Transcripts associated with microRNAs

Question

[haiying.kong]

I have identified some mutations in a region that is annotated as a specific miRNA by the software Oncotator.

My questions:

1. Does this mean the specific miRNA is going to bind to this region, or this region will transcribe the specific miRNA?

2. Is there any way to find out which gene this miRNA is regulating?

3. In the region which is annotated as this specific miRNA, can I get some information for the sequences? For example, if I have a mutation in this region, how to decide if this mutation is functional or not?

Answer 1

[Thomas]

1. If the software you are using annotated that region as a miRNA (and the software and annotation is reliable) then that means that that region will transcribe the specific miRNA

2. Which kingdom are you working with here? Whether it is plant or animal, then you are going to get a much different response:

   My expertise is with the animal kingdom, so if that is what you are working with:

   It depends - is this a known, well-annotated miRNA? Does it exist in miRBase?

   If it does, you can use the official TargetScan website to get predicted targets quite easily

   If the miRNA is not included already in Targetscan databases, your best bet at the moment would be to use TargetScan. However, this will require a fair bit of work downloading and running the necessary perl scripts (and their dependencies) along with the relevant text files

   There are other target prediction algorithms that exist such as miRanda at microrna.org. However, as far as I am aware, Targetscan is the best target prediction software currently out there.

3. Again, it depends on the specific kingdom and species you are working with.

   For animal miRNA, there is something known as the 'seed region' - roughly around nucleotides 1-8 of the miRNA (it is dependent on the specific mRNA target site as well). If any nucleotide in the seed sequence is mutated it will likely have a strong effect on binding. If the mutation is outside the seed region, then it is much less likely to have a great effect on binding.

   I guess the only way to know for sure, would be to have clear knowledge of the miRNA targets, then perform some form of experiment and experimental analysis to judge whether there is a significant effect on binding to the target depending on whether the miRNA is mutated or not.

Other than that, I am not sure how you would be able to tell

[haiying.kong]

Thank you so much, Thomas. This is very helpful.

I am working on human, so the miRNA database is quite complete.

The mutations from different patients are spread more than 20bp. So some of the mutations are most likely not on the seed region, but as you suggested, this would need experimental verification.

[Thomas]

No problem, if you have any more questions, please let me know

[haiying.kong]

Non of the mutations we have identified on the microRNA are in the seed region. In fact, the region where the mutations are is all spliced out. The mutations are not in the ~22 base region that will be in the mature miRNA. It is interesting that 11 out of 35 patients are having somatic mutations that are annotated as for this specific microRNA by the software tool Oncotator. My background is not in biology. I am still learning biology. I apologize if I am asking too obvious questions. Would the mutations in the regions that will be truncated play any functional role to the expression level of mature miRNA? I tried to find some literature, but it seems like not much this kind of study is done.

Thank you very much in advance.

[Thomas]

I am afraid that we have reached beyond the limit of my expertise at this point:

However, it could be the case that the mutations affects expression of the mature miRNA:

A mutation in the pri-miRNA might affect recognition of the pri-miRNA by Drosha. A mutation in the pre-miRNA might affect recognition of the pre-miRNA by Dicer

Fundamentally, to solve this problem we would need an understanding of how drosha and dicer recognises pri-miRNA and pre-miRNA respectively

I did a quick google search, and found this article which may be a good primer:

"While the sites of Drosha cleavage are determined largely by the distance from the terminal loop, variations in stem structure and sequence around the cleavage site can fine-tune the actual cleavage sites chosen."

Please let me know what you find, as I would be interested to know about this myself

[haiying.kong]

I used Oncotator for annotating the mutations I have identified.

I found a couple of things interesting.

We used SureSelectHumanAllExonV5_UTRs protocol for whole exom sequencing. The genomic region that captured the mutations for the microRNA does not have any exom. In the annotation data provided by the company website there is only chromosome, start, and end site for this genomic region.

There are 2 mature microRNAs encoded in different strand, different genomic regions within this target genomic region. The 2 mature microRNA are named mirxxxxA and mirxxxxB. I think they both are spliced from the 2 complementary strands. Mutations that I have identified are in the genomic region encoding the mature mirxxxxA. But the software annotated all mutations as mirxxxxB