One to rule them all

The rules that dictate the three-dimensional shapes of RNA molecules are primarily based not on complex chemical interactions but simply on geometry, according to a new biophysical study. The findings circumvent the fact that NMR spectroscopy and X-ray crystallography are not always effective tools in the analysis of complex biomolecules with potentially myriad conformations.

Chemist and biophysicst Hashim Al-Hashimi of the University of Michigan and colleagues describe the geometric rules of RNA in the 8th January issue of the journal Science.

"RNA is a very floppy molecule that often functions by binding to something else and then radically changing shape," explains Al-Hashimi. "These shape changes, in turn, trigger other processes or cascades of events, such as turning specific genes on or off."

However, understanding RNA in detail is rendered more difficult than for small molecules, because RNA cannot be defined as having a single structure. "It has many possible orientations, and different orientations are stabilized under different conditions, such as the presence of particular drug molecules," adds Al-Hashimi.

A major goal in structural biology and biophysics, nevertheless, is to be able to predict not only the complex three-dimensional conformations that RNA assumes, which are dictated fundamentally, by the order of its building blocks, but also the various shapes the molecule adopts once other molecules have bonded to it, whether those are proteins or small-molecule drugs. A new detailed understanding of RNA's conformational responses might lend itself to our being able to manipulate the three-dimensional structure of RNA, by tweaking those drug molecules with which it interacts.

Al-Hashimi draws a parallel with the "conformations" of the human body in action and just how RNA changes shape as it interacts with its chemical environment. "Your body has a specific shape that changes predictably when you are walking or when you are catching a ball," he says, "we want to be able to understand these anatomical rules in RNA."

RNA has been ascribed countless vital cellular roles and the list of diseases with which RNA malfunction is associated continues to grow: spinal muscular atrophy, amyotrophic lateral sclerosis, Wolcott-Rallison syndrome, prostate cancer... As such, methods that allow us to control and manipulate RNA effectively are keenly sought in molecular biology and drug design. In one sense, RNA performs many of its roles by serving as a switch. It changes shape in response to specific cellular signals and it is this switching that triggers other reactions and responses in the cell. It is worth noting, that retroviruses, including HIV, have no DNA and rely instead on RNA for their genetic invasions and replication by host cells.

Previously, Al-Hashimi and colleagues had discovered that RNA does not necessarily change conformation in direct response to its encounters with drug molecules, but rather follows a predictable course of shape shifting on its own. The drug molecules simply wait for the right conformation to emerge and then find that they can bind to the RNA when it is in this form, the particular drug's preferred orientation. So, what are the rules that control the apparently predictable shape-shifting of RNA? And might those rules be universal among all kinds of RNA molecules? Those were the next questions Al-Hashimi's team hoped to answer.

The structure of RNA can be seen as analogous to the structure of the human body in that it has limbs connected at joints, Al-Hashimi explains. The prevailing view, however, was that interactions among the looped structures at the tips of the RNA limbs somehow played a role in defining the molecule's overall 3D conformation, much as a handshake defines the orientation of two arms, but Al-Hashimi's work offers an entirely different perspective on RNA orientation.

"We wondered if the junctions themselves might provide the definition," Al-Hashimi explains. "If you look at your arm, you'll notice that you can't move it, relative to your shoulder, in just any way; it's confined to a certain pathway because of the joint's geometry. We wondered if the same thing might be true of RNA."

To investigate this possibility, the researchers turned to a database of RNA structures (RNA FRABASE) and the Protein Data Bank (PDB). "RNA FRABASE is database of RNA fragments that draws from the structures initially deposited in the PDB," explains graduate student Max Bailor. "It provide a means for researchers to search data, primarily structural, on RNA motifs that may be of interest," he told us, "We used it to conduct a general search for all two-way junctions, and then analysed those junctions with our own software to determine the three topological angles (alpha - twist of helix 2, beta - interhelical bend and gamma - twist of helix 1).

We also used the PDB to do a search of all RNA-ligand complexes. In general, anytime anyone ever solves a structure the three-dimensional coordinates for those structures are deposited here. Using the search capabilities of the PDB I was able to search all RNA-ligand structures to find the Asite, DIS and TPP structures that were reported in the paper."

The researchers discovered that all structures with two helices linked by a particular type of junction called a trinucleotide bulge fell along the same pathway. The researchers then explored structures of RNA molecules with other kinds of junctions and found that those too were confined to similar pathways, but the precise pathway of a given RNA depended upon structural features of its junction.

"With these findings, it now should be possible to predict gross features of RNA 3D shapes based only on their secondary structure, which is far easier to determine than is 3D structure," Al-Hashimi concludes. "This will make it possible to gain insights into the 3D shapes of RNA structures that are too large or complicated to be visualized by experimental techniques such as X-ray crystallography and NMR spectroscopy."

The anatomical rules of RNA also provide a new model against which small molecules might be tested for their ability to manipulate RNA in specific ways and perhaps lead to novel drugs against HIV and other diseases involving RNA.

"The timescale for targeting some of these RNA diseases is now," Al-Hashimi told SpectroscopyNOW, "although it may take a few years to see results."