Genetic Redundancy Allows Cells to Optimize mRNA Sequences Against Aggregation

Messenger RNA, or mRNA, is best known for carrying genetic instructions from DNA to the cellular machinery that makes proteins. But all RNA molecules have another, less appreciated property: they are naturally sticky.

When RNA is removed from cells and studied in the laboratory, the molecules readily interact with one another and can clump together, or aggregate. This creates a biological puzzle. Cells contain many thousands of mRNA molecules crowded into a tiny space, yet their mRNAs do not routinely form the large aggregates that their physical properties would seem to favor. This puzzling behavior is important for cell function: unwanted RNA interactions can prevent mRNAs from being accessible to make proteins, and large RNA aggregates can be toxic to cells.

Now, researchers at Whitehead Institute have uncovered one way cells may have evolved to avoid these sticky situations. Their findings suggest that evolution has shaped mRNA sequences to reduce unwanted interactions with other mRNAs. The work could potentially inform the development of RNA therapeutics, allowing drug designers to learn from evolution when selecting RNA sequences.

The study, led by Whitehead Institute Member Ankur Jain, also an associate professor of biology at MIT, and Marco Todisco, a postdoctoral researcher in his lab, reveals a previously unrecognized constraint on the evolution of genetic sequences: DNA must not only encode functional proteins, but also produce mRNAs with physical properties that help keep them soluble inside the cell.

The researchers' findings were published in the Proceedings of the National Academy of Sciences on September 14.

Scientists studying purified RNA have known for decades that the molecules can readily aggregate. But if the chemistry of RNA makes these interactions so favorable outside cells, Todisco wondered, why shouldn't the same thing happen inside them?

To investigate, Todisco and colleagues focused on Escherichia coli, or E. coli, a bacterium whose biology has been extensively studied. Researchers have detailed information about which mRNAs are present in an E. coli cell, how many copies of each are present, and what those molecules look like - making it possible to model the behavior of an entire collection of cellular mRNAs, known as the transcriptome.

Todisco developed computer simulations that tracked individual mRNA molecules and predicted how they would behave at concentrations similar to those found inside a cell. Based on the physical chemistry of RNA alone, the simulations indicated that the molecules should aggregate. Longer mRNAs were particularly prone to joining these clusters.

The team then tested that prediction experimentally. With help from Christalyn Ausler, a research technician in the Jain lab, they purified mRNA from E. coli cells. The authors found that without proteins and other components normally surrounding it in the cell, the purified mRNA readily aggregated. Using RNA sequencing, the researchers identified which mRNAs were enriched in the aggregates and found that their properties closely matched the simulations.

Those findings strengthened the original puzzle: If mRNA has such a strong physical tendency to associate with other RNA molecules, how have cells managed that problem?

The researchers found part of the answer written into the genetic sequences themselves.

DNA carries the instructions for building proteins using three-letter sequences called codons, each of which specifies an amino acid, one of the building blocks of proteins. But the genetic code contains redundancy: most amino acids can be encoded by more than one codon. That means an enormous number of different DNA and mRNA sequences can produce exactly the same protein. Evolution therefore has some freedom in which sequence it uses.

The researchers took advantage of that flexibility to computationally create alternative versions of E. coli mRNAs. They changed the RNA sequences while preserving the proteins they encoded - in effect creating alternative evolutionary paths in which an organism could make the same proteins from different mRNAs.

When the researchers compared these randomized sequences with naturally occurring E. coli mRNAs, they found that the natural sequences were less prone to interacting with one another. Native mRNAs tended to fold onto themselves and to avoid exposing sticky stretches, reducing opportunities to form stable and unwanted interactions with other mRNA molecules.

In other words, stickiness is unavoidable, but evolution appears to have favored not just DNA sequences that make the right proteins, but sequences that produce less sticky mRNAs along the way.

The researchers found evidence that the phenomenon extends beyond bacteria. When they performed sequence analyses on a set of abundant human mRNAs, they found similar signatures: naturally occurring human RNA sequences also showed a reduced potential for aggregation compared with alternative sequences. 

The discovery adds another dimension to the familiar "central dogma" of molecular biology, in which information flows from DNA to RNA to protein. Scientists have traditionally thought about genetic sequences largely in terms of their ultimate product: the protein. The new findings suggest that evolution also has to contend with the physical behavior of the mRNA intermediate.

That may not be the whole solution. Although naturally occurring mRNA sequences are less prone to aggregation, purified mRNA still readily clumps together. The fact that widespread aggregation is not normally seen inside healthy cells suggests that proteins or other cellular components may provide an additional layer of protection by buffering inappropriate RNA-RNA interactions. These findings help build a broader picture of how cells may use multiple mechanisms to manage RNA interactions and prevent inappropriate aggregation. 

The findings could also have implications for the growing field of mRNA therapeutics. When researchers design synthetic mRNAs for vaccines and other treatments, they can choose among different codons that ultimately produce the same protein. The new work suggests that how the resulting RNA folds and interacts with other RNAs may be another important consideration when selecting those sequences.

What this gives us is a new lens for thinking about how genomes evolve. Sequences aren't evolving just to encode functional proteins; there is an additional constraint to produce mRNAs that are less likely to stick to one another. These findings could also provide guiding principles for designing the sequences used in mRNA vaccines and other RNA therapeutics."

Ankur Jain, Whitehead Institute