Yeast positive-stranded virus-like RNA replicons. 20 S and 23 S RNA terminal nucleotide sequences and 3' end secondary structures resemble those of RNA coliphages.
Saccharomyces cerevisiae strains carry single-stranded RNAs called 20 S RNA and 23 S RNA. These RNAs and their double-stranded counterparts, W and T dsRNAs, have been cloned and sequenced. A few nucleotides at both ends, however, remained unknown. These RNAs do not encode coat proteins but their own RNA-dependent RNA polymerases that share a high degree of conservation to each other. The polymerases are also similar to the replicases of RNA coliphages, such as Qbeta. Here we have determined the nucleotide sequences of W and T dsRNAs at both ends using reverse transcriptase polymerase chain reaction-generated cDNA clones. We confirmed the terminal sequences by primer-extension and RNase protection experiments. Furthermore, these analyses demonstrated that W and T dsRNAs and their single-stranded RNA counterparts (i) are linear molecules, (ii) have identical nucleotide sequences at their ends, and (iii) have no poly(A) tails at their 3' ends. Both 20 S and 23 S RNAs have GGGGC at the 5' ends and the complementary 5-nucleotides sequence, GCCCC-OH, at their 3' ends. S1 and V1 secondary structure-mapping of the 3' ends of 20 S and 23 S RNAs shows the presence of a stem-loop structure that partially overlaps with the conserved 3' end sequence. Nucleotide sequences and stem-loop structures similar to those described here have been found at the 3' ends of RNA coliphages. These data, together with the similarity of the RNA-dependent RNA polymerases encoded among these RNAs and RNA coliphages, suggest that 20 S and 23 S RNAs are plus-strand single-stranded virus-like RNA replicons in yeast.
Departamento de Microbiología y Genética/Instituto de Microbiología Bioquímica, Universidad de Salamanca/Consejo Superior de Investigaciones Científicas, Salamanca 37007, Spain.
N
N
Rodríguez-Cousiño
A
A
Solórzano
T
T
Fujimura
R
R
Esteban
D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012331:RNA, Fungal / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D012093:Replicon / Q000235:genetics*; D012260:Ribonucleases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017423:Sequence Analysis, RNA
D005656:Fungal Proteins; D012331:RNA, Fungal; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D012260:Ribonucleases