clusters. Some that I looked at are very close on the genome!! So how can I reliably predict whether the region I select upstream of the predicted location is actually the promoter of that specific miRNA gene or something else? I havent begun trying to read about what other published results say! I was just wondering if anyone had any suggestions regarding what the best/most acceptable way to go about it is!
Thanks.
I think that your approach is fine, although I took 5 kb (and in retrospect wish I had taken more - for example the promoter of miR-34a is 30 kb upstream!) and it worked out quite well. For your second question, I think that 1 promoter will drive expression of an entire cluster of miRNAs, assuming they're not too far apart I suppose. You can specifically take a look at the miR-17-92 cluster, Myc binds the promoter of this cluster and regulates expression of all the miRNAs.
I agree with miRNA man. 5kb should be ok but is just a "start" sometimes. The intriguing fact with the clusters is that you could have a regulatory step also when it comes to "produce" a mature miRNA. Sometimes all miRNAs from a cluster are processed toghether, other times you can have that only a part of the miRNAs inside a cluster are processed. This is very intriguing to me, but quite an open field
Fizban
I agree with miRNA man. 5kb should be ok but is just a "start" sometimes. The intriguing fact with the clusters is that you could have a regulatory step also when it comes to "produce" a mature miRNA. Sometimes all miRNAs from a cluster are processed toghether, other times you can have that only a part of the miRNAs inside a cluster are processed. This is very intriguing to me, but quite an open field
Fizban
Hey...
thanks, the two of you! I will start looking at 5kb now on!!
Also, that was sort of another problem I had!! Is there a way one could predict/ascertain with any amount of confidence that the region that we select (1, 3, 5, 10kb) IS THE ACTUAL promoter (not too much more, not too less) region for that specific gene/miRNA. I understand that it would be different for different genes, but where do you say... ok, this is not it, I need to take a bigger chunk; or vice-versa, how can you realise that you have taken too much, and most of the 'interesting' stuff is really false positive random junk!!
Thanks in advance for any advice!!
[/quote]
Hey...
thanks, the two of you! I will start looking at 5kb now on!!
Also, that was sort of another problem I had!! Is there a way one could predict/ascertain with any amount of confidence that the region that we select (1, 3, 5, 10kb) IS THE ACTUAL promoter (not too much more, not too less) region for that specific gene/miRNA. I understand that it would be different for different genes, but where do you say... ok, this is not it, I need to take a bigger chunk; or vice-versa, how can you realise that you have taken too much, and most of the 'interesting' stuff is really false positive random junk!!
Thanks in advance for any advice!!
[/quote]
I'm not an expert in the field but to my knowledge there's no way to be sure using prediction programs.
The only way is to clone your region upstream a reporter gene and try with serial deletions until you find the core promoter. If you are lucky 5kb will be enough.
Obviously, you CAN'T be sure that there's only ONE regulatory region 
That's a lot of work, but it's not avoidable unluckily.
Don't know if anyone has a better suggestion.
let us know if you're lucky!
Fizban
I think you're right Fizban, there's no easy reliable way. If you only have a few samples, maybe you could try RACE. I've never done it and don't know how much effort it would take (although if I were a betting man, I'd put my money on a lot of effort 
). But it would give you the start site.
Alternatively, you can check out this recent Cell paper and see if your miRNA is in their supplementary data of ChIP-Seq data. This will be much easier that RACE 
http://www.cell.com/content/article/abstra...092867408009380