Diversity and evolution of viroids and viroid-like agents with circular RNA genomes revealed by metatranscriptome mining

Abstract Viroids, the agents of several plant diseases, are the smallest and simplest known replicators that consist of covalently closed circular (ccc) RNA molecules between 200 and 400 nucleotides in size. Viroids encode no proteins and rely on host RNA polymerases for replication, but some contain ribozymes involved in replication intermediate processing. Although other viroid-like agents with cccRNAs genomes, such as satellite RNAs, ribozyviruses and retrozymes, have been discovered, until recently, the spread of these agents in the biosphere appeared narrow, and their actual diversity and evolution remained poorly understood. Extensive, targeted metatranscriptome mining dramatically expanded the known diversity of cccRNAs genomes. These searches identified numerous, diverse viroid-like cccRNAs, many found in environments devoid of plant and animal material, suggesting replication in unicellular eukaryotic and/or prokaryotic hosts. Several cccRNAs are targeted by CRISPR systems, supporting their association with bacteria. In addition to small cccRNAs in the viroid size range, a broad variety of ribozyviruses and novel viruses with cccRNAs genomes, with genomes reaching nearly 5 kilobases, were discovered. Thus, metatranscriptome mining shows that the diversity of viroid-like cccRNAs genomes is far greater than previously suspected, prompting reassessment of the relevance of these replicators for understanding the primordial RNA world.