Rearchers from PTC Therapeutics, South Plainfield, N.J., identified an enzyme complex that plays a part in the production of transfer ribonucleic acid (tRNA) and RNA processing. The complex also shows a link between the pathways involved in regulating messenger RNA (mRNA). The finding, according to the company, could lead to new therapies that target the post-transcriptional mechanisms of RNA processing events.

"The interesting thing about this study is that one of these enzymes brings down proteins that were thought to be part of a completely separate pathway in RNA synthesis," says David Engelke, PhD, director of the Program in Biomedical Sciences at the University of Michigan, Ann Arbor, who studies earlier enzymes in the same pathway and other areas related to RNA processing.

"The proteins identified are known to affect the end processing, called 3´ end processing, of mRNA," says Engelke. "As far as I know, no one has ever suspected that there was any link in the two pathways that process messengers and transfers. They are treated as completely separate entities."

In addition to identifying the tRNA-splicing endonuclease, the study shows the enzyme has polypeptides associated with it, says Engelke, which creates the initial link between the two pathways. "[PTC Therapeutics] goes on to show that when the level of this enzyme is reduced, there is an effect on the messenger processing pathway. This provides not only a physical link between the two pathways, but a pseudo-genetic link as well," he says.

PTC (derived from "Post Transcriptional Control") Therapeutics is a small drug discovery and development company that develops technologies for seeking out small molecules with oral bioavailability. These molecules modulate post-transcriptional control processes to ultimately increase or decrease protein expression. The company is working in multiple therapeutic areas with a concentration on oncology and inflammation.

Regulating how RNA and tRNA mature is an effective target for regulating cell proliferation, says Stuart Peltz, PhD, president and CEO of PTC Therapeutics. "By regulating [RNA and tRNA] abundance, you can regulate cellular proliferation as well as protein synthesis."

"We identified a complex of endonucleases involved in processing tRNA, which regulates tRNA abundance levels," says Peltz. "We also found that this complex is not only involved in regulating tRNA levels, but is also involved in regulating mRNA levels. We have, in essence, found an enzyme complex that is involved in the processing of multiple RNA targets."

In tRNA splicing, the introns are removed differently than in mRNAs, says Peltz. When their researchers identified the tRNA-splicing endonucleases, they found those enzymes were part of a larger complex. "When we characterized that complex, we realized it had enzymes and proteins that were involved in not only tRNA splicing, but in mRNA polyadenylation."

The complex formed an endonucleasome involved in multiple aspects of post-transcriptional control, says Peltz. "That endonucleasome is a target in which you have a specific set of enzymes that you can use to identify new therapeutics."

The PTC researchers started with trying to identify similar enzymes involved in tRNA splicing in mammalian cells that were previously identified in yeast. Once they had the complex, they identified the other proteins involved through biochemical and mass spectroscopy approaches. PTC researchers then used siRNAs to knock out these RNAs in human cells and show that the proteins were involved in tRNA splicing and mRNA production.

"[The company] demonstrated that associations seen with the enzyme are real and serve a function in the cell because when you disrupt what was thought to be a tRNA enzyme, it has an effect on messenger processing," says Engelke. "Any process that affects a large number of different pathways provides a potential drug target. If you can inhibit the cell's use of this [process] for active metabolism, you have a chance [for developing a drug]."

PTC Therapeutics developed the assay wherein the enzyme finds an inhibitor for tRNA splicing and mRNA polyadenylation. The company then matches the target against their chemical library of more than 200,000 compounds that would selectively inhibit that reaction. "We identified a new set of targets in which we will look for small molecules that can treat diseases such as cancer and inflammatory diseases where we can use a small-molecule approach," says Peltz.

By Elizabeth Tolchin