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Johns Hopkins' Joshua Mendell on microRNA Localization

Name: Joshua Mendell
Position: Assistant professor, pediatric/molecular biology and genetics, Johns Hopkins University School of Medicine
Background: Postdoc, Johns Hopkins University School of Medicine — 2003-2004
MD, Johns Hopkins University School of Medicine, — 2003
PhD, genetics, Johns Hopkins University School of Medicine — 2001
BA, biology/genetics/development, Cornell University — 1996

In the Jan. 5 issue of Science, Joshua Mendell and colleagues at Johns Hopkins University School of Medicine published research showing that at least one microRNA, miR-29b, contains sequence elements that direct its subcellular localization — in this case to the nucleus — raising the possibility of modifying small RNAs in order to direct them to specific places in the cell.
This week, RNAi News spoke with Mendell about his findings.
Let’s start with a little background on your lab and its focus.
We’ve been interested in the mechanisms through which microRNAs are regulated, both transcriptionally and post-transcriptionally. We are [also] interested both in the links between those mechanisms, diseases [associated with those mechanisms], and the basic science involved.
We think those are important because it’s clear that microRNA expression is very tightly regulated during development and across tissues and that this regulation is critical for their appropriate functions. Additionally, it’s clear that microRNA expression is nearly ubiquitously disregulated in cancer.
One part of my lab has been studying the transcriptional regulation of microRNAs in cancer, In particular we’re interested in an oncogene called myc, which is a transcription factor that is widely over-expressed in cancer, and we’ve identified microRNAs that are regulated by [this oncogene].
We are also interested in other mechanisms that contribute to microRNA expression and other settings through which microRNAs that are relevant to cancer are dynamically regulated — in particular the cell cycle. That was actually the initial experiment we did that led to the studies [detailed in Science].
Can you give some more detail on the initial experiments and how they transformed into the work [in Science?]
As I said, there is a lot of evidence that microRNA expression is abnormal in cancer, and we were interested in determining whether microRNAs might be involved in regulating the cell cycle. We had developed arrays to monitor microRNA expression globally — obviously a lot of other labs have done that, as well — and one of the things we decided to use the arrays for was to ask whether there are microRNAs that are differentially expressed during different stages of the cell cycle.
We simply isolated populations of cells passing through each of the cell cycle phases and looked at microRNA expression. Really the most dramatic thing we observed was up-regulation of this microRNA called miR-29b in mitotic cells specifically.
At this point, how much do you know about miR-29 and its family members? Do you know their targets and what they’re involved in, or is it still kind of a mystery?
Other groups have begun studying the miR-29s for other reasons, and we’re learning more. I would definitely say we’re still at the beginning of knowing their functions and targets. I know there was a recent paper from Carlo Croce’s group [at Ohio State University] where they have found an important target of miR-29b in a particular kind of leukemia. They’ve also identified some mutations in the miR-29 transcript in leukemia, as well.
So there is some evidence that these microRNAs could be important in cancer, which is another reason why it’s of interest to us that this is a cell cycle-regulated microRNA. But we haven’t yet been able to identify a target; we haven’t really taken that part of the study far enough to really be confident about anything [like that] yet.
Now in this work, you found miR-29b in an unexpected place.
That finding grew out of our observation that it was up-regulated in mitosis. But there were a couple of things about that observation that were immediately of interest to us. First, miR-29B is actually co-transcribed along with another highly related microRNA called miR-29a. They’re part of the same primary transcript but show very different expression patterns. Where miR-29a is constitutively expressed through the cell cycle, miR-29b is at very low levels except at mitosis.
Because we have this interest in microRNA regulation, we immediately were intrigued by that observation because it suggested that there is a post-transcriptional mechanism that prevents miR-29b from being expressed even though it’s transcribed.
Basically, we went on to show that, somewhat surprisingly, miR-29b is actually very unstable in cells that are constitutively cycling through the cell cycle. It only becomes stabilized in mitosis. So there is a mechanism that we don’t yet know that is able to recognize miR-29b specifically and degrade it. That has not been seen before for microRNAs.
Obviously, mRNA turnover is a very important way of regulating messenger RNA levels, but microRNA turnover has not yet been observed.
Having observed it in this case, do you think it was a matter of people not knowing what to look for, and now having seen it, [researchers] might find this in multiple situations?
