Ribozyme Reversal of Fortunes

By Jason Socrates Bardi

"The one duty we owe to history is to rewrite it."

—Quote attributed to Oscar Wilde (1854–1900)

Ten years ago, a group of scientists who were all working in the field of RNA processing met at the annual meeting of the RNA Society in Madison, Wisconsin to discuss their latest research.

Their field had gone through a lot in the previous decade. RNA processing encompasses the biochemical mechanisms through which RNA chains are edited—cut and pasted into new RNA chains, much as individual shots in a movie are spliced together by an editor to make a scene. And until the early 1980s, scientists had assumed that only proteins spliced RNA. For that matter, scientists had assumed that only proteins could carry out any enzymatic reaction at all.

In 1982, however, Tom Cech at the University of Colorado Boulder made the completely unexpected discovery that RNA from a unicellular protozoan called Tetrahymena thermophila could perform auto-splicing. Cech shared the Nobel Prize in Chemistry in 1989 with Sidney Altman for his discovery of RNA molecules with catalytic function, and this opened up a whole new field for scientists: the structure, function, and mechanism of action of RNA enzymes.

Scripps Research Institute Associate Professor Martha Fedor was a postdoctoral fellow in the Boulder laboratory of Olke Uhlenbeck in 1989, and was next door to Cech when the significance of Cech's discovery was acknowledged by the Nobel committee. She did some of the first quantitative measurements of the kinetic parameters of RNA enzymes, which soon began to be called "ribozymes."

Fedor, who is now a member of The Skaggs Institute for Chemical Biology at Scripps Research, was still investigating RNA enzymes 10 years later when leading scientists in the ribozyme field gathered for this 1994 meeting. The mood at the meeting was one of growing confidence and elation. Dozens of ribozymes had been characterized, and the way in which they catalyzed reactions was known: ribozymes had to bind to metal cofactors to function. In fact, some scientists were so confident of this that they had taken to calling ribozymes "metalloenzymes."

But this confidence was misplaced.

Only a few years later, Fedor demonstrated that merely positively charged ions were needed to enable ribozymes to assemble into functional structures. Metal ions need not participate directly in catalytic chemistry.

Fedor and her colleagues saw that ribozymes can react in alternative buffers like cobalt hexamine or ammonium acetate instead of conventional metal cations like magnesium or manganese. Ammonium acetate buffers contained no metal ions at all. In cobalt hexamine, the metal remains bound to its amine ligands and cannot facilitate reactions by binding ligands associated with the reactive phosphate in the way that magnesium or manganese ions would. Cobalt hexamine does contain metal, but the metal is complexed in such a way that it cannot facilitate reactions the way that traditional metal cations would.

"Our claim to fame was that our ribozyme [functioned] perfectly well in an environment with no [metal] cofactors," she says.

Now, says Fedor, a decade after that Madison meeting, ribozymes have undergone a complete reversal of fortune in terms of being characterized as metalloenzymes—in fact, now scientists wonder whether any ribozymes use metal cofactors.

"Our field is a wonderful one," says Fedor. "People are very good about saying, 'Everything we thought we knew was wrong.'"

RNA Minimalism

The discovery of catalytic RNA in 1982 altered what had been known as the central dogma of molecular biology—that genetic information was stored in DNA molecules and that biological action (catalysis) was taken by proteins.

RNA had largely been assumed to be an intermediary in this process, unable to participate directly in catalysis.

Perhaps for good reason.

Protein enzymes are polymer strings made up of the 20 different amino acid building blocks, many of which have reactive side chains that can participate in biocatalysis. RNA polymers are made up of only four different building blocks, the RNA nucleotides, which lack the chemical versatility and reactivity of the protein functional groups.

After the discovery that RNA could act as a biocatalyst, many scientists began to theorize and discuss the possibility of an RNA world—the notion that at one time the world was ruled by RNA-based life forms in which RNA enzymes were the chief catalytic molecules and RNA nucleotides were the building blocks that stored genetic information. This raised a number of questions, such as where catalysis came from and how the world transitioned from an RNA-based form to the current DNA-RNA-protein form.

Another group of scientists, including Fedor, became interested in the fundamental question of how. How do ribozymes do it? What is it about RNA structure that confers unique properties on the nucleotides that allows them to do catalytic chemistry?

Today, half of Fedor's group at Scripps Research works on understanding the mechanisms of RNA catalysis using a simple ribozyme as a model—a shortened, "minimal" form of catalytic RNA that's about 50 nucleotides long.

Called the hairpin ribozyme, this minimal RNA enzyme was discovered by botanists in plants infected by tobacco ring spot virus and a number of other viruses. It is what is known as plant satellite RNA.

Satellite RNAs are parasitic pieces of RNA that are not exactly viruses because they don't encode for proteins. Instead, they catalyze simple cut-and-paste reactions in order to replicate themselves, exacerbating or ameliorating diseases caused by plant viruses. A plant that has tobacco ring spot virus, for instance, will be more diseased if it also has this satellite RNA.

Since the hairpin ribozyme is so short, it's easily handled in the laboratory, which allows Fedor and her colleagues to manipulate individual parts of the ribozyme to see which ones are most important for catalysis.

