Persistent yeast single-stranded RNA viruses exist in vivo as genomic RNA.RNA polymerase complexes in 1:1 stoichiometry.

Yeast narnavirus 20 S and 23 S RNAs encode RNA-dependent RNA polymerases p91 and p104, respectively, but do not encode coat proteins. Both RNAs form ribonucleoprotein complexes with their cognate polymerases. Here we show that these complexes are not localized in mitochondria, unlike the closely related mitoviruses, which reside in these organelles. Cytoplasmic localization of these polymerases was demonstrated by immunofluorescence and by fluorescence emitted from green fluorescent protein-fused polymerases. These fusion proteins were able to form ribonucleoprotein complexes as did the wild-type polymerases. Fluorescent observations and cell fractionation experiments suggested that the polymerases were stabilized by complex formation with their viral RNA genomes. Immunoprecipitation experiments with anti-green fluorescent protein antibodies demonstrated that a single polymerase molecule binds to a single viral RNA genome in the complex. Moreover, the majority (if not all) of 20 S and 23 S RNA molecules were found to form complexes with their cognate RNA polymerases. Since these viral RNAs were not encapsidated, ribonucleoprotein complex formation with their cognate RNA polymerases appears to be their strategy to survive in the host as persistent viruses.

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.

An import signal in the cytosolic domain of the Neurospora mitochondrial outer membrane protein TOM22.

TOM22 is an integral component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The protein is anchored to the lipid bilayer by a central trans-membrane segment, thereby exposing the amino-terminal domain to the cytosol and the carboxyl-terminal portion to the intermembrane space. Here, we describe the sequence requirements for the targeting and correct insertion of Neurospora TOM22 into the outer membrane. The orientation of the protein is not influenced by the charges flanking its trans-membrane segment, in contrast to observations regarding proteins of other membranes. In vitro import studies utilizing TOM22 preproteins harboring deletions or mutations in the cytosolic domain revealed that the combination of the trans-membrane segment and intermembrane space domain of TOM22 is not sufficient to direct import into the outer membrane. In contrast, a short segment of the cytosolic domain was found to be essential for the import and assembly of TOM22. This sequence, a novel internal import signal for the outer membrane, carries a net positive charge. A mutant TOM22 in which the charge of the import signal was altered to -1 was imported less efficiently than the wild-type protein. Our data indicate that TOM22 contains physically separate import and membrane anchor sequences.

Yeast viral 20 S RNA is associated with its cognate RNA-dependent RNA polymerase.

Most Saccharomyces cerevisiae strains carry in their cytoplasm 20 S RNA, a linear single-stranded RNA molecule of 2.5 kilobases in size. 20 S RNA copy number is greatly induced in stress conditions such as starvation, with up to 100,000 copies per cell. 20 S RNA has coding capacity for a protein of 91 kDa (p91) with sequences diagnostic of RNA-dependent RNA polymerases of (+) strand and double-stranded RNA viruses. We detected p91 in 20 S RNA-carrying strains with specific antisera. The amount of p91 in growing cells is higher than that of stationary cells and similar to the one in 20 S RNA-induced cells. Although 20 S RNA is not encapsidated into viral particles, p91 non-covalently forms a ribonucleoprotein complex with 20 S RNA. This suggests a role of p91 in the RNA to RNA synthesis processes required for 20 S RNA replication. Although the strain analyzed also harbors 23 S RNA, a closely related single-stranded RNA, 23 S RNA is not associated with p91 but with its putative RNA polymerase, p104. Similarly, 20 S RNA is not associated with p104 but with p91. These results suggest that 20 S RNA and 23 S RNA replicate independently using their respective cognate RNA polymerases.

Identification and initial characterization of the cytosolic protein Ycr77p.

