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Biochemistry professor Aaron Hoskins (left) and graduate student Joshua Larson use lasers to study how cells process messenger RNA. Photos by Sevie Kenyon BS'80 MS'06

For people who know about RNA mostly from its place in the central dogma of biology—DNA➙RNA➙Protein—this story may hold a number of surprises.

That handy equation, taught in Biology 101 courses around the globe, sums up the flow of genetic information in living organisms: how our DNA gets copied into RNA, which then gets converted into proteins, the building blocks of our cells, our bodies.
Originally, the RNA referred to in this equation—messenger RNA, or mRNA, the type that codes for proteins—was the only kind known to science. However, over the years, it has become clear that there are many, many other kinds.

“The world of RNA has proven to be a big and fascinating place,” says Marv Wickens, a CALS professor of biochemistry and leading pioneer in RNA research. “I’ve come to think of it as a Fellini movie, full of strange and unexpected characters.”

These Felliniesque characters are all the non-coding RNAs that exist in nature—the kinds that don’t code for proteins. They go by names like small interfering RNA, piwi-interacting RNA, microRNA, long non-coding RNA, small nuclear RNA—the list goes on and on. Together they far outnumber messenger RNAs in the cell; while only 3 percent of the human genome gets made into proteins (via messenger RNA), a full 80 percent gets copied into RNA.

What are all of these other RNAs doing? Lots of important and surprising things, scientists are discovering.

Over the past few decades, RNA, a close chemical cousin of DNA, has proven itself to be a much more versatile molecule than originally thought—far more than just a passive messenger.

The first big surprise came in the 1980s when it was shown that RNA can have catalytic activity, meaning that it can perform chemical reactions inside the cell. Originally assumed to be inert, like DNA, scientists found RNA molecules that could edit their own sequence—expunging a segment of their own genetic code.

Later, RNAs were discovered at the heart of important cellular machines, or enzymes, performing critical catalytic reactions, including those at the heart of the cell’s information transfer system. Previously only proteins were thought capable of such enzymatic feats.

These findings, it’s interesting to note, support the idea that RNA may be the original material of life. With them, RNA has two things going for it: it’s made of heritable genetic material and it’s chemically active.
“You could imagine you have a little RNA molecule that develops the capacity to copy itself—and now you’re off,” says Wickens.

Biochemistry professor Marv Wickens serves as the hub of UW – Madison’s RNA community. Photo by Wolfgang Hoffmann BS’75 MS’79

More recently, scientists were surprised to discover that microRNAs, which are short pieces of non-coding RNA, play a major role in regulating gene expression. They do so by binding directly to messenger RNAs and altering the amount of protein the messenger RNAs produce. Scientists now believe that microRNAs may be regulating as many as 60 percent of human genes this way.

“It was a revolution. These microRNAs had been completely invisible to us,” says Wickens. “We now know that there are hundreds of these RNAs that affect gene expression. They’re involved in cancer and in other diseases—they’re everywhere.”

Yet many RNA mysteries remain. And CALS researchers, as they have been for decades, are at the forefront of efforts to explore these unknowns, working from a variety of angles to shed light—sometimes quite literally, using lasers—on the next big questions in the field.

“For a while, RNA was kind of like the dark matter inside the cell. Everything was below the radar,” says CALS biochemistry professor Sam Butcher. “Now we’re at the point where we know it’s there, and we’re working to figure out what’s going on.”

UW’s RNA community

The University of Wisconsin–Madison has a proud legacy of RNA research that goes back to the late 1960s, when members of the so-called “Ribosome Group” began to hit their stride. The group included CALS biochemist Masayasu Nomura, who figured out how to assemble the ribosome—the RNA-powered enzyme that converts messenger RNA into proteins—in a test tube, and biochemist Julian Davies, who discovered that many antibiotics work by interfering with the ribosome. Around the same time, Howard Temin, a professor of oncology in the School of Medicine and Public Health (SMPH) made a breakthrough discovery about how RNA retroviruses work—research that won him a Nobel Prize in 1975.

