October 02, 2014
Have you ever noticed that the length of your hair is just right for a small window of time? Too short, and you feel a little exposed – too long, and it gets in your face.
Like your hair, the telomeres at the ends of your chromosomes have a length that is just right for them (and you). Telomeres are non-coding sequences of DNA added to the ends of chromosomes that protect the important DNA there from being lost. Telomeres that are too long may contribute to cancer; while telomeres that are too short are associated with aging.
It makes sense, then, that telomere length should be carefully regulated (just as, in a perfect world, you might want to keep your hair the perfect length all the time). The enzyme that adds the non-coding DNA sequences to the end of chromosomes, telomerase, is composed of highly conserved subunits. In yeast, this enzyme is a quaternary complex composed of the regulatory subunits Est1 and Est3, the catalytic subunit Est2, and the RNA template component, TLC1.
Previously, it was thought that telomerase was primed for action whenever it was needed in the cell. But this does not seem to be the case.
In a recent publication in Genes & Development, Tucey et al. showed that, in addition to the active quaternary telomerase complex, two subcomplexes – a preassembly complex and a disassembled complex – were also present in the cell. Each of these subcomplexes lacked one subunit compared with the active telomerase, and the missing subunit was different for each subcomplex.
So, how did the authors discover these subcomplexes? Using a special immunoprecipitation protocol, one of the first things the researchers noticed was that Est1, Est2, and the TLC1 RNA were associated with each other throughout G1 and S phase, but that Est3 was missing from the complex during G1 phase and present only at very low levels throughout S phase. They called this Est1-TLC1-Est2 complex the “preassembly complex.”
In fact, Est3 was only appreciably associated with the preassembly complex during G2/M and late in the cell cycle. And, even then, only 25% of the preassembly complex was associated with Est3 to form the active quaternary complex. So the presence of the active holoenzyme was regulated with respect to the cell cycle.
Since Est3 was not always bound to the preassembly complex, the authors set out to determine what was required for Est3 binding. They found that neither Est1 nor Est2 could bind Est3 if it was unable to bind TLC1 RNA. Thus, the RNA component of the presassembly complex was necessary for its two other subunits to form an active telomerase by binding to Est3.
In addition, they determined that both Est1 and Est2 contained binding surfaces for Est3, and that both of these surfaces were required for the full association of Est3 with the preassembly complex. So Est3 interacts directly with each of the other protein subunits to form the active telomerase.
There was still one more question that intrigued Tucey and coworkers – Est3 appeared to exist in excess compared with the other subunits, so why did it have such limited association with the rest of the telomerase subunits? This suggested some sort of inhibitory regulation of Est3.
Indeed, the authors uncovered an Est3 mutant, Est3-S113Y, which appeared to have lost the ability to be inhibited. This mutant exhibited elongated telomeres and increased association with telomerase, and was associated with the preassembly complex in G1 phase rather than being restricted to late in the cell cycle. This mutation lies directly adjacent to Est3’s binding domain, leading the authors to conclude that Est3 is regulated by a “toggle switch” that specifies whether or not it can bind to the preassembly complex.
As mentioned previously, the authors also saw evidence of a second subcomplex during their studies. When they drilled down on the components, they identified a “disassembly complex” that lacks only Est2, in contrast to the preassembly complex that lacks Est3. They determined that this subcomplex is inactive and requires the prior formation of the quaternary complex since its formation requires Est2 binding to TLC1, just as is observed for the preassembly complex.
Given the cell’s desire to have telomeres that aren’t too long and aren’t too short, it makes sense that the enzyme that lengthens them is regulated. There is a toggle switch that signals formation of the active complex and, once formed, the active complex is transient, dissociating its catalytic subunit to become inactive. This regulation ensures that telomerase is active only late in the cell cycle. Just as you might work to keep your hair just the right length, the cell regulates telomerase to keep the lengths of telomeres from getting out of control.
by Selina Dwight, Ph.D., Senior Biocurator, SGD
Categories: Research Spotlight
Tags: aging, protein complex, telomerase, telomere