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Columbia University s Oliver Hobert Discusses His Work with microRNAs

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At A Glance

Name: Oliver Hobert

Position: Assistant professor, Columbia University

Background: Postdoc, Harvard Medical School — 1996-1999; PhD, signal transduction, Max-Planck Institute — 1995; Msc, biochemistry, University of Bayreuth — 1992

While the role of microRNAs in the nervous system was not an area of research for Oliver Hobert when he joined the faculty of Columbia University, it has become increasingly apparent that these small RNAs play a major part in regulating biological processes.

Recently, Hobert took time to speak with RNAi News about his microRNA work and where he sees the field heading.

Could you give an overview of your lab and what you do there?

We study the genetics of nervous system development in the nematode C. elegans. We’re trying to identify genes that are required for the nervous system to develop and function appropriately. Basically, neuronal cell fate and patterning.

We use genetic tools, meaning we basically identify mutants in which specific aspects of pattern formation and fate determination in the nervous system are defective. Then, we try to identify the molecular nature of these mutations. That’s how we came across microRNAs. Part of what the lab is doing is trying to understand how microRNAs are involved in nervous system patterning.

You recently published a paper in Nature. Can you talk about the findings there to give some background?

The paper shouldn’t be seen in isolation. There are actually two paper in Nature, one [published] in 2003 and one in 2004. They both deal with a very similar process, both involving the microRNA species and both being a differentiation process where two different classes of chemosensory neurons require certain gene-regulatory factors to differentiate appropriately, meaning to express the correct set of chemoreceptors that allow them to sense chemosensory input.

What we found in those two papers is that, first of all, there are two microRNAs involved in that, and secondly, that one microRNA affects the other microRNA’s expression, [which] eventually leads to the specific expression of the class of chemoreceptor genes.

The special thing about those papers was they were the first to define a role for microRNAs in nervous system development. They still are, actually — even now there’s no other examples of other microRNAs being involved in nervous system development. There’re lots that have been described to be expressed in the nervous system, but what they really do is still not known. We have been putting [the microRNAs] very firmly through a specific regulatory context, meaning we have shown that they act in a specific developmental context in chemoreceptor differentiation, and we have identified their target genes through which they exert their function.

One of the two [microRNAs] is one of only five total microRNAs whose function has been studied by loss of function approaches where you knock out the gene and then determine the effect of that. There’s only the two classic C. elegans examples and two examples in flies. The first microRNA we described in the 2003 paper was only the fifth example for which we had a loss of function phenotype.

This is important because it’s easy to identify a microRNA, and it’s also not so terribly difficult to over-express a microRNA somewhere and get some effect, or to try to screw around with its expression level. But the most conclusive way to prove what a gene does — any gene in any organism — is to knock out the gene and ask what the consequence for the organism is if you don’t have the gene.

Where does that bring you now?

First of all, we’re trying to identify by expression pattern analysis whether there’re more microRNAs that are expressed in the neurons we study, in chemosensory neurons. The other thing is we’re trying to identify more targets for those microRNAs that we study in this developmental context.

We’re also continuing our genetic screens to identify more factors that are involved in this developmental context. Specifically, we hope to get genes out that are required for the microRNAs to function.

One central problem that exists, and is a little underappreciated, is that microRNAs do not work in isolation on their targets. There’s obviously the whole enzymatic machinery that processes microRNAs, there’s obviously some intersection with the translational machinery, but in addition there’re most likely going to be proteins that bind to the microRNA/mRNA target duplex to determine specificity, and to provide the link to translational repression. Those proteins are completely unknown, and we hope that with biogenetic screening approaches we will get these additional protein factors out.

What’s the time frame for a project like that?

A couple of years. This genetic screening approach — to get mutants out and screen them — is a specialty for C. elegans because they’re relatively easy to deal with. But then to map and clone those genes, and determine their interactions, that takes a span of years.

Are you doing this on your own, or are you working with other labs that might have more experience with microRNAs?

We do have a collaboration with bioinformatic labs who have been attempting over the past few years to predict targets for microRNAs. That has been, so far, relatively unsuccessful and you can see that by the simple fact that different labs that have developed different prediction algorithms have come up with completely different sets of targets they’ve predicted.

Basically, we are working with bioinformaticists to experimentally validate targets they predict.

Which labs are you working with?

Debbie Marks at Harvard [Medical School] and Chris Sander at Memorial Sloan-Kettering.

Do you have any projections on where all this might be leading?

It’s very clear that microRNAs are going to be an extremely prominent and extremely important class of regulators of gene expression in general, be that in development or be that in homeostasis. That is clearly a surprise; nobody would have anticipated that three or four years ago.

We’re not just dealing here with a handful of new regulatory molecules, which comes up every once in a while — a new transcription factor here, a new transcription factor there. It’s a whole class, and this is a very big problem right now. We don’t even know how many microRNA genes there are in the genome. There have been predictions, but more recent work suggests those predictions are all underestimates, so we still don’t know if we’re dealing with hundreds or thousands of microRNAs.

Then, the target predictions, as erroneous as they may be at the moment, also suggest that a large number of genes — possibly a quarter, a third, or half of all genes — are under microRNA control.

In the next few years a lot of groundwork needs to be done to really determine the targets for the microRNAs and how functionally relevant they really are. But … it’s very clear that they wouldn’t exist or be so abundantly expressed if they didn’t have an important function.

Are you willing to take a guess on how many?

My guess is it’s going to be thousands.

Everything depends on how narrowly you define microRNAs. The people who have originally defined them — David Bartel [from the Massachusetts Institute of Technology] for example — have given very strict definitions of what constitutes a microRNA. That has been an extremely useful and laudable starter for the field, but as we learn more about microRNAs, I find those definitions becoming too restrictive.

If one loosens this restrictive definition to say that a microRNA is any small regulatory RNA that can regulate gene expression and that is processed from a good or not so good hairpin, I suspect you’ll find thousands of them.

What’s this strict definition?

The general idea is that [microRNAs] have to be coming from an energetically stable hairpin, they have to be processed by Dicer, and they have to release a microRNA, which is thought to be 21 to 23 nucleotides long.

Each of those steps is a rather narrow definition. One of the unpublished results we seem to be having is that there are microRNAs that do not come from very good hairpins, meaning these hairpins are not very energetically stable.

We have no idea about whether there may be other enzymes that process microRNAs, so this Dicer criteria may not be very good either.

Lastly, the size restriction may also not be the last word because some [microRNAs] may be larger or some may be smaller.

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