Discovered in 1986, the puzzling cellular structures known as vaults are now being used to record gene activity.N. KEDERSHA AND L. ROME, JOURNAL OF CELL BIOLOGY (1986) 10.1083

The barrel-shaped structures found by the thousands in most animal cells are one of biology’s biggest mysteries. But although researchers haven’t figured out the function of these “vaults,” they now report a new use for the puzzling particles. A team has engineered vaults to scoop up RNA and record nearly all gene activity in groups of cells for up to 7 days, providing an unprecedented look into their past.

The new method is “superpowerful” and a “very innovative idea,” says chemist Jiahui (Chris) Wu of the University of Massachusetts Amherst. He and others predict it could help investigate cellular processes that unfold with time, such as why some tumor cells become resistant to cancer drugs.

“It’s so cool,” adds University of Washington geneticist Jay Shendure. “You’re engineering vaults, something we barely understand, [to track RNA]. I love the leveraging of something endogenous and mysterious.”

Existing ways to record the gene activity of cells have limitations—tracking just one or a few genes, for example, or capturing only the genes that happen to be active right when cells are broken open. Fei Chen, a biomedical engineer at the Broad Institute, recently tasked a Ph.D. student in his lab with developing a simpler, more complete method for tracking gene activity over days. The idea was to capture messenger RNAs (mRNAs), the protein-building instructions made by working genes, and store them so they could be read out later.

Unprotected, mRNAs are normally broken down within minutes or hours. To record gene activity over a longer period, Chen’s team sought to connect Poly (A) binding protein (PABP), which cells naturally attach to one end of mRNA, to more stable molecules that might protect the strands. They first tried linking PABP to bacterial proteins, but the system didn’t work well. Then, the Broad’s Yu-Kai (Kevin) Chao, who was leading the project, suggested using vaults after reading a profile of Leonard Rome, the University of California, Los Angeles biologist who co-discovered them in 1986 and has been trying to unlock their secrets ever since. Chen was intrigued. Unlike many biologists, he had actually heard of the strange objects and knew their properties could be just what the lab needed.

Vaults are composed of 78 copies of a long molecule known as the major vault protein (MVP), which form a sturdy shell that is largely empty inside except for an RNA of unknown function and some other proteins that bind to the interior. To make mRNAs lodge inside a vault, the team fused the gene for PABP with DNA encoding the MVP-binding subunit of one of those interior proteins. When the altered PABP gene was added to cells along with extra copies of the MVP gene, captured mRNAs ended up inside newly made vaults (see graphic, below). To read out the gene activity, the team chemically pried open the vaults and sequenced the mRNAs to identify their genes of origin.

“The vaults worked really well,” says Chen, who likens the system to a time capsule and calls it a TimeVault.

A genetic time capsule

A new method for tracking gene activity captures and stores cells’ protein-building instructions, strands of messenger RNA (mRNA), inside structures called vaults. Scientists added a protein domain that binds to the major vault protein (MVP) to Poly (A) binding protein (PABP), which naturally attaches to mRNA. This fusion protein (left panel) then secures captured mRNAs to the interior of the vault (right panel).

The method proved itself by confirming the well-known genetic responses of cells to heat and low oxygen, Chen’s group reports. The team also used TimeVaults to identify genes that enable some lung cancer cells to withstand an approved drug. By adding the mRNA-recording vaults to cancer cells and then exposing the cells to the drug, Chen’s team pinpointed protective genes that were active before treatment—previous methods had only spotted genes that turned on in response. Adding a second drug targeting one newly identified gene wiped out most cells resistant to the first therapy.

Rome is pleasantly surprised by the new work. He says his group had little success stuffing mRNA or genes into vaults using other molecular fasteners.

“We tried for years,” he says ruefully.

The difference, he suspects, is he wanted to fill as many vaults as possible to deliver potential vaccines or therapies. Chen’s method, Rome says, may store mRNA only in a small percentage of vaults, but that seems to be enough to capture nearly all gene activity, especially when studying thousands of cells at a time.

Chen’s lab now plans to see whether TimeVaults can record all the genes active in a single cell. They also want to engineer the genes for the system into mice, in hopes it can capture the symphony of genes turning on and off in a living animal.

The strategy offers a new kind of “molecular selfie,” says Michael Elowitz, a California Institute of Technology bioengineer whose group has developed other methods to track gene activity.

“TimeVaults provide a major advance in a long-standing quest to understand cells as dynamic entities, continually changing and developing over time.”

Source – American Association for the Advancement of Science