When the high-resolution structure of the hairpin ribozyme appeared a few years ago, determining which parts are important for catalysis became easier because the structure pointed to particular bases that might have a role in the catalysis by virtue of their proximity to the ribozyme's active site.

By eliminating these bases one by one within the active site of the ribozyme, Fedor and her colleagues were able to determine which ones were important for catalysis. Eliminating some nucleotides resulted in no change in catalysis, suggesting that these RNA bases played no significant role. Eliminating others resulted in hairpin ribozymes with defects, suggesting that these bases contribute to normal activity.

The scientists have concentrated on these latter nucleotides, hoping to learn something about the ribozyme catalytic mechanism through a technique called exogenous nucleobase rescue of abasic substitutions—a way they found to delete particular nucleobases from the active site of an RNA enzyme without disrupting its structure and restore activity by providing nucleobases in solution.

"It's a cool way of investigating the structural and functional requirements for a particular nucleotide," says Fedor, adding that the technique was a big breakthrough. "Before we found that abasic substitutions could be rescued by exogenous nucleobases, any time we touched [the ribozyme], we destroyed the structure and couldn't tell what was important for holding it together and what was important for catalytic chemistry."

Exogenous nucleobase rescue involves substituting RNA nucleotides of interest with abasic residues that maintain the continuity of the RNA chain but have a single hydrogen atom in place of a critical nucleobase. Then the researchers add in small molecules that can rescue the activity that was lost due to the missing nucleobase.

Using this technique, Fedor has been developing a new picture of the RNA enzyme reaction mechanism.

She is proposing that rescued nucleobase ribozymes bind the reactive phosphate through the same functional groups that normally engage in Watson-Crick base pairing in conventional RNA structures. In the context of the ribozyme active site, however, these interactions promote catalysis by stabilizing a highly energetic transition state in which five oxygen atoms are associated with one central phosphorous atom—a highly electronegative situation. A good way to stabilize this transition state would be to have a positive charge close by, a charge that might come from adding a proton to a nearby nuclotide. Data showing that rescue activity increases as reaction buffers are made increasingly acidic supports this idea, she says.

The way in which RNA functions as an enzyme is of even more interest now that the ribosome structure has been solved. The ribosome, a huge nucleoprotein complex, is the RNA machine that strings together amino acids to make proteins in cells, and its structure reveals that there are no proteins or metal ions in its active site. It is the RNA alone that performs the peptide bond formation.

"This is one of the most well-studied reactions on the planet, but we still don't know how the reaction is catalyzed," says Fedor.

In Vivo Approaches and Therapy

The other half of Fedor's group uses the ribozyme as a model for RNA folding in vivo. Using yeast as a model system, they look at ribozyme activity and ask how their experiments carried out in vitro will translate to living systems.

Interest in ribozyme activity in vivo has grown in the last few decades because of its potential for use in therapeutic applications to human diseases. There are many diseases that are associated with aberrant RNA expression that could be addressed by modifying RNA levels—from knocking out mRNAs expressed by cancerous oncogenes to getting rid of viral RNA inside infected cells.

"Ribozymes that recognize targets through complementary base pairing allow you to develop a therapeutic reagent without knowing anything about the structure or the function of your protein target—you just need to know the gene," says Fedor.

Fedor and her group have developed a way of measuring ribozyme cleavage of RNA targets in vivo, based on the intrinsic rate of RNA degradation. By correlating the rate at which the RNA disappears in the presence or absence of the ribozymes, they can determine the cleavage rate of the ribozymes.

"All RNAs turn over at a characteristic rate," says Fedor. "We use this as a clock."

This is important in the context of therapeutic RNA because the cleavage rate determines the potential efficacy of the RNA. Only if the cleavage rate is much greater than the intrinsic degradation rate will the RNA catalysis be worthwhile. If the cleavage rate is not much greater than the intrinsic degradation rate, then subjecting cells with it probably will not make a difference in the abundance of the target.

Inactivation of target RNA is most effective against a stable, slowly decaying RNA, such as a chronic viral infection like hepatitis where the RNA persists for a long time in cells.

On the other hand, the stability of the ribozyme structure is a factor as well, and Fedor has done experiments with the natural, long form of ribozyme to compare its stability to the short form.

"One of the things we've learned over the last few years that has made a big difference is that the natural form assembles in a four-way helical junction."

This four-way helical junction is far more stable than the two-way helical junction of the short hairpin ribozyme and is completely functional at intracellular ionic strength, whereas the minimal, two-way junction ribozymes require high concentrations of divalent cations for its activity.

Fedor's results suggest that four-way junction ribozymes may be more amenable to therapeutic applications because the four-way junction ribozymes are more stable in vivo.

Data from Fedor lab studies also show that the four-way junction ribozymes are very efficient ligases—that is, they can stitch nucleotides together as well as they can pull them apart. This suggests that the four-way junction ribozymes could be used as RNA repair enzymes.

Such findings may help the therapeutic dreams of ribozyme enthusiasts become a reality.


Send comments to: jasonb@scripps.edu



Associate Professor Fedor in interested in a fundamental question: How can RNA molecules perform catalytic chemistry?






Hairpin ribozymes are found in the satellite RNA of tobacco ringspot virus where they mediate self-cleavage and ligation reactions that are needed for satellite RNA replication. Click to enlarge





Model of electrostatic stabilization by a cationic nucleobase. Click to enlarge