Who needs the philosopher’s stone to live forever when we already have telomerases?

Chromosomes contain all the genetic information a cell needs. Replicate the chromosomes and you can replicate the cell. So when an old cell dies, the new one can still do its job. If you could indefinitely replicate your cells, then you could (theoretically) live forever; every cell in your body could be young and fresh and working properly, and any time a cell got old, you could just copy it to make a new one. There’s just one problem: your bodies can't replicate the entire chromosome. Chromosomal replication has a functional limitation that means it can’t copy the tail ends of chromosomes. The ends of chromosomes possess repetitive, non-coding DNA in a structure called a telomere. It isn’t a big deal that a bit of telomeric DNA is lost every time the chromosomes replicate. But eventually, the telomeres will run out, and the cell has to choose between losing important genetic information, or not replicating itself at all. At this point, referred to as the "Hayflick limit," most cells simply stop replicating. However, some cells can achieve cellular immortalization by regenerating their telomeres, which effectively allows them to continue replicating and dividing forever.

You may remember that most of the DNA in your cells is copied by an enzyme called DNA polymerase. Telomeres are elongated by telomerase, a special type of DNA polymerase. Telomerase is made up of TR, an RNA molecule that serves as a template for telomerase elongation, and hTERT, a protein with polymerase activity. It reverse transcribes the RNA template into the DNA that extends the telomeres. In most cases, DNA is synthesized by DNA polymerase from an existing DNA template strand, and RNA is transcribed from a DNA template. In this case, the reverse is occurring. The RNA TR is serving as a template to extend one DNA strand of the telomeres. It is only once this occurs that the DNA extension serves as the template to synthesize the second strand of the telomere, creating the complete, double-stranded DNA telomere. Telomerase expression is connected to several human diseases. Telomerase activity is tuned to just the perfect level in healthy cells. Defective telomerase results in a genetic condition called dyskeratosis congenita, which affects rapidly reproducing cells such as skin or gut cells, since they have shorter telomeres than they should. A lack of telomerase activity is connected with sun-damage to skin, leading to chromosomal instability and frequently cancer. On the other hand, excessive telomerase activity is linked to many different types of cancer, since it allows for unlimited replication of tumor cells.

The telomerase RNA (TR) component is extremely variable in different species, but it has several conserved secondary structural elements. The 3’ element is responsible for stabilizing the TR and consists of two hairpins, an H box, and an ACA region. The H/ACA motif forms a complex with dyskerin and other proteins, without which the TR would degrade soon after synthesis. Two more conserved regions (CR4/CR5) form a stem-loop that binds to TERT, the protein required to complete the telomerase complex. TERT also binds to a conserved pseudoknot (where a single strand of RNA participates in two different secondary structural elements) and the template for reverse transcription, which together make up the core domain of TR.

Secondary structure elements of hTERC. Black lines represent the backbone of RNA. Colored ovals represent proteins.

Once telomerase assembles, it is recruited to telomeres by other cellular complexes. This is necessary because there are relatively few telomerase complexes and telomeres in the cell, so they are unlikely to encounter each other without a specific recruitment mechanism. Once telomerase binds to telomeres, the protein element uses the TR as a template to catalyze the extension of telomeres by synthesizing the same sequence for addition repeatedly. Once the telomerase extends one DNA strand with repetitive DNA, DNA polymerase can use that DNA as a template to synthesize the complementary strand. This results in the complete extension of double-stranded telomeres at the end of chromosomes.

Diagram of repetitive sequence addition by telomerase. Telomeres consist of the nucleotide sequence "TTAGGG" and its complement on the other strand, repeated over and over again. Telomerase has the template for addition of TTAGGG to the end of telomeres, so this same 6-nucleotide sequence can be added over and over again to make telomeres longer.

Since telomerase activity is linked to malignant tumor cell replication, telomerase inhibition is a current area of active research as potential treatments for cancer. One study by researchers Min Chen and Li-Na Xing, researchers in the Department of Cancer Radiotherapy at the Harbin Medical University in Harbin, China, looked specifically at the effect of TR inhibition in cervical cancer cells. Many cancer cells are resistant to radiation, so Chen and Xing hoped that inhibiting TR and corresponding telomerase activity could make cancer cells more sensitive to radiation therapy. They designed an siRNA (small interfering RNA, a small RNA molecule that induces the sequence-specific degradation of other RNAs) to target TR for degradation in vivo using Hela cells. Once they introduced the siRNA to Hela cells, they measured a decrease in TR, indicating that their siRNA was effective at knocking down (degrading or inhibiting) the RNA component of telomerase.

However, since telomerase forms complexes with cellular elements including hTERT, the researchers wanted to see if decreasing the RNA TR was enough to reduce telomerase activity, or whether the catalytic component, the protein hTERT, could continue telomerase elongation just as effectively even with fewer RNA components. To measure this, the researchers used TRAP-PCR, which stands for Telomere Repeat Amplification Protocol Polymerase Chain Reaction. PCR amplifies specific DNA sequences of interest. TRAP amplifies specifically the telomerase extension products (ie the repeat sequences added to the end of telomeres). The amount of telomerase DNA product as measured by TRAP-PCR provides a way to measure telomerase activity. In cells treated with the siRNA, and therefore with reduced hTERC, the researchers measured a decrease in telomerase activity, indicating that the catalytic protein component of telomerase could not function as effectively if the RNA component was inhibited.

Measurements show decreased levels of TR (labeled here as hTERC) with siRNA designed to target TR as opposed to controls that include non-specific siRNA treatments and DNA other than TR. Though the image was distorted in the original publication, there is still a noticeable change in RNA levels: the controls and the first attempted siRNA show high TR RNA levels, but the second and third designed siRNAs show greatly reduced TR RNA levels. siRNA III appears to be most effective at targeting degradation of TR, so that was the siRNA used in future experiments.

Fewer colonies survive when treated with radiation after being treated with TR-specific siRNA  (yellow) than when just treated with radiation (blue).

The last step of the experiment was to see if this reduced telomerase activity could increase the sensitivity of cells to radiation therapy. Individual colonies were less efficient at forming when treated with siRNA and radiation, as compared to colonies just treated with radiation. And xenografts (tissue transplants of tumor cells) in mice showed reduced tumor size both after treatment with siRNA and after treatment with siRNA and radiation. Most prior research on telomerase inhibition as a method of cancer treatment focused on the protein component, but this study showed that focusing on the TR RNA is a promising area for cancer treatment research.

References:
Cong, YS, Wright, WE, and Shay, JW. (2002). Human Telomerase and Its Regulation. Microbiol Mol Biol Rev, 66(3): 407-425.
Schmidt, JC, and Cech, TR. (2015). Human telomerase: biogenesis, trafficking, recruitment, and activation. Genes Dev, 29(11): 1095-1105.
Zhang, Q, Kim, NK, and Feigon, J. (2011). Architecture of human telomerase RNA. PNAS, 108(51): 20325-20332.
Chen, M, and Xing, LN. (2012). siRNA-mediated Inhibition of hTERC Enhances Radiosensitivity of Cervical Cancer. Asian Pacific J Cancer Prev, 13(12): 5975-5979.
Mender, I, and Shay, JW. (2015). Telomerase Repeated Amplification Protocol. Bio Protoc, 5(22): e1657.


About the author:
Caley is a senior at Mount Holyoke College majoring in biochemistry and French. Outside of classes, Caley enjoys working in Professor Kathryn McMenimen's research lab studying small heat shock proteins, playing piano, running, and cooking. Following graduation, she is going to medical school at Columbia University Vagelos College of Physicians and Surgeons.