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UCSD s Amy Pasquinelli on microRNAs and Where the Field is Heading

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At A Glance

Name: Amy Pasquinelli

Position: Assistant professor, biology, University of California, San Diego

Background: Postdoc, Harvard Medical School — 1999-2003; PhD, biomolecular chemistry, University of Wisconsin, Madison — 1998; BA, biology, Bucknell University — 1993

Having just completed her post-doctoral work in the lab of Harvard’s Gary Ruvkun, Amy Pasquinelli is currently putting together her own lab, where she and her colleagues plan to conduct research into gene regulation and regulatory RNA.

Recently, she took time to talk with RNAi News about her work and where she sees it going.

How did you get started with RNAi?

My graduate work had been studying how RNAs are exported from the nucleus to the cytoplasm. From there, I really wanted to get my hands wet with some genetics and developmental systems, and decided that C. elegans was a good choice for me. I wasn’t necessarily committed to staying in the RNA world.

I looked around at different projects in different labs and I had read some work from the early ‘90s from Gary Ruvkun’s lab studying a tiny RNA gene that regulated development. I thought: Maybe with my RNA background this might be a good way to get my foot in the door to a C. elegans lab.

I ended up joining Gary’s lab with the interest of pursuing the role of what, at the time, seemed to be a weird worm RNA, in development. Fortunately, when I joined the lab, a postdoc and grad student in the lab had just found another small RNA in the same pathway in C. elegans. So I came to the lab hoping to work on that and figure out the role of that in development in a worm.

Fortunately, during my first year there, we discovered that these types of RNAs weren’t actually unusual worm gene regulators. Instead, we found evidence that at least the let-7 RNA — which was the second one found in worms — seemed to be present in a wide variety of animals. That kind of helped set the stage for people becoming aware that these types of tiny RNA genes were present in organisms outside of just nematodes.

Around that same time was when a lot of breakthrough work was being done in the RNAi field. A lot of it stemmed from Craig Mello and Andy Fire’s initial paper reporting that double-stranded RNA can initiate this RNA interference effect in worms, and also the fact that it can spread throughout this multi-cellular organism. That motivated a variety of different labs — particularly the Sharp, Bartel, Tuschl, [and] Zamore labs — to get at the molecular mechanism of how RNAi might work.

As more information was being gleaned from in vitro systems, as well as some genetics in C. elegans, and it started to coincide with what had been known about post-transcriptional gene silencing in plants, there came to be a picture that initial long double-stranded RNAs could be cleaved into tiny small interfering RNAs that would then regulate the expression of protein-coding genes.

Of course, that sounded very familiar to us, at least what we thought about these small RNAs in C. elegans that were naturally encoded, and how they seemed to function as 22-nucleotide RNAs. They also regulated targets via antisense-based pairing.

So, with these correlations, as a postdoc in Gary’s lab I ended up collaborating with Craig Mello’s lab to look at whether the RNAi pathway and the pathway that makes these small RNAs — now known as microRNAs — in C. elegans would use similar factors and mechanisms. I also collaborated with Phil Zamore’s lab to address similar questions in Drosophila and human cells.

It did turn out to be more than just a coincidence that these RNAs are similar in size and seem to come from similar size precursors. Some of the factors we know that are essential for the RNAi pathway, such as the Dicer RNase, seem to play what I like to think of as an endogenous role … in actually making these microRNAs. We now know there are hundreds of these types of genes in animals, as well as plants.

Where does that bring us to in terms of what you’re working on now?

I’m continuing to focus on using the C. elegans system to try and really understand everything there is to know about the microRNA pathway. So, we’re focusing on some of the better understood microRNAs, such as the let-7 gene, and just really trying to address some basic questions that so far we don’t know a lot about, such as: How does this tiny RNA gene turn on at the right time of development to control developmental processes? What are the steps it needs to go through to go from an initial transcript to this tiny functional 22-nucleotide RNA? How does it find its targets and when it does, it looks like the mechanism of action is different from the RNAi pathway — RNAi very efficiently causes degradation of its targets. With the targets we know about for let-7, it doesn’t look like [miRNAs] affect their stability, but certainly can turn off the expression of the protein. A lot of labs have provided evidence that these RNAs seem to work at the translational level, but mechanistically, how that works and what other factors are involved, we’re just starting to be able to explore.

