Molecules that accumulate at the tip of chromosomes are known to play a key role in preventing DNA damage. Research led by a team at the École Polytechnique Fédérale de Lausanne (EPFL), in Switzerland, has now generated new insights into how these molecules are at home in specific sections of chromosomes. Their findings, reported in Nature, “RAD51
Stretches of DNA called telomeres form protective caps at the ends of chromosomes that act like a shoelace’s bead, preventing the lace end from flaking. “Telomeres – repetitive, non-coding nucleotide motifs and associated proteins found at the ends of eukaryotic chromosomes – mediate genome stability and determine cellular lifespan,” explained first author Marianna Feretzaki, PhD, at EPFL and colleagues. However, as cells divide, telomeres gradually become shorter, making the protective cap less effective. When telomeres eventually become too short, the cell stops dividing. Telomere shortening and dysfunction have been linked to cell aging and age-related diseases, including cancer.
Scientists have known that RNA species called TERRA (telomere-repeatable RNA) help regulate the length and function of telomeres. Discovered in 2007 by postdoctoral fellow Claus Azzalin, PhD, in the team of EPFL professor Joachim Lingner, PhD, TERRA belongs to a class of molecules called long non-coding RNAs. These are RNA molecules that do not translate into proteins, but instead function as structural components in chromosomes. TERRA accumulates at the chromosome ends and signals that telomeres need to be elongated or repaired. “Telomeric repeat RNA (TERRA) is a class of long non-coding RNAs (lncRNAs) that are transcribed from the chromosome ends,” the authors further explained. “… these RNAs in turn regulate telomeric chromatin structure and telomerase maintenance through the telomer-extending enzyme telomerase and homologous DNA repair.”
However, it has not been clear how TERRA is directed at the tips of chromosomes and remains there. “The mechanisms by which TERRA is recruited for chromosome ends remain poorly defined,” the investigators said. As Lingner pointed out, “The telomere makes up only a tiny bit of total chromosomal DNA, so the question is ‘how does this RNA find its home?'” To address this question postdoc Marianna Feretzaki and others in Lingner’s team at EPFL and Lumir Krejci, ph .d., at Masaryk University in the Czech Republic, set out to analyze the mechanism by which TERRA accumulates in telomeres, as well as the proteins involved in this process.
By visualizing TERRA molecules under a microscope, the researchers found that a short stretch of RNA is essential to bring it to telomeres. Their experiments showed that when TERRA reaches the tip of chromosomes, more proteins regulate its attachment to telomeres. Among these proteins, RAD51 plays a particularly important role, Lingner said.
RAD51 is a well-known enzyme involved in the repair of damaged DNA molecules. The protein also appears to help TERRA adhere to telomere DNA to form an ‘RNA-DNA hybrid’ molecule. Researchers believed that this type of reaction, which leads to the formation of a three-stranded nucleic acid structure, mainly occurred during DNA repair. The recently reported results indicate that it can also occur at chromosome ends when TERRA binds to telomeres. “This is paradigm shifting,” Lingner commented.
The researchers also found that short telomeres recruit TERRA much more efficiently than long telomeres. “Cells carrying short telomeres recruited TERRA much more efficiently than cells with long telomeres, seen by transient or stable expression of TERRA,” they commented. And while the mechanism behind this phenomenon is unclear, researchers assume that when telomeres become too short, either due to damage or because the cell has split too many times, they recruit TERRA molecules. DNA “… short telomeres need to be more accessible for the recruitment or retention of TERRA; alternatively, long telomeres may contain active systems exhibiting TERRA, ”they wrote. This recruitment is mediated by RAD51, which also promotes elongation and repair of telomeres. “TERRA and RAD51 help prevent accidental loss or shortening of telomeres,” Lingner added. “It’s an important feature.”
The authors say that the observed base pairing between TERRA and telomeric DNA provides a mechanism for directing TERRA to its main site of action at chromosome apexes. “Our data reveal the mechanism by which TERRA is recruited for chromosome extensions through RNA strand invasion,” they noted. “… the recruitment of TERRA appears to be through a process very similar to the strand invasion and homology search mechanism utilized in all living organisms during DNA repair by HDR, and which is also characteristic of telomere stabilization by the ‘alternative elongation of telomeres’ (ALT) mechanism in cancer cells. ”
Given the role of telomeres in health and disease, it will be important to see how the newly discovered mechanism, derived from observations in living cells and reproduced in test tubes, is regulated in the highly complex cellular environment, Lingner noted. “We put forward a model that is supported by the data we have – but often in science it turns out that the model needs to be changed. There can certainly be further surprises. ”
Next, the team plans to address other key issues, including whether RAD51 mediates association of other non-coding RNAs with chromosomes. The researchers also aim to better characterize the machinery that mediates TERRA’s association with chromosomes and to develop the functions that this association enables. “There are many questions that remain open,” Lingner acknowledged.