Viral genomes evolve to maximise the potential to overcome the defence mechanisms of the host they are infecting. The genomes of RNA viruses, such as the human immunodeficiency virus (HIV) and coronaviruses, are single stranded, and can fold upon themselves to form complex secondary structures. Riboswitches are highly structured RNA segments binding to small-molecule ligands. They have recently been increasingly understood to play a crucial role in the infectiousness and severity of viral diseases. The latest advances in the field of RNA therapy already allow for the manufacturing of small molecules capable of disrupting specific RNA secondary structures. Soon virus-specific RNA switches could be re-coded to express products able to block viral replication. As such, the RNA structurome constitutes an exciting target for new drug discovery.
However, the structurome, while once considered a fixed feature, is now understood to be a very complex dynamical system, with multiple structural conformations often available to the RNA. Riboswitches are thought to make use of this structural heterogeneity to regulate key genes expression. To identify and exploit riboswitches, it is therefore essential to draw a full picture of their structural landscape.
Thanks to the emergence of new experimental technologies, we were able to develop a software package, DREEM, to precisely identify and characterise RNA structural heterogeneity. Using DREEM, we were able to identify a potential riboswitch in HIV, which uses two alternative structures to regulate the translation of Tat, a protein which controls viral gene expression.
In parallel to the structurome, the epitranscriptome is known to also play a crucial role in various regulatory mechanisms. As such, both RNA structures and RNA methylation have gained recent interest for the role they play in RNA viruses, especially in the context of RNA therapeutics, but very little has been done so far to understand how they work together. To remedy this lack of knowledge we have combined the power of DREEM with long read Nanopore sequencing. Using manufactured riboswitches, we were able, for the first time, to fully characterise heterogeneous structural systems using long read sequencing. We were also able to simultaneously detect secondary structures and methylation sites at single molecule level, enabling a comprehensive picture of both the epitranscriptome and structurome. We applied our novel approach in-vivo to the HIV candidate riboswitch we previously identified, to determine if methylation at key sites near the region of interest associates with a change in structure.