Novel coding,translation, and gene expression of a replicating covalently closed circular RNA of 220 nt

The highly structured (64% GC) covalently closed circular (CCC) RNA (220 nt) of the virusoid associated with rice yellow mottle virus codes for a 16-kDa highly basic protein using novel modalities for coding, translation, and gene expression. This CCC RNA is the smallest among all known viroids and virusoids and the only one that codes proteins. Its sequence possesses an internal ribosome entry site and is directly translated through two (or three) completely overlapping ORFs (shifting to a new reading frame at the end of each round). The initiation and termination codons overlap UGAUGA (underline highlights the initiation codon AUG within the combined initiation-termination sequence). Termination codons can be ignored to obtain larger read-through proteins. This circular RNA with no noncoding sequences is a unique natural supercompact “nanogenome.”Viroids and virusoids (viroid-like satellite RNAs) are typically small (220–450 nt) covalently closed circular (CCC) RNAs with no coding capacity (i.e., no genetic information) (1–3) and are the smallest replicating circular RNA pathogens (3, 4). Because of their circular nature, they usually replicate through a rolling circle model to produce larger concatemers (4, 5) which are then processed into monomeric forms with a self-splicing hammerhead ribozyme (virusoids and viroids in the Avsunviroidae family) (6, 7) or by cellular enzymes (8). We have previously reported (9) the characterization and nucleotide sequence of the smallest circular virusoid (220 nt), that of the rice yellow mottle virus (sobemovirus) (RYMV). Like other known virusoids, the small circular satellite of RYMV (scRYMV) depends on a helper virus RYMV for replication and packaging (9, 10).In silico translation of scRYMV revealed the presence of an unusual ORF capable of initiating translation from the AUG in the sequence UGAUGA of the 220-nt circular RNA by internal ribosome binding site (IRBS). As 220 is not an integer multiple of 3, after the first round of translation, the same circular sequence (or possibly the linear head-to-tail concatemers generated in vivo during rolling-circle replication) would be read in a different frame register. After the second round of translation, termination at the same initiation–termination sequence UGAUGA would result in the production of a highly basic 16-kDa protein with the N- and C-terminal halves of the protein encoded by the same 220-nt sequence but read in two distinct, totally overlapping frames. This virusoid could also suppress the leaky tandem termination codons (UGAUGA) to read the same sequence in a third frame and produce a new (18 kDa) read-through protein with an 18-aa C-terminal extension ended by a UAG codon. However, even the latter UAG termination codon may occasionally be ignored to generate longer proteins.In this report, we present evidence demonstrating that the putative ORF(s) deduced from the 220-nt circular RNA sequence as described above are indeed operational.Although this scRYMV RNA is classified as a virusoid, we report here that this virusoid is the only one so far found to encode for a protein. Modalities of initiation of translation (e.g., overlapped initiation and termination codons), the distinct N- and C-terminal halves of the 16-kDa protein translated from the same 220-nt circular sequence and the generation of read-through proteins were examined. We discuss the evolutionary implications of the genetic information and biological functions densely packed into a 220-nt nanogenome.