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Telomerase reverse transcriptase (TERT), an enzyme associated with nearly all malignant human cancers, is even more diverse and unconventional than previously realized, new CU Boulder research finds.听

Telomeres, the protective ends of chromosomes, help to maintain genomic stability. In most normal adult human cells, the telomeres eventually shorten beyond a critical length, bringing a cell鈥檚 life to its natural end. In almost all human cancers, however, telomerase reactivates, leading to cell proliferation and tumor formation. This is a key part of what makes cancer cells 鈥渋mmortal.鈥�

As TERT is the component of telomerase that is required for cancer development, it has become an attractive target for cancer therapeutics in recent decades.听

鈥淭ERT has great importance in cancer progression and hence there is a great interest in studying its expression regulation,鈥� said Gabrijela Dumbovic, a post-doctoral researcher in the Department of Biochemistry and co-lead author of the CU Boulder study.

The study, , used powerful, high-magnification imaging techniques to reveal differences in how TERT is produced on the single-cell level, in individual cancer cells. Previously, studies relied on general averages of TERT production within whole tissue samples or large cell populations, without investigating potential cell-to-cell variation.

91制片厂 a year ago, researchers at the BioFrontiers Institute began discussing ways to apply ribonucleic acid (RNA) localization imaging protocols to better classify how TERT RNA was being made within individual cancer cells. (RNA typically serves as a messenger for turning DNA genetic information into proteins, though it is also important for coding and regulating gene expression and catalyzing certain biochemical reactions.) What began as a casual conversation grew into an interdisciplinary effort, combining experience in RNA imaging from the laboratory of Professor John Rinn of CU Boulder鈥檚听Department of Biochemistry听and the听BioFrontiers Institute听with innovative telomerase research led by Distinguished Professor Thomas Cech, a听听Investigator and Nobel laureate.

鈥淲e have a great community of scholars in the Caruthers Biotechnology Building,鈥� Cech explained. 鈥淥ur students and postdoctoral fellows are encouraged to talk freely about their work, so collaborations are forged naturally and frequently.鈥�

The researchers used a microscopy technique known as single-molecule RNA fluorescent in situ hybridization (鈥渟mFISH,鈥� for short) to visualize individual RNA molecules that coded for TERT in separate cancer cells. Looking at the microscope images, they counted the number of TERT RNA molecules 鈥� which appeared as tiny fluorescent spots 鈥� in different locations within each cell.听

鈥淧revious studies were mostly focused on studying TERT expression in a population of cells. We took a different approach and actually could visualize TERT RNA levels, and RNA distribution on a single-cell level,鈥� said Dumbovic.

The authors also found that while most cells in our bodies have two copies of a given gene (one from our mother and one from our father), the cancer cells frequently had more than two copies of the TERT gene. Such gene amplification is common in cancer cells, which have relatively unstable genomes.听

鈥淭he TERT gene has made all these extra copies of itself,鈥� Rinn said. 鈥淪elfishly, it wants to replicate itself, and cancer wants to hijack that mechanism to keep the its cells alive indefinitely. That鈥檚 something we can only see with this kind of imaging.鈥�

Intriguingly, the study also found that when looking at where TERT messenger RNA resides within a given cell, a high amount (over 80 percent in some instances) stays quarantined in the nucleus, rather than the expected cytoplasm, raising yet another mystery for future study. Typically, messenger RNA, which is made in the cell鈥檚 nucleus, is exported from the nucleus to be turned into protein in the cell鈥檚 cytoplasm.听

鈥淩NA imaging has continually shed new insights into biology by providing an important layer of information of听飞丑别谤别听a gene is in the cell. When we took the 鈥榤olecular picture鈥� of the TERT gene, we鈥檙e struck by the unexpected and sometimes substantial amount of TERT RNA in the nucleus where it can鈥檛 function to make protein,鈥� explained Rinn. 鈥淭his opens up a new layer of regulation where the molecules of TERT RNA are when considering its abundance in cancer and other disease states.鈥�

This surprising pattern of nuclear localization was also observed in healthy cells that produce TERT, specifically human induced pluripotent stem cells (iPSCs). 鈥淭his suggests that the nuclear localization is not a behavior specific to the diseased cancer cells,鈥� said Teisha Rowland, co-lead author of the study and former post-doctoral research in the lab of Thomas Cech. Rowland is now Director of the new听Stem Cell Research and Technology Resource Center听in the Department of Molecular, Cellular, and Developmental Biology.

鈥淚t was generally assumed that TERT mRNA localizes to the cytoplasm, which is needed for it to make a protein product, but now we have a different perspective. Now, we want to understand why TERT RNA is retained in the nucleus,鈥� Dumbovic said. 鈥淚s there a stimulus that causes it to move, or to stay?鈥