These figures drew other powerhouses to the UW campus—including RNA researcher Jim Dahlberg, now an emeritus professor of biomolecular chemistry in the SMPH—who then drew others.

“I came to the UW because of the RNA community that existed, because of the virology and the ribosome work being done here. So that’s what made the job attractive—and I think we’ve kept that going,” says Dahlberg.

Indeed, UW–Madison’s RNA research community is considered among the top in the world. Faculty working in RNA hail from more than a handful of CALS departments, including bacteriology, biochemistry, genetics, nutritional sciences and plant pathology, as well as a number of SMPH units. Many focus on eukaryotic systems, while some stick to bacteria. Some use genetics-based approaches, while others apply the tools of biochemistry, chemistry, biophysics and even physics. Some are lifelong RNA researchers, while others “bumped into” RNA in the course of their work.

It’s a very diverse group, yet they don’t stick to their silos. Beginning in 1988, they started coming together regularly as members of the RNA MaxiGroup, an organization founded by Wickens to help foster scientific discussion and research collaborations among this far-flung group. Wickens, who is widely seen as the hub of the UW’s RNA research community, has been running things ever since.

“He’s been the main driver in keeping things going,” says Dahlberg.

Through Wickens’ vision and effort, the group has helped transform the UW’s RNA researchers into a cohesive community. The group gathers for monthly seminars that draw top RNA researchers from around the globe to speak on campus, bringing in fresh ideas and increasing the visibility of UW research. These gatherings have also helped spark numerous cross-department and cross-college collaborations.

“There’s a really wonderful environment here for RNA researchers,” says Butcher, who is involved in a longstanding collaboration with an SMPH colleague.

Collectively the group is exploring some of the most pressing questions in the RNA field: how messenger RNAs are regulated; what the spliceosome—the enzyme that edits messenger RNA—looks like and how it works; the phenomenon of RNA interference; the role microRNAs and other non-coding RNAs play in the cell; and others. This work is helping to push back the dark edges in our understanding of RNA and pave the way for important medical breakthroughs down the line.

The following is a selection of exciting RNA projects to emerge from CALS.

Insights into mRNA regulation

Figure 1 mRNA Structure

Ever since Wickens joined the CALS biochemistry faculty in 1983, he’s been in the vanguard of efforts to understand how messenger RNAs (mRNAs) are regulated—how their protein production is controlled—with a particular interest in how that regulation impacts embryonic development, cell growth and memory.

Early on he focused on how the ends of mRNAs are formed, which at the time was largely a mystery. His team made a major discovery about the poly(A) tail, the long string of adenine (“A”) residues at the end of mRNA molecules (see Figure 1). They found that cells can tack on more “A” residues to the end of the poly(A) tail to ramp up an mRNA’s protein output, and then remove them to dial things back—and that animal embryos regularly rely on this trick during early development to quickly boost production of important proteins at key moments.

Next, Wickens began exploring another regulatory region found in mRNA molecules known as the 3’ untranslated region—or 3’ UTR—which is full of binding sites for regulatory proteins and non-coding RNAs that impact protein production. Wickens started hunting for these binding sites—and their corresponding binding factors—but was frustrated by the lack of tools available to do this kind of work. So he helped devise one: the Yeast Three Hybrid System, a powerful test that is now widely used in labs around the world and has facilitated a string of major discoveries.

Wickens and University of Washington biologist Stan Fields came up with the idea over dinner one evening. The test, in which a motley assortment of molecules must line up to give a positive result, gives researchers a way to find out which proteins bind to a particular piece of RNA, or vice versa.

“It seems so Rube Goldberg, and it’s kind of preposterous that it works—but it does,” says Wickens. “And all you want to know is if these two guys—an RNA molecule and a protein—interact.”

One early and high-impact use of the Yeast Three Hybrid System took place in the form of a collaboration between Wickens and CALS stem cell researcher Judith Kimble, even though—at the time—working together was something that both Wickens and Kimble, who are married, had hoped to avoid.

