A new study by EPFL researchers shows how RNA species called TERRA muster at the tip of chromosomes. [EPFL / Demcon-Nymus3D]

Molecules that accumulate at the tips of chromosomes are known to play a key role in preventing damage to DNA. Research headed by a team at the École Polytechnique Fédérale de Lausanne (EPFL), in Switzerland, has now generated new insights into how these molecules home in on specific sections of chromosomes. Their findings, reported in Nature, “RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops,” could help scientists better understand the processes that regulate cell survival in aging and cancer.

Stretches of DNA called telomeres form protective caps at the ends of chromosomes, which act akin to the aglet of a shoelace that prevents the lace end from fraying. “Telomeres—repeated, noncoding nucleotide motifs and associated proteins that are found at the ends of eukaryotic chromosomes—mediate genome stability and determine cellular lifespan,” explained first author Marianna Feretzaki, PhD, at the EPFL, and colleagues. But as cells divide, the telomeres become gradually shorter, making the protective cap less effective. When the telomeres eventually become too short, the cell stops dividing. Telomere shortening and malfunction have been linked to cell aging and age-related diseases, including cancer.

Scientists have known that RNA species called TERRA (telomeric-repeat-containing RNA) help to regulate the length and function of telomeres. Discovered in 2007 by postdoc Claus Azzalin, PhD, in the team of EPFL professor Joachim Lingner, PhD, TERRA belongs to a class of molecules called long noncoding RNAs. These are RNA molecules that are not translated into proteins, but instead function as structural components of chromosomes. TERRA accumulates at chromosome ends, signaling that telomeres should be elongated or repaired. “Telomeric-repeat-containing RNA (TERRA) is a class of long noncoding RNAs (lncRNAs) that are transcribed from chromosome ends,” the authors further explained. “… these RNAs in turn regulate telomeric chromatin structure and telomere maintenance through the telomere-extending enzyme telomerase and homology-directed DNA repair.”

However, it has not been clear how TERRA is directed to the tips of chromosomes and remains there. “The mechanisms by which TERRA is recruited to chromosome ends remain poorly defined,” the investigators stated. As Lingner pointed out, “The telomere makes up only a tiny bit of the total chromosomal DNA, so the question is ‘how does this RNA find its home?'” To address this question, postdoc Marianna Feretzaki and others in the teams of Lingner at EPFL and Lumir Krejci, PhD, at Masaryk University in the Czech Republic, set out to analyze the mechanism through which TERRA accumulates at 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 the RNA is crucial to bring it to telomeres. Their experiments showed that when TERRA reaches the tip of chromosomes, several proteins regulate its association with telomeres. Among these proteins, RAD51 plays a particularly important role, Lingner said.

RAD51 is a well-known enzyme that is involved in the repair of broken DNA molecules. The protein also seems to help TERRA stick to telomeric DNA, to form an ‘RNA-DNA hybrid’ molecule. Scientists thought this type of reaction, which leads to the formation of a three-stranded nucleic acid structure, mainly happened during DNA repair. The newly reported results indicate that it can also happen 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, as seen upon transient or stable expression of TERRA,” they commented. And while the mechanism behind this phenomenon is unclear, the researchers hypothesize that when telomeres get too short, either due to damage or because the cell has divided too many times, they recruit TERRA molecules. DNA “… short telomeres must be more accessible to recruitment or retention of TERRA; alternatively, long telomeres might contain active systems that expel TERRA,” they wrote. This recruitment is mediated by RAD51, which also promotes the elongation and repair of telomeres. “TERRA and RAD51 help to prevent accidental loss or shortening of telomeres,” Lingner added. “That’s an important function.”

The authors say the observed base-pairing between TERRA and telomeric DNA provides a mechanism for directing TERRA to its major site of action at chromosome tips. “Our data reveal the mechanism by which TERRA is recruited to chromosome ends through RNA strand invasion,” they noted. “ … the recruitment of TERRA seems to occur through a process that strongly resembles the strand-invasion and homology-search mechanism exploited in all living organisms during DNA repair by HDR, and which is also characteristic of telomere stabilization by the ‘alternative lengthening 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, which was deduced from observations in living cells and reproduced in test tubes, is regulated in the very complex cellular environment, Lingner noted. “We put forward a model, which is supported by the data we have—but often in science, it turns out that the model must be modified. There can certainly be additional surprises.”

Next, the team plans to address other key questions, including whether RAD51 mediates the association of other noncoding RNAs with chromosomes. The researchers also aim to better characterize the machinery that mediates the association of TERRA with chromosomes, and work out the functions that this association enables. “There are a lot of questions that remain open,” Lingner acknowledged.

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