That’s a lot. Can you comment in a narrower, more specific way of how you’re answering these broad questions?

Our lab just started up, so we’re just getting our lab up and running, but basically the idea is to use a combination of genetic approaches, as well as molecular and biochemical types of assays, to ask specific questions.

The genetic approach is kind of a broad net to ask: If we have a particular phenotype that we know is because a microRNA isn’t expressed correctly, can we enhance or suppress that phenotype by another genetic mutation? Then, if we can uncover that gene, it might tell us something about this pathway, and, of course, that … any sort of factor can be discovered in a genetic approach.

In some more detailed molecular approaches, we’re simply asking, for example: What does the initial transcript of the let-7 microRNA look like? It’s still a pretty wide open question — even what polymerase makes these microRNA genes initially, and beyond that how are they regulated? It’s very clear that a number of microRNAs are only expressed at certain times of development, as well as even in certain tissues. There has to be important levels of regulation that are making sure these RNAs are only made at the right time and in the right place.

So, we think one important approach is to identify what the initial transcript of a microRNA, for example the let-7 gene in C. elegans, looks like before it gets processed down to its functional form.

Going into this, do you have any theories on what you think might be going on in terms of regulation?

We really think that, as it stands now, the production of the functional form — which we believe is a tiny 22-nucleotide form — can actually be regulated at a number of different levels. So even though we often think about gene expression being regulated at the transcriptional level, and certainly it’s probably true that many of these microRNAs are going to be regulated by transcriptional processes — what sits on their promoter and makes the initial transcript. The fact that we can see stable accumulation of precursors — different processing intermediates of these microRNAs — indicates that there is potential for the cells to regulate the production of the mature form at the processing level, as well.

In fact, a number of groups have recently reported a specific export factor that takes the precursor form of the microRNA to the cytoplasm. And so, there can even be regulation at the level of transport, you can imagine. It looks like this export factor can even distinguish among functional precursors as opposed to ones that may be incorrectly made.

So, perhaps the reason there might be this elaborate level of transcriptional, processing, [and] transport steps could be a way that the cell can really regulate the making of these types of RNAs at a number of different levels. I think we’re really just at the brink of understanding when and how the cell might actually do that.

What areas do you see this work carrying into down the road?

I think that one of the most important implications of the discovery of the microRNA world is that, because of the fact that there are hundreds of these tiny RNA genes that we weren’t really even aware of couple years ago, really emphasizes how little we understand about what a functional gene product is.

Certainly, in this dawn of genomics, where we can go in and look at full sequences and predict genes, the reality is there’s probably lots of genes and different kinds of genes we just can’t recognize right now, many of which are probably going to turn out to be other types of non-coding RNAs.

I think a family of microRNAs that we consider to be these 22-nucleotide RNAs are just one size and one type of non-coding regulatory RNA. It really helps a variety of different fields to think about this idea that when they have an interesting phenotype, and they’re trying to track down the gene that is responsible for it, if there’s not an obvious open-reading frame in their area of mapping, they won’t just discard it as not being the correct region, but they’ll be able to look for other types of genes that we just aren’t even aware exist right now.

I think it really adds to the entire field of gene finding, and kind of humbles us in a way as to thinking that there are going to be a lot of new ways that nature has evolved to regulate gene expression; it’s most certainly not all going to be at the transcriptional level. RNAi is certainly a powerful example of post-transcriptional regulation.

Understanding how RNAi works, as well as these microRNAs genes, affords a lot of potential for ... therapeutics or new targets for drugs, or looking for disease-causing genes.

 

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