“I was over at Bock Labs running my lab, completely separate from Marv,” explains Kimble. “As a woman professor, it was important to be independent, especially of my husband, but then my group stumbled into an interesting 3’ UTR, and Marv was the 3’ UTR expert.”

Kimble’s team had found what they believed to be a key binding site in the 3’ UTR of an mRNA that was important in cell fate decisions in the worm C. elegans. Kimble wanted to figure out what protein was binding to the site and collaborated with Wickens’ group to fish it out using the Yeast Three Hybrid System. The test identified FBF, a member of the now widely studied PUF family of proteins.

Although they made this discovery in C. elegans, PUF proteins have turned out to play important roles in organisms from yeasts to humans. It’s now clear that these proteins, which bind to and regulate a broad spectrum of mRNAs, are important in early development, stem cell maintenance, and even learning and memory. Groundbreaking work in this area by Kimble and Wickens has helped spark interest among other scientists.

“Now there’s a little cottage industry of people trying to figure out what these proteins do,” says Wickens.

Cracking the spliceosome

One holy grail for the international RNA community is to crack the spliceosome—to figure out what it looks like and how it works. Two CALS scientists are participating in this effort.

The spliceosome, the enzyme responsible for editing messenger RNA (mRNA), is an amazing cellular machine. Its job is to process “pre-messenger RNA” into “mature” mRNA. To do so, the spliceosome removes unwanted segments in the pre-messenger RNA, called introns, and stitches together the keepers, called exons.

Figure 2 Alternative Splicing

The enzyme, remarkably, is able to mix and match the exons. So, from the same gene, it creates different mRNA products in different tissues, as called for (see Figure 2). This process, known as alternative splicing, is extremely prevalent in humans, affecting more than 90 percent of our genes.

This phenomenon is partly responsible for making humans the complex creatures that we are, notes Aaron Hoskins, a CALS professor of biochemistry. While humans don’t have all that many more genes than C. elegans or fruit flies, he points out, “One thing we do a lot more than any of those organisms is edit our RNA.”

The spliceosome itself is a massive structure, with small nuclear RNA (snRNA) at its catalytic core performing the splicing reaction. It’s composed of five major subunits that assemble step-wise to form a whole, functioning enzyme. Once assembled, the spliceosome performs a single splicing reaction and then falls apart.

Although the spliceosome is a large enzyme, it’s still too small to see with a microscope. And because it’s in constant motion—building up and falling apart—it’s proven to be very difficult to study, particularly for scientists who’d like to crystallize it and figure out its three-dimensional shape, known as “solving” its structure.

But CALS structural biologist Sam Butcher is undeterred. He’s tackling the challenge one piece at a time. As part of a long-standing collaboration with SMPH biomolecular chemistry professor David Brow, the two recently crystalized and solved the 3-D structure of U6, the snRNA believed to be the spliceosome’s key catalytic component, bound to a nearby protein (see Figure 3—and you can see an animated illustration of U6 at http://www.news.wisc.edu/22907).

Figure 3 3-D Structure of U6* (RNA shown in red)

“It’s a beautiful crystal that we’re really excited about,” says Butcher. “And we found something about the structure that we never expected, that no one has ever seen before, in fact—the RNA and the protein are intimately entwined.”

Down the line, explains Butcher, this section of the spliceosome could be combined with other sections—like pieces of a puzzle—to assemble a full picture of the enzyme.

“The idea is if we can finally see it, then we can start asking better questions about how it works,” says Butcher.

For his part, Hoskins is shedding light on how the spliceosome assembles—quite literally—by zapping it with lasers. Hoskins uses an approach he helped develop called co-localization single molecule spectroscopy (CoSMoS) and was the first to apply it to complex cellular machines such as the spliceosome.

