Former orphan riboswitches reveal unexplored areas of bacterial metabolism, signaling, and gene control processes.

Comparative sequence analyses have been used to discover numerous classes of structured noncoding RNAs, some of which are riboswitches that specifically recognize small-molecule or elemental ion ligands and influence expression of adjacent downstream genes. Determining the correct identity of the ligand for a riboswitch candidate typically is aided by an understanding of the genes under its regulatory control. Riboswitches whose ligands were straightforward to identify have largely been associated with well-characterized metabolic pathways, such as coenzyme or amino acid biosynthesis. Riboswitch candidates whose ligands resist identification, collectively known as orphan riboswitches, are often associated with genes coding for proteins of unknown function, or genes for various proteins with no established link to one another. The cognate ligands for 16 former orphan riboswitch motifs have been identified to date. The successful pursuit of the ligands for these classes has provided insight into areas of biology that are not yet fully explored, such as ion homeostasis, signaling networks, and other previously underappreciated biochemical or physiological processes. Herein we discuss the strategies and methods used to match ligands with orphan riboswitch classes, and overview the lessons learned to inform and motivate ongoing efforts to identify ligands for the many remaining candidates.