For the first time, we can discern the structure of individual RNA molecules in living plant cells. This new method ‘unlocks a second, hidden layer, of the RNA’, researchers from the UK’s John Innes Centre write in Nature.

RNA molecules are important for many biological processes. To understand how RNA molecules influence these processes, knowledge of their structure is necessary. That structure consists of double helix pieces, where two parts of the RNA molecule bind to each other, and single-stranded RNA pieces. Yet researchers have a poor understanding of the structure. To determine the structure of RNA molecules, researchers blindly apply modifications to the pieces of single-stranded RNA. They then read the sequence of the RNA molecules to find out where the modifications are. The researchers can then use the observed modifications to determine the most suitable structure.

Longer molecules

With the current method, using the Illumina platform, researchers can read only 100 nucleotides at a time. To determine the structure of longer RNA molecules, researchers merge the information obtained from multiple fragments. Thus, according to Yiliang Ding of the John Innes Centre, principal investigator of the Nature study, they ‘see the structure of RNA molecules as averages, without knowing whether there is large or small variation’.

Ding’s group has now developed a method that overcomes this problem. Using the PacBio platform, the researchers can read much longer RNA molecules, of several thousand base pairs. ‘In this way, they can see at once from the entire RNA molecule which nucleotides are not paired with another base,’ says researcher RNA structure determination Danny Incarnato of the University of Groningen, who was not involved in the study. ‘This allows the researchers to determine the structure for each individual RNA molecule.’

Regulation of flowering

Using this technique, the researchers, together with colleagues from Caroline Dean’s group, also from the John Innes Centre, analysed the structure of the RNA molecule COOLAIR in hot and cold environments. COOLAIR is involved in the regulation of a plant’s flowering. The first results were surprising, says Ding. ‘We expected a lot of change as a result of cold treatment. But this was not the case, we saw changes in the ratio of certain RNA structures. This is something we are really pleased about.’

One of the changes in the ratio was the emergence of a cold-specific structure. And this structure, the researchers discovered, is necessary for disabling the flowering-suppressor gene in colder conditions. Mutations that only change the cold-specific structure made the plant flower earlier or later.

Danny Incarnato is very excited about the dynamics exhibited by RNA structures. ‘It implies that the structure can change in response to changes from the environment.’ The greater resolution at which RNA structures are now visible will certainly contribute to a better understanding of how cells regulate processes such as gene expression.