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Pitt’s Kyle Orwig on Identifying microRNA in the Rhesus Macaque Genome

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NAME: Kyle Orwig
 
POSITION: Assistant professor, obstetrics, gynecology, reproductive sciences/molecular genetics, biochemistry — University of Pittsburgh School of Medicine’s Magee-Women’s Research Institute
 
BACKGROUND:
Research assistant professor, University of Pennsylvania — 1999-2003; Postdoc, University of Kansas Medical Center — 1994-1998; PhD, biochemistry, biophysics, animal sciences, Oregon State University — 1994; BS, biology, Whitlow College — 1990
 

 
Earlier this month, Kyle Orwig and colleagues from the University of Pittsburgh published a report in BMC Genomics identifying 383 novel microRNA genes in the rhesus macaque genome.
 
According to the authors, the work is expected to help create a miRNA array for the rhesus, which is one of the most common non-human primates used in biomedical research due to its genetic and physiologic similarity to humans.
 
This week, RNAi News spoke with Orwig about the paper.
 
Could you provide an overview of your lab?
 
We’re primarily an animal biology lab, so we do research in mice, rats, and monkeys. The reason we cover that spectrum of mammalian biology is because it represents various stages of the evolutionary tree. The monkey part, because of its similarity to human physiology, is sort of a translation from our basic studies in rodents … to potential applications in humans.
 
Had you explored the world of microRNA before [the work described in the paper]? When did they come into the picture?
 
I should say that the area we research is male reproduction, specifically the stem cells in the testes that make sperm. We’re interested in, at a basic level, the molecular mechanisms that control sperm production in the testes. In that regard, we’re interested in microRNAs, first, as a basic research tool because you can use microRNAs to knock down specific genes and see whether that gene is important for function by observing what happens in the animals.
 
In that sense, microRNAs are just a research tool for us — particularly in those species where the technologies don’t exist to produce a knockout animal or to knock out a specific gene. So in mice, we can knock out a gene to find out whether it is important, but because of the lack of ES cells we can’t do that in a rat or monkey — but we can use microRNAs to knock down gene expression.
 
You’re talking about microRNA mimics?
 
Basically, you can design a microRNA with a sequence that targets a specific messenger RNA in a cell … [and deliver it using] a vector that expresses [the miRNA].
 
And how did that lead to the rhesus macaque work?
 
That’s a completely different story. The roles of small RNA in normal cell physiology are becoming more appreciated, whether you are talking about microRNAs or piRNAs or short hairpin RNAs. They have sort of risen from being something that was just a phenomenon to something people appreciate, including us, as molecules that play important parts in normal cell physiology.
 
So while we’re using it as a tool, we’re also paying attention to what’s going on in the literature. One of the things that we do in the laboratory is try to gain insights into molecular mechanisms that regulate spermatogenesis. Historically, that has meant DNA, RNA, and proteins. Now, we have broadened our thinking to include these small RNA molecules.
 
Just like as if we were evaluating gene expression by asking which RNAs get expressed [using] microarrays that represent all the genes that might be expressed in the genome … now arrays are becoming available that have all the microRNAs that might be expressed. With those available, we can ask not only about what genomic genes are expressed but what microRNAs are expressed under different conditions and whether they affect our system of interest.
 
But as you note in the paper, for the rhesus macaque there hasn’t been too much work done on identifying microRNA.
 
Right. There is a place — the Wellcome Trust Sanger Institute miRBase — where you can find databases of microRNAs for different species, but the rhesus isn’t very complete at this point. It has 71 microRNAs listed in it, and because rhesus is very similar to humans we suspected that that list was incomplete.

 

So we simply used human sequences to search the entire rhesus genome to see if we could find similar sequences in the rhesus genome, and in most cases we did.
 
What were the key findings from that effort?
 
The key finding, first of all, is that indeed the rhesus genome is similar to the human genome, and indeed it has these genes that are most likely microRNAs. We’ve evaluated that, at this point, just based on homology. Certainly, we have not tested these functionally.
 
But we did take a few of them and ask if the sequences were actually expressed in rhesus cells. We picked eight examples at random and found that they were all expressed. And not only were they expressed, but their levels of expression changed depending on what tissues [we studied], suggesting that they are regulated … in a way that may be important for some function.
 
Are there plans to follow up with any sort of functional validation?
 
There is not an immediate plan because we do lots of things, but one of the next steps will probably be to produce a microarray that contains all the microRNAs from the rhesus genome. You would use that array to ask how the microRNA transcriptome changes in one condition versus another condition.
 
The first thing we need to do is create the tool, and once the tool is there then we can begin assaying microRNAs the exact same way as we assay gene expression now using microarray technology.
 
Is that the kind of thing you would do in-house or would look to do that through some sort of industry collaboration?
 
It would most likely happen through an industry collaboration. And because we’re not really a genomics lab, that’s not something we would do ourselves; we would [likely] find a collaborator [in another lab] on campus and … it would be their decision whether they wanted to do it in-house or contract with a company.
 
Have the data you pulled from this work been submitted to miRBase?
 
We have not done it yet, but it is something I need to follow up on. As long as we did the work, we need to make sure that it’s distributed through this public database.

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