Pumby – The Artificial Name-Taking, RNA-Controlling Protein
Scientists at MIT report that they have developed a building-block approach that simplifies the assembly of RNA-binding proteins. With this approach—which the scientists call Pumby, for Pumilio-based assembly—it is possible to not only attach fluorescent labels to selected RNA sequences, but also to enhance RNA translation. The scientists have already demonstrated that Pumby can be deployed in living cells to image and monitor RNA species of interest (“taking names,” to borrow a phrase) and even control RNA activity.
Pumby adapts naturally occurring proteins called Pumilio homology domains (PumHDs), which consist of amino acids that bind to each of the four different ribonucleotide bases that make up RNA sequences. Native PumHDs normally help guide embryonic development. In bioengineering, however, native and modified PumHDs have been used to target selected RNA sequences.
To date, the PumHD proteins that have been synthesized for experimental use have tended to be one-off products. That is, even when PumHD proteins were assembled by means of a programmable, modular approach, some tweaking and customization was still necessary.
Hoping to make the assembly of RNA-binding proteins less of a trial-and-error and more programmable task, the MIT researchers tested many amino acid
combinations. Ultimately, the researchers found a particular set of amino acids that will bind each of the four bases at any position in the target sequence. Using this system, the researchers effectively targeted RNA sequences varying in length from 6 to18 bases.The details appeared online the week of April 25 in PNAS, in an article entitled, “A Programmable RNA Binding Protein Composed of Repeats of a Single Modular Unit.” The article described how Pumby modules can be “concatenated in chains of varying composition and length to bind desired target RNAs.”
“We validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs,” the article’s authors continued. “We further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells.”
“Modularity is one of the core design principles of engineering,” noted Edward Boyden, an associate professor of biological engineering and brain and cognitive sciences at the MIT Media Lab. “If you can make things out of repeatable parts, you don’t have to agonize over the design. You simply build things out of predictable, linkable units.”
In experiments in human cells grown in a lab dish, the researchers showed that they could accurately label mRNA molecules and determine how frequently they are being translated. First, they designed two Pumby proteins that would bind to adjacent RNA sequences. Each protein is also attached to half of a green fluorescent protein (GFP) molecule. When both proteins find their target sequence, the GFP molecules join and become fluorescent—a signal to the researchers that the target RNA is present.
Furthermore, the team discovered that each time an mRNA molecule is translated, the GFP gets knocked off, and when translation is finished, another GFP binds to it, enhancing the overall fluorescent signal. This allows the researchers to calculate how often the mRNA is being read.
This system can also be used to stimulate translation of a target mRNA. To achieve that, the researchers attached a protein called a translation initiator to the Pumby protein. This allowed them to increase dramatically translation of an mRNA molecule that normally wouldn’t be read frequently.
The researchers are now working toward using this system to label different mRNA molecules inside neurons, allowing them to test the idea that mRNAs for different genes are stored in different parts of the neuron, thus helping the cell to remain poised to perform functions such as storing new memories. “Until now it’s been very difficult to watch what’s happening with those mRNAs, or to control them,” Boyden said.
These RNA-binding proteins could also be used to build molecular assembly lines that would bring together enzymes needed to perform a series of reactions that produce a drug or another molecule of interest.
