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Study resolves decade-long argument over protein’s role at telomeres.

Mice and humans share a lot more than immediately meets the eye, and their commonalities include their telomeres, protective ends on chromosomes. But in recent years, the role of one particular protein at telomeres has puzzled scientists.

New work at Rockefeller University has solved the contradiction regarding the protein, Rap1, a component of the shelterin complex that binds to telomeres to help guard and maintain them, so they can perform their protective function. Previous studies of its role in mice versus humans had turned up contradictory results. In human cells, Rap1 appeared crucial, whereas mouse Rap1 seemed to only play a minor role. Given the close evolutionary relationship between mice and humans, both being mammals, this discrepancy was hard to explain.

The current study has proven that Rap1 contributes next to nothing to the protection of human telomeres. The opensource research, published in Cell Reports, contradicts a number of studies from the past decade.

It seemed unlikely that Rap1’s role would have changed so dramatically between mice and humans, and now the medical community knows that it didn’t. Human Rap1 is in fact quite mouse-like.  But this raises another question, since Rap1’s evolutionary lineage extends back to budding yeast. If Rap1 doesn’t protect telomeres, why has evolution maintained it in mice and humans?.

Telomeres are DNA-protein complexes formed when shelterin binds to specific repetitive DNA sequences that protect the integrity of chromosomes. Dysfunctional telomeres have been linked to cancer and aging, therefore, it is imperative that telomeres are properly maintained, which means maintaining their length. Shelterin helps regulate telomere length and also protects them from misguided cellular processes, including erroneous DNA repair efforts that can have disastrous repercussions.

Rap1 is one of six shelterin components in humans and mice, and one that evolution has maintained for a very long time, all the way back to yeast and single-celled parasites called trypanosomes. Beginning about a decade ago, research suggested that human Rap1 was important for telomere function. Rap1 was shown to be involved in controlling the length of telomeres, and also preventing a repair process that fuses telomeres together, which would ultimately result in cell death.

Meanwhile, studies in which Rap1 was inactivated in mice produced not only apparently normal cells but also apparently normal mice, so clearly erroneous DNA repair was not fusing chromosomes together. Furthermore, no changes in mouse telomere length were detected. However, because mice have much longer telomeres than humans, subtle changes are difficult to detect.

Studies from both sides of this human-mouse discrepancy included those from the lab itself. After working on experiments showing that Rap1 was not needed to prevent telomere fusion in mice the team set out to investigate the conflicting reports by inactivating Rap1 in humans. To do so the researchers the gene encoding Rap1 by using a site-specific nuclease called TALEN, a special enzyme designed to cut DNA at a specific location.

The team state that they gained a lot of negative data looking for effects of removing Rap1 from human telomeres. They observed no fusions, no significant changes in telomere length, no turning on of DNA damage signalling, the list goes on.

In considering what might account for the discrepancy between the current results and the previous studies, the team summise that the techniques used may have produced results that were difficult to interpret for some studies, while others employed artificial systems that may not accurately represent the normal situation in a cell.

While researchers cannot dismiss the possibility that Rap1 has a redundant function that overlaps with other shelterin components, it is most likely that Rap1 must fill some other, unrelated role. Otherwise, why would it be so strongly conserved?.

Evidence for that other function was already there, Rap1 was first identified not as a component of shelterin but as a protein that turns genes on or off. In fact, studies in mammalian cells have shown that it has a strong influence on genes related to body weight and metabolism. Using three different types of cells, the team then compared gene expression profiles between cells with and without Rap1 in those cell lines. They found large changes in the expression levels for a number of genes, whose identities varied depending on the cell line.

The team can now state with certainty that Rap1 may only have a minor role in protecting telomeres in mammals, but it appears evolution has maintained it for an unrelated, but important reason; as a factor that regulates the expression of many genes.

Source:  The Rockefeller University

 

The conserved protein Rap1 functions at telomeres in fungi, protozoa, and vertebrates. Like yeast Rap1, human Rap1 has been implicated in telomere length regulation and repression of nonhomologous end-joining (NHEJ) at telomeres. However, mouse telomeres lacking Rap1 do not succumb to NHEJ. To determine the functions of human Rap1, we generated several transcription activator-like effector nuclease (TALEN)-mediated human cell lines lacking Rap1. Loss of Rap1 did not affect the other components of shelterin, the modification of telomeric histones, the subnuclear position of telomeres, or the 3′ telomeric overhang. Telomeres lacking Rap1 did not show a DNA damage response, NHEJ, or consistent changes in their length, indicating that Rap1 does not have an important function in protection or length regulation of human telomeres. As human Rap1, like its mouse and unicellular orthologs, affects gene expression, we propose that the conservation of Rap1 reflects its role in transcriptional regulation rather than a function at telomeres.  TALEN Gene Knockouts Reveal No Requirement for the Conserved Human Shelterin Protein Rap1 in Telomere Protection and Length Regulation.  Lange et al 2014.
The conserved protein Rap1 functions at telomeres in fungi, protozoa, and vertebrates. Like yeast Rap1, human Rap1 has been implicated in telomere length regulation and repression of nonhomologous end-joining (NHEJ) at telomeres. However, mouse telomeres lacking Rap1 do not succumb to NHEJ. To determine the functions of human Rap1, we generated several transcription activator-like effector nuclease (TALEN)-mediated human cell lines lacking Rap1. Loss of Rap1 did not affect the other components of shelterin, the modification of telomeric histones, the subnuclear position of telomeres, or the 3′ telomeric overhang. Telomeres lacking Rap1 did not show a DNA damage response, NHEJ, or consistent changes in their length, indicating that Rap1 does not have an important function in protection or length regulation of human telomeres. As human Rap1, like its mouse and unicellular orthologs, affects gene expression, we propose that the conservation of Rap1 reflects its role in transcriptional regulation rather than a function at telomeres. TALEN Gene Knockouts Reveal No Requirement for the Conserved Human Shelterin Protein Rap1 in Telomere Protection and Length Regulation. Lange et al 2014.

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