It’s possible. I will say that to my knowledge, it certainly hasn’t been published. There hasn’t been a global survey of microRNA stability.
I think the general idea, not to speak too broadly, is that when microRNAs are incorporated into the RNA-induced silencing complex, [the result] is a relatively stable entity. What’s mysterious and intriguing is why this microRNA, which we know does get loaded into RISC, is somehow recognized as different from other microRNAs and is targeted for degradation. It’s also, as the paper points out, targeted for this unusual trafficking [into the nucleus].
The way that this research led to the [localization] observation [stemmed from our trying to] understand what this [miRNA] could be doing to mitosis. To try to characterize it better, we thought we should look at the sub-cellular localization because mitosis is unique in terms of the cell cycle in that the nuclear membrane is broken down. So we asked, “In cycling cells, is this nuclear or cytoplasmic?” Amazingly, it turned out in fractionation experiments to be predominantly nuclear, which for animal microRNAs has not been observed before.
And why do you think it’s primarily nuclear?
We were lucky in that [we were able to] go back to the miR-29a example, which is co-transcribed with [miR-29b]. As the names suggests, they’re very similar to each other in sequence, but they behave dramatically differently: miR-29a is stable throughout the cell cycle, while miR-29b is degraded very rapidly except during mitosis; miR-29a is primarily cytoplasmic like other microRNAs that have been studies, where as miR-29b is predominantly nuclear, which is unique.
But the advantage that we had was that miR-29a and b only differ at a couple of positions in their sequences. We first synthesized the fully processed versions of miR-29 a and b, put them into cells, and they behaved just like the endogenous microRNAs. That told us the sequences that are dictating these behaviors had to be just within the mature microRNA sequence itself. That really narrowed it down and allowed us to define the sequence.
There are only two regions that are different between miR-29a and b. There is a central position, single nucleotide change, and then the last six nucleotides are different. We did swapping experiments where we found that if we took the last six nucleotides of miR-29b and stuck them on a, it directed a into the nucleus where it behaved like b. That central position difference had no effect on the sub-cellular localization.
Do you expect that these six nucleotides may also play a role in why miR-29b gets degraded?
That’s an excellent question and obviously something we’re very interested in figuring out. Our original idea was that this might target [the miRNA] for degradation. It’s not quite that simple, we found. One of the things we were able to show is that we can put these last six nucleotides on unrelated small RNAs and it will alter their trafficking, as well, and they can go to the nucleus. We’ve done this for several examples now, but don’t know how completely generalizable that is.
To some extent, these [six nucleotides] function as a nuclear-localization element, and at this point we’re testing how generalizable that is. We thought that these other small RNAs that we tagged with this motif may also be degraded like miR-29b, but that turned out not to be the case. They were stable.
That was surprising to us, but it is somewhat useful; if we’re able to develop this motif as a tool, and we still really need to show that we can, these nuclear small RNAs we create won’t be unstable.
We think there is a complex interplay between the nuclear localization of miR-29b and some of the sequences within the small RNA that can target it for degradation, but within the nucleus only. We’re trying to work that out … but it’s not quite as simple as we originally thought.
So you’re trying to see if this localization feature can be applied to small RNAs. If it can be, what do you see as the applications [for the technology]?
One of the things we’re interested in and currently testing is whether we can enhance the efficiency of using small RNAs to target nuclear events in gene expression such as transcription. There have been somewhat variable reports on the ability of using siRNA to silence transcription by targeting promoter regions. At least in one paper, it was suggested that a rate-limiting step in getting that to work is getting the siRNAs to the nucleus. There is a paper where they actually permeabilized the nuclear membrane and that enhanced the efficiency. So perhaps by targeting small RNAs to the nucleus with this motif, we can enhance the efficiency of that process.
It also would be of interest to determine whether we can use small RNAs to affect later steps in nuclear gene expression such as RNA processing.
Another obvious question that we’re actively pursuing is: How is this [six-nucleotide] motif recognized and what is the mechanism of import. There isn’t really a basis in our current … understand of RISC assembly to account for how this microRNA is recognized as being different from the others. So understanding that mechanism is a priority for us.

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