Polyadenylation: Now there’s a word to conjure with. If you can pronounce it. (polly–ah’-denill-ayshen) Who’s to know (besides biologists) that it’s one of the most important things that keeps you – and everything else living – alive? It’s a process in living cells, one that heretofore was extremely difficult to study because almost any kind of mucking with it or the proteins involved, kills the cells under study. One protein in particular with the typically memorable name of CstF-64 (cleavage stimulation factor-64, yes, biologists make jokes about it) is the focus of important new research.
The key point about polyadenylation is that it is crucial to making mRNA, messenger ribonucleic acid, the specialist RNA carrying DNA encoded instructions that travels throughout the cell to where proteins assemble cell materials. The protein CstF-64 plays an important role in the polyadenylation process, but it’s been something of an enigma. It exists, mostly, in the cytoplasm (the area outside the cell nucleus), but to do its work it needs to be inside the nucleus. The question was how does it get there?
To answer that question, it was first necessary to overcome the problem of killing the studied cells. This where research at Texas Technical University (USA) comes in. The work represents the inventive side of molecular biologists, when confronted with difficulties in getting answers to important questions.
“Previously, it had been very hard to examine the functions of most of the polyadenylation proteins in cells because polyadenylation is essential for ‘life as we know it.’ If we perturbed polyadenylation in any way, the cells died, and we could not measure anything,” says Hockert, who is now an assistant professor at the University of the Cumberlands in Williamsburg, Ky.
To make his observations, Hockert had to employ what co-investigator Clinton MacDonald calls “a trick.”
“Andrew realized we can make a version of the protein that is different than the regular version already in the cell. We can mutate it,” says MacDonald, an associate professor at Texas Tech who oversaw Hockert’s work. “And, if you put that mutated version of the protein in the cell, it only works on the genes we tell it to work on and not the rest. So, it doesn’t kill the cell.”
Having come up with a clever way to study and measure different aspects of the protein in a living cell, MacDonald says, the team then had to pick one in particular on which to focus.
“The feature Andrew chose to examine was how CstF-64 interacted with another polyadenylation protein and how that interaction allowed both those proteins to work inside the nucleus,” MacDonald says.
As important as CstF-64 is to gene expression, it doesn’t exactly have “VIP” status when it comes to gaining access to the nucleus. Lacking what is known as a nuclear localization signal, it has to rely on its partner protein, CstF-77, to lead the way to and get in the door.
“We already knew the sequence of our protein, CstF-64, and so we knew it didn’t have a special signal to get it in the nucleus. So, we hypothesized something else was dragging it in, and the most likely thing was a partner protein working alongside it,” Hockert explains.
With the mutant version of the protein in place, the team soon discovered their hypothesis was correct: CstF-64 had to bind with CstF-77 to get into the cell’s command center. Furthermore, MacDonald says, the team was able to report which piece of CstF-64 binds with its partner — “the hinge domain.”
The development of a mutant protein to ‘fool’ and control the cell’s processes is one of those techniques that become ‘obvious’ afterwards, and are taken up by numerous other studies. “Tricks” such as this one are vital to getting past the inherent sensitivity of living cells in order to study the most fundamental processes of life.