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UCHC s Brenton Graveley on Using RNAi to Explore Alternative Splicing

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At A Glance

Name: Brenton Graveley

Position: Assistant professor, genetics and developmental biology, University of Connecticut Health Center

Background: Postdoc, Harvard University — 1996-1999; PhD, microbiology and molecular genetics, University of Vermont — 1996; BS, molecular, cellular, and developmental biology, University of Colorado at Boulder — 1991

Although it was the work of others that piqued his interest in RNAi, Brent Graveley quickly incorporated the gene-silencing technology into his work studying alternative splicing and has published multiple papers on the subject.

He recently spoke with RNAi News about his work.

How did you get started with RNA interference?

I had seen a lot of papers where people had started doing RNA interference, and at the time I had just started working a little bit in the lab in Drosophila cells — my lab is primarily focused on alternative splicing.

I saw that people could easily do RNAi in Drosophila cells and it seemed like a great opportunity to use the power of RNA interference to try and dissect the mechanisms by which alternative splicing is working. So, we started a project where we were essentially knocking out different RNA-binding proteins and asking: What effects [did that have] on the alternative splicing of several different genes?

That’s essentially how I got started in it.

Up until you had adopted RNAi, what sort of approaches were you taking?

Prior to that, the approach that I was taking was pretty much what everybody else does in the field of splicing, which is you take radioactive RNA, you throw it into a HeLa nuclear extract, you do UV cross-linking, and you find some 50-kilodalton protein that binds to it. Then you have to figure out what it is.

That was limited in the sense that you had to know what proteins existed and you had to have antibodies available to proteins to be able to identify them. In a sense, it’s a useful approach, but it’s limited also. I saw RNAi as being a way of doing the reverse of that — in a sense, doing genetics, which hasn’t really been applied to the study of alternative splicing very much.

In terms of what you’re working on right now in your lab incorporating RNAi, can you talk about those?

Sure. We have two main projects that involve RNAi. The first is what I alluded to: Knocking out different RNA-binding proteins. We’re actually preparing a paper to send out now, where we’ve knocked out 75 percent of the RNA-binding proteins in the Drosophila genome and then we examined the alternative splicing of three different genes simply by doing RT-PCR.

In this very limited study, we’ve identified almost 50 splicing factors that have an effect on alternative splicing, and 26 of these proteins had never been shown to be involved in alternative splicing at all. So, in the course of several months, we discovered many, many new proteins that can regulate splicing. Since this was only looking at the alternative splicing of three genes, [it] suggests to me that we’ve just touched the tip of the iceberg of what’s possible.

Do you mind me asking what publication you’re submitting the paper to?

We’re submitting a paper to Science, [but] anyone can do that. [The paper will] be somewhere, hopefully, soon.

The next step of this [project] is to use microarrays that can detect changes in alternative splicing of most of the genes in the genome, and then analyze the changes in splicing on a genome-wide level when we knock out all the different RNA binding proteins. We’re hoping in that way to use this technology to dissect the splicing regulatory networks that exist in the fly.

That’s one project. The second project that involves RNAi is using what we call exon-specific RNAi, [in] which … you basically design your double-stranded RNA to a specific alternative exon. At least in fly cells, that specifically degrades only RNAs that contain that exon, but not RNAs that lack that exon.

In that way, we’re using [RNAi] as a tool to address the functional relevance of different alternative spliced isoforms in the biology of the fly.

What are the long-term implications of that project?

Where I would like that project to go is to do this on a genome-wide level, because one of the big mysteries of biology is … with Drosophila, why they have so few genes. Drosophila has 14,000 genes or so, and worms have about 20,000. Yet, flies are significantly more complex than worms.

One of the main explanations I have for this is that flies have a lot of alternative splicing — they just generate many, many more proteins than worms do. So, the important thing is not determining what all the genes do; the important thing is determining what all the proteins do. The only way to get at that is to be able to test what … the proteins [encoded by] specific alternative-spliced mRNAs … are doing.

People have done knockouts of all the genes and all these things are essential, but I think you’ll get different answers when you look at specific isoforms that are synthesized by those genes.

It gets really complicated because there are lots of genes in the genome that encode two different proteins, but one of the genes we work on, the Dscam gene, encodes 38,000 different proteins — it’s a very large problem.

Can you talk about Dscam a little bit?

Dscam is a gene that was initially discovered in Larry Zipursky’s lab at UCLA, and it’s a gene that encodes an axon guidance molecule. The interesting aspect of it to me is [its] extent of alternative splicing — it contains 95 alternative exons, and based on the combinatorial possibilities in which they can get spliced, you can generate 38,016 different possible isoforms.

Each of those isoforms has different protein interaction properties and those interactions are important for determining the specificity of wiring of the neurons. So, then, alternative splicing of this gene and how that is regulated really plays a key role in the wiring of the fly brain.

The main focus of my lab is trying to figure out how the splicing of this gene is regulated, and that’s where we’re using the RNAi to knock out different proteins and see what proteins are involved in controlling the splicing of this gene, and then using the exon-specific RNAi to analyze what the functions of some of these 38,000 different isoforms are.

Do you have an comment on challenges you see associated with RNAi? Are there aspects of the technology that could be improved upon?

I think one of the challenges still, and it’s not a major problem, is specificity. Even if you’re using an siRNA, there’s several studies out suggesting that you can get indirect effects. That becomes more of a problem in the fly where you’re using larger RNA, because you have a greater probability that one sequence can indirectly affect others. But, if you do your computer searches correctly, you can eliminate a lot of that.

I think that in most cases, probably the biggest hurdle is doing this in a high-throughput manner in the animal. In worms it’s easy because you can just soak them, but that doesn’t apply to any of the other organisms.

For all the other organisms you essentially have to make transgenic [animals]. The bottleneck there, I think, is more in the technology that exists to make transgenic animals in a high-throughput manner, as opposed to getting the RNAi to work — once you get those guys, it’ll work.

Do you make your own RNA?

We use long double-stranded RNA … and we synthesize all of those ourselves enzymatically.

We just design primers to the cDNA that we’re interested in and make sure that the sequences that we’re going to amplify don’t exist elsewhere in the genome. Then we amplify those and clone those into vectors. We have a plasmid library of all the genes we’re knocking out, and that way we … know what we’re trying to knock out. Then, what we do is, from that library, we use the phage promoter that exists in there as PCR primers. Then we transcribe those PCR products.

Going forward, are there other programs that you anticipate getting involved in where RNAi might play a role?

I would say that I would be interested in doing some of the similar things we’ve been doing of knocking out different splicing factors and RNA binding proteins, and analyzing alternative splicing. Doing that in other organisms such as worms and mice and humans, and seeing what the differences are — what are the different types of splicing factors in these different organisms and what not — [interests me].

At the same time, [I’d like to try] using the exon-specific RNAi in these other organisms, as well. [This] actually probably won’t work in the worm, because they have RNA-dependent RNA polymerases, which cause all sorts of issues you don’t want. But, at least in the mouse and human genomes, where there’s tons and tons of alternative splicing, trying to use that technology to figure out what the different isoforms are doing, would be useful.

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