UC San Francisco researchers have discovered a region in the telomerase enzymethat they say could prove to be a target for killing cancer cells and regenerating damaged cells, and could also lead to a possible target forattacking HIV.
The enzyme -- brought to popular fame two years ago by studies showing that it could be manipulated in cell culture to increase the life span of cells - has the capacity to replenish the tips of chromosomes, known as telomeres, which lose their final fragments with each cycle of cell division. When activated, telomerase replenishes telomeres by copying the RNA folded within it into telomeric DNA and assembling it on the ends of the chromosomes.
Telomeres maintain the stability of chromosomes in numerous ways and, much like chemical bookends, prevent them from unwinding. But in a fine illustration of nature's ingenuity, they also play a role in regulating the life span of a cell, because the tips of telomeres drop off chromosomes with each cycle of cell division.1 As the telomeres gradually shorten, the probability that they will signal the cell to stop dividing gradually increases. As a result, after many cycles of cell division have occurred, all cells in a population have died.
While the telomerase enzyme has the ability to replenish the telomeres on the ends of chromosomes, it is inactive in many adult human tissues. However, it is active when massive amounts of cell division are underway - as in self-renewing adult cells of the immune system, during the development of an embryo, and in cancer.
In the UCSF study, reported in the May 5 issue of Science, the researchers determined that a small structure within the RNA molecule of yeast telomerase controls the precision with which the enzyme carries out its key function -- spinning out the repeated sequences of telomeric DNA that bind the ends of chromosomes.
When they disrupted this small region of RNA, the enzyme began to spin out telomeres uncontrollably, until they stalled out like a car run into a ditch. This result occurred in culture and in the yeast cells themselves. Cell death soon ensued.
As the human version of telomerase appears to have a structural region that is similar to that examined in the yeast enzyme, the region could prove to be a target for killing cancer cells, says the senior author of the study, Elizabeth Blackburn, PhD, UCSF professor of microbiology and immunology and biochemistry and biophysics, who co-discovered telomerase in 1985. (The function of the human version of the region has not been determined.) Moreover, she says, it could prove a target for regenerating cells that have been damaged through injury or wear and tear.
Telomerase and other so-called reverse transcriptases, including HIV, both contain RNA enfolded in protein. As such, they are known as ribonucleoproteins. Most enzymes, by contrast, contain only protein.
Much of the excitement regarding the current finding stems from the fact that, to date, research on telomerase and HIV has focused only on the protein components, as they make up a central part of the enzymes' active sites.
"This discovery represents the first time anybody has shown a mechanistic role for a structure of RNA in the action of telomerase, and we think this is probably a universal kind of feature of telomerase," says Blackburn.
The finding also adds weight to recent evidence that the RNA component of HIV, traditionally disregarded in retroviral therapies as passive, plays an integral role in HIV replication, and it therefore should be closely re-examined as a possible target for therapy, says Blackburn.
"This discovery should turn researchers' spotlights back to the RNA components of both telomerase and HIV," she says.
The discovery may also offer a glimpse into the evolutionary past. Some researchers have believed that during an earlier period in evolutionary history the RNA molecule in telomerase might have deferred functional power to the protein component.
The discovery that the seemingly archaic RNA retains a key mechanistic role in telomerase function builds support for the theory that telomerase -- and reverse transcriptases such as HIV -- represent an intermediate step in the evolution of enzymes from strictly RNA sequences to strictly protein sequences, says Blackburn.
"Such a direct function for the RNA structure in the enzymatic action of telomerase supports an evolutionary scheme in which RNA enzymes acquired protein components evolving into ribonucleoprotein enzymes," she says. "The RNA components then gradually lost their functional roles in catalysis and were subsequently dispensable."
Telomerase and the other reverse transcriptase enzymes function by synthesizing copies of DNA from the RNA folded within their protein. Nearly all organisms have an enzyme that can stimulate the conversion of DNA, which contains an organism's genetic code, or genes, into messenger RNA, the first step on the road to developing protein. Only the reverse transcriptases do the opposite.
But telomerase and HIV diverge in a key aspect of their transcription mechanism. While HIV draws thousands of bases of RNA through its catalytic site, spinning out long sequences of viral DNA, telomerase draws in only a very small portion of its long RNA sequence to the catalytic site, and copies this one segment, known as the template, over and over into telomeric DNA, which it then assembles and adds to the ends of chromosomes.
Until now, researchers have not understood what specifies the enzyme's template boundaries, preventing unbridled spinning of telomeric DNA onto the ends of chromosomes. In the current study, the researchers determined that the limitation on DNA synthesis is controlled by a small segment of RNA nestled up adjacent to the downstream end of the RNA template. The region, made up of base pairs of RNA that are "zipped up" together within a larger segment of RNA, acts as a boundary for the replication process.
When the researchers altered this RNA region, the buffer "unzipped," providing a long strip of RNA - up one side of the zipper -- for the enzyme to continue converting into DNA. With free reign, the enzyme drew more and more of the ribonucleotides into its active site, until it synthesized so much telomeric DNA that eventually, says Blackburn, the telomerase RNA may have bunched up, halting telomere synthesis and causing cell death.
Such abnormal, almost ceaseless, replication, says Blackburn, is reminiscent of the behavior of normal reverse transcriptases such as those found in retroviruses like HIV.
"Our finding in telomerase gives strength to the importance of looking at the RNA component of HIV," says Blackburn, "because by making just a simple change in telomerase RNA we can make it act more like HIV, and this suggests that HIV is actually like telomerase -- when it acts in cells to make new viruses, it really is acting together with its RNAs. I think this is something one should be thinking about for drug targets."
The finding adds weight to a recent UCSF study led by Tristram Parslow, MD, PhD, professor of pathology, and Thomas James, PhD, professor and chair of pharmaceutical chemistry, who reported (Nature Structural Biology, vol. 5, p. 432, 1998) that a different "zipped up" region of RNA base pairs is essential for replication of HIV. When the base pairs were mutated, HIV lost its ability to infect a cell.
Co-authors of the study were Yehuda Tzfati, PhD, postdoctoral researcher, Tracy B. Fulton, graduate student and Jagoree Roy, PhD, postdoctoral researcher, all of the UCSF Department of Microbiology and Immunology.
The study was funded by the National Institutes of Health.
Background: 1. This built-in limit on cell division is known as the "Hayflick limit" and was discovered in 1961 by Leonard Hayflick, PhD, an adjunct professor of anatomy at UCSF before any one knew about the molecular nature of telomeres or telomerase was discovered.
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