Study provides insight into cellular defenses against genetic mutation

October 25, 2005

With their latest discovery, researchers have significantly advanced the understanding of how human cells protect themselves from constant and potentially destructive changes in gene expression. According to an article published in this month's Nature Structural & Molecular Biology, the research is important because the protection itself can contribute to disease, and the ability to side-step it may lead to new treatments for hundreds of genetic disorders.

The blueprint for the human body is encoded in genes, many of which hold the information necessary for the building of one or more proteins. Gene expression is the process by which information stored in genes is coverted into proteins that make up the body's structures and carry out its functions. While genetic instructions are stored in chains of deoxyribonucleic acids (DNA), they are put into practice by ribonucleic acids (RNA). Messenger RNA (mRNA), a modified copy of DNA, is transported to cellular factories called ribosomes that receive instructions for building proteins by "reading" mRNA templates.

Over time, genes evolve to show changes in their makeup. Some changes, or mutations, have no impact, some provide advantages making organisms more likely to survive, and others cause disease. One frequently occurring, damaging class of mutation is the inclusion of premature "stop reading" signals (stop codons) within mRNAs. Called "frameshift" or "nonsense" mutations, they order the genetic process to stop reading part way through the instructions, resulting in the building of incomplete proteins. Affected mRNAs create shortened, disabled proteins that can sabotage natural processes by competing for spots usually held by their full-length counterparts, or by simply not working.

In recent years, researchers at the University of Rochester Medical Center have revealed the existence of a natural surveillance system that determines which mRNAs pass muster as legitimate templates, and that sees to the destruction of the rest. They also found that the screening process, called nonsense-mediated mRNA decay (NMD), can nearly eliminate the supply of an mRNA template for an essential protein because the mRNA has acquired a nonsense codon.

"Our study is important because we have determined for the first time that the mRNA-binding protein CBP80 tells the NMD system which mRNAs to review for nonsense codons," according to Lynne E. Maquat, Ph.D., professor of Biochemistry and Biophysics at the Medical Center, and senior author of the Nature piece together with post-doctoral associate Nao Hosoda, Ph.D. "That is critically significant because, knowing the structure and role of CBP80, we can now seek to develop drug-based gene therapies that interfere with it in cases where NMD contributes to disease-causing protein shortages." Researchers may be able to convert mRNA quality control from NMD over to a more flexible system that "overlooks" flaws, and provides more templates for building functional proteins.

Genetic Decision Maker

For two decades, researchers have made intuitive leaps in the understanding how NMD works with translation, the second phase of gene expression where RNAs direct the building of proteins. From studying genetic diseases, Maquat theorized four years ago that there must be two types of translation. An early "pioneer" round checks all new mRNAs for errors, and initiates NMD when errors are detected. A second "steady-state" round translation then directs the mass production of normal proteins based on "NMD-approved" mRNAs. Recently, Maquat's team has worked to identify the roles of proteins involved in NMD, the focus of the Nature paper.

During translation, mRNA chains are read in one direction due to the nature of their building blocks, called nucleotides, starting with the 5-prime end, and ending with the 3-prime end. As mRNA chains are synthesized, a 7-methylguanosine cap is attached to the 5-prime end and a tail of repeating adenosine molecules to the 3-prime end. The cap and the tail protect the mRNA from enzymes that would otherwise destroy it from both ends.

Along with its protective role, the 5-prime cap is designed to bind to a complex of two linked cap-binding proteins (CBPs), CBP80 and CBP20. The cap-binding complex is part of the larger pioneer translation initiation complex, which enables mRNAs to be read by ribosomes during quality control review. Previous studies in the Maquat lab revealed two vital facts about cap-binding proteins. First, any mRNA with a CBP80 attached to its 5-prime cap can be translated by ribosomes. Secondly, mRNAs linked to CBP80 are targeted for NMD when a ribosome finds an early stop codon. Most recently, the Nature paper demonstrated for the first time that CBP80 works by activating a protein factor known to drive NMD called human up-frameshift (hUpf) protein 1.

A Matter of Spacing

Immature mRNA is made of both "must-read" pieces of RNA called exons that code for the building of proteins, and pieces called introns that do not code for proteins. For mRNA to mature, it must be cut and pasted by cellular machines that re-assemble end to end only exons in the final template for protein construction. In recent years, Maquat's team has confirmed that complexes that identify early nonsense codons form at exon-exon junctions the places where each "must read" section is joined to the next in the mature chain.

What Maquat and colleagues are learning is that much of the regulatory action of NMD depends on the physical spacing of molecules in the RNA chain. If a stop reading signal occurs too far ahead of the final exon in the chain, as marked by an exon-exon junction complex (EJC), the cell concludes that the stop codon has mistakenly fallen in the middle of a set of instructions. Specifically, Medical Center researchers now believe a stop codon is labeled premature when it occurs more than 55 nucleotides upstream (toward the 5-prime end) of an exon-exon junction.

If these spatial requirements are met, CBP80 interacts with human up-frameshift proteins, factors that come together at exon-exon junction complexes to drive NMD review. The three up-frameshift proteins work together to trigger decay once an early nonsense codon is detected, most likely by recruiting enzymes that remove the tail and cap from suspect mRNAs and digest their chains. If nonsense codons are not detected, CBP80 and its partner CBP20 are replaced by another cap-binding protein that renders mRNA immune to further notice by NMD.

"NMD is an elegant process that offers a number of targets for future medical therapies," Maquat said. "Beyond its role in screening out faulty mRNAs through immediate decay, we have also learned that the NMD factor Upf1 is part of a related process that regulates the expression of many normal genes by controlling the timeframe in which their encoded mRNAs decay. Evolution often borrows a useful mechanism from one process and puts it to work in another."
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University of Rochester Medical Center

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