The RNA proximity ligation method reveals how different regions of an RNA molecule interact to form structures. In this "map" of an RNA component, red regions show where the molecule folds back onto itself to create a 3D structure.
Researchers at UW Medicine have developed a new technique to decipher the 3-dimensional shape of RNA molecules. A report of their work was published online August 3 in Nature Biotechnology.
In the study, lead author Vijay Ramani, a graduate student; Ruolan Qiu, a research scientist; and senior author Jay Shendure, UW associate professor of genome sciences, looked at RNA molecules involved in a variety of tasks in yeast and human cells.
RNA molecules are made of long, single chains of ribonucleic acids (RNA) that play a key role in such essential cellular tasks as gene regulation and protein synthesis. In order to do these tasks, these chains must fold and twist into complex 3-dimensional shapes. Understanding the 3-dimensional structure of these molecules is key to understanding how they work.
Currently, scientists have a number of tools to study the structure of these RNA molecules—such as electron microscopy, crystallography and spectroscopy. But these approaches are labor-intensive and time-consuming. Other techniques, such as computer modelling, often do a poor job at predicting the structure of the molecule when the folding is complex.
In the new study, researchers adapted a technique, called proximity ligation, that has been used to analyze the structure of other large molecules, such as DNA and proteins. In this process, an enzyme, called an endonuclease, is allowed to make random cuts in the RNA molecules. Then a second enzyme is introduced, called a ligase, which ties loose ends of RNA strands together.
In some cases, the ligase will rejoin the ends that were originally together. In this case the sequence of that segment of the RNA chain will remain unchanged. But some of the time, the ligase will join ends from snipped segments that are typically far apart on the RNA chain but, because the RNA chain has folded back on itself, are now in close proximity. When this happens, the sequence of the RNA chain will be changed where the two separate segments have been newly joined.
By analyzing the sequences of these altered chains and identifying the spots where the sequences are being changed, it is possible to work out which segments of a folded RNA molecule must be close together in 3-dimensional space.
“To be joined together the two segments must have been close together,” explained Ramani.
From this information it may eventually be possible to create a complete model of an RNA molecule’s 3-dimensional structure, said Shendure.
“Essentially this information puts limits on what structures are possible, because to be accurate any model must have these segments in close proximity,” he said.
To test this approach, the researchers looked at a number of RNA molecules whose 3-dimensional structure has been worked out with other methods and found that that the proximity ligation method reliably predicted the molecules’ structures. The new approach will supplement, not replace other techniques, Shendure said. “This method doesn’t solve everything, but it will provide us with information that can greatly improve our understanding of the structure and function of these molecules.”
This research was funded by a National Institutes of Health (NIH) Director’s Pioneer Award (1DP1HG007811) and by a NIH National Human Genome Research Institute NHGRImGenome Training Grant (5T32HG000035).