The approach involves attaching fluorescent dyes of various colors to molecules of interest—such as spliceosome subunits and RNA molecules—and then watching them interact by shining lasers on the sample. Different lasers—blue, green, red and orange—cause the different dyes to emit light. The way it works, Hoskins’ team can actually focus in on a single molecule at a time and get a kinetic picture of how it’s interacting with the other dye-labeled molecules.

This research is revealing valuable information about the order of splicing events and how the spliceosome “knows” which mRNA to make. Understanding how this process occurs is essential for understanding how one version of an mRNA can be produced in lung tissue, for instance, and a different form in heart tissue, despite both RNAs originating from the same gene.

“We’re using this approach primarily to study how all the pieces of the spliceosome come together at the right location at the right time,” says Hoskins, who is also developing methods to perform similar experiments inside live cells in real time. “That’s where this project is going.”

Applications in human health

It’s not hard to imagine that many basic RNA research projects could one day lead to medical breakthroughs—and in many cases, including on the UW campus, they already are doing so.

We live in a time when antibiotics are losing their power, and new ones are desperately needed. This could come from the kind of work being done by CALS bacteriology professor Karen Wassarman, who studies a small RNA in bacteria known as 6s RNA. Wassarman figured out how 6s protects bacterial cells from stress, helping them slow their growth and conserve energy during times of nutrient scarcity.

In biochemistry professor Marv Wickens’ lab, graduate student Shruti Waghray injects RNA into frog eggs to study the workings of the poly(A) tail.
Photos by Nicole Miller MS’06

The work, which revealed a novel mechanism of action for bacterial small RNAs, is just about as basic as it gets, notes Wassarman—yet it could one day reveal entirely new classes of RNA-based targets for antibiotics.

“Lots of small RNAs in bacteria are involved in stress response, which is exactly the type of thing we would want to exploit,” she says.

There’s also the prospect of capitalizing on RNA interference, the phenomenon where the introduction of small interfering RNAs (siRNAs)—which are a type of short, double-stranded RNA—into a cell causes the cell to “turn off” the expression of genes that look similar to the inserted siRNA.

“These siRNAs could be extraordinarily useful. Say someone has a disease that’s caused by having too much of gene X. Well, if you could give them a little RNA trigger to turn off gene X—problem solved,” says SMPH genetics professor Scott Kennedy, who studies the heritability of RNA interference. “siRNAs could be very potent drugs. That’s something people are working on right now.”

MicroRNAs, on the other hand, are already starting to prove themselves useful in diagnosing, understanding and monitoring cancer. In limited cases, doctors have begun screening cancer biopsies for their microRNA profile, looking for information about the nature of the cancer, including where it originated in the body, its aggressiveness and whether it’s responding to treatment.

And this heralds a much larger medical advance on the horizon: the screening of a person’s transcriptome, the full complement of RNAs in their various tissues. While genome sequencing tells a person what genes they have, the transcriptome could reveal much more, including information about what forms of messenger RNA are being made from a given gene in various tissues (via alternative splicing) and how that expression is being regulated.

Closer to home, a number of UW labs have made RNA-related discoveries that are being developed into commercial products by biomedical companies. CALS biochemistry professors Laura Kiessling and Ron Raines founded Quintessence in 2005 to develop a cancer-fighting compound. The drug candidate, an altered enzyme, preferentially destroys RNA in cancer cells, killing the cells. Phase I clinical trials of the drug recently wrapped up, with promising results.

Companies that have arisen from RNA discoveries made in SMPH labs include Third Wave Technologies, which was co-founded by Dahlberg in 1993—and later bought by Hologic in 2008—to commercialize a method for quantifying microRNAs. Another is PTC Therapeutics, a New Jersey-based company that’s developing basic discoveries made in a UW oncology lab by alumnus Stuart Peltz into drugs that target RNA to treat a wide range of diseases.

And, across campus, there may well be more.

It’s impossible to know when the next big RNA discovery or medical application will occur—or what it will be. But as long as our scientists continue to probe the remaining dark matter of RNA, we can rest assured that more discoveries and more applications will come.

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