The nucleotide sequence of yeast chromosome III encompassing the previously the previously described open reading frames (ORFs) YCR80w, YCR77c and YCR78c (Oliver et al., 1992) has been updated. In the corrected sequence, these ORFs are replaced by two new ORFs, YCR80w (453 bp) and YCR77c (2391 bp). In addition, the orientation of Ycr79c is reversed to give ORF Ycr79w, which has an unaltered nt sequence. The predicted translation products do not exhibit significant homology to known proteins. ORF Ycr77p encodes an 88 kDa, cytosolic protein. A fraction of the protein is associated with small membranous structures in a salt-sensitive fashion. Initial characterization revealed that the protein is not essential for yeast viability, growth on non-fermentable carbon sources, mating and sporulation. The chromosome III DNA sequence that was used for the analysis has the Accession Number X59720 in the GenBank/EMBL database.

Both yeast W double-stranded RNA and its single-stranded form 20S RNA are linear.

Most yeast strains carry a cytoplasmic double-stranded RNA (dsRNA) molecule called W, of 2.5 kb in size. We have cloned and sequenced most of W genome (1), and we proposed that W (+) strands were identical to 20S RNA, a single-stranded RNA (ssRNA) species, whose copy number is highly induced under stress conditions. Recently it was proposed that 20S RNA was circular (2). In this paper, however, we demonstrate that both W dsRNA and 20S RNA are linear. Linearity of W dsRNA is shown by the stoichiometric labelling of both strands of W with 32P-pCp and T4 RNA ligase. The last 3' end nucleotide of both strands is about 70 to 80% C and 20 to 30% A. Linearity of 20S RNA is directly demonstrated by a site-specific cleavage of 20S RNA with RNase H, using an oligodeoxynucleotide complementary to an internal site of 20S RNA. The cleavage produced not one but two RNA fragments expected from the linearity of 20S RNA.

T double-stranded RNA (dsRNA) sequence reveals that T and W dsRNAs form a new RNA family in Saccharomyces cerevisiae. Identification of 23 S RNA as the single-stranded form of T dsRNA.

Some strains of the yeast Saccharomyces cerevisiae harbor a double-stranded RNA (dsRNA) molecule, called T. We obtained T cDNA clones by random priming of denatured T dsRNA followed by reverse transcription. Sequence data of T show that only one strand ((+)-strand) has coding capacity for a protein with 940 amino acids which spans almost the entire length of the molecule (2.9 kilobases). Within this protein we found a sequence pattern characteristic of RNA-dependent RNA polymerases of (+)-strand and double-stranded RNA viruses. Although T has no homology with other dsRNAs found in S. cerevisiae, such as L-A, L-BC, M1, or W, the T-encoded protein shows a high degree of conservation with the W-encoded protein. This conservation extends beyond a region that contains the consensus sequences for RNA-dependent RNA polymerases, suggesting that both T and W are evolutionarily related. With a (+)-strand-specific probe for T we identified 23 S RNA, a new single-stranded RNA (ssRNA) species with a sedimentation coefficient of 23 S. T and 23 S RNA have the same mobility under denaturing conditions with glyoxal, suggesting that 23 S RNA is, in fact, the (+)-single-stranded RNA form of T dsRNA. 23 S RNA synthesis is induced under stress conditions such as heat shock and starvation. The relationship between T and 23 S RNA clearly resembles the one between W and its single-stranded derivative form, 20 S RNA. Thus T and W dsRNAs (and their respective single-stranded species) constitute a new RNA family in S. cerevisiae.

Molecular cloning and characterization of W double-stranded RNA, a linear molecule present in Saccharomyces cerevisiae. Identification of its single-stranded RNA form as 20 S RNA.

Most strains of the yeast Saccharomyces cerevisiae harbor a double-stranded RNA (dsRNA) molecule, called W. We obtained W cDNA clones by random priming of denatured W dsRNA followed by reverse transcription. Sequence data of W shows that only one strand ((+)-strand) has coding capacity for a protein with 829 amino acids which spans almost the entire length of the molecule (2.5 kilobases). Within this protein we found a sequence pattern characteristic of RNA dependent RNA polymerases of (+)-strand and double-stranded RNA viruses. W has no homology with other dsRNAs found in S. cerevisiae, such as L-A, L-BC or M1. However, a (+)-strand-specific probe for W hybridized with 20-S RNA. Furthermore, W (+)-strands comigrated with 20 S RNA in strand separation gels. These results suggest that 20 S RNA is a (+)-single-stranded RNA form of W dsRNA itself or a closely related molecule.