Hiromitsu Moriyama

[URE3] Prion Propagation in Saccharomyces cerevisiae: Requirement for Chaperone Hsp104 and Curing by Overexpressed Chaperone Ydj1p

ABSTRACT The [URE3] nonchromosomal genetic element is an infectious form (prion) of the Ure2 protein, apparently a self-propagating amyloidosis. We find that an insertion mutation or deletion of HSP104results in inability to propagate the [URE3] prion. Our results indicate that Hsp104 is a common factor in the maintenance of two independent yeast prions. However, overproduction of Hsp104 does not affect the stability of [URE3], in contrast to what is found for the [PSI+] prion, which is known to be cured by either overproduction or deficiency of Hsp104. Like Hsp104, the Hsp40 class chaperone Ydj1p, with the Hsp70 class Ssa1p, can renature proteins. We find that overproduction of Ydj1p results in a gradual complete loss of [URE3]. The involvement of protein chaperones in the propagation of [URE3] indicates a role for protein conformation in inheritance.

The dsRNA-binding protein DRB4 interacts with the Dicer-like protein DCL4 in vivo and functions in the trans -acting siRNA pathway

Arabidopsis thaliana encodes four Dicer-like (DCL) proteins and five dsRNA-binding (DRB) proteins. We have previously demonstrated that DCL4 specifically interacts with DRB4 in vitro. Here we describe the interaction between DCL4 and DRB4 in vivo. The phenotype of a mutant with a defect in DCL4 (dcl4-2) was similar to that of a mutant with a defect in DRB4 (drb4-1): both mutant plants had elongated and downwardly curled rosette leaves and over-accumulated anthocyanin. In immunoprecipitation experiments with either anti-DCL4 or anti-DRB4 antibody and crude extracts of wild-type Arabidopsis plants, co-immunoprecipitation of DCL4 and DRB4 was detected, indicating that DCL4 interacts with DRB4 in vivo. This interaction was confirmed by immunoprecipitation experiments using extracts from dcl4-2, drb4-1, or transgenic plants expressing the hemagglutinin-tagged version of DCL4 or DRB4. The results of immunoprecipitation experiments also suggest that most DCL4 is associated with DRB4, but that some DRB4 is free or associated with other proteins. Reduced accumulation of the TAS1 and TAS3trans-acting siRNA (ta-siRNA) and over accumulation of their target mRNAs (At5g18040 and auxin response factors ARF3 and ARF4) were detected in both drb4-1 and dcl4-2 mutants. These results indicate that DRB4, together with DCL4, functions in the ta-siRNA biogenesis.

Mycoviruses related to chrysovirus affect vegetative growth in the rice blast fungus Magnaporthe oryzae

Mycoviruses causing impaired growth and abnormal pigmentation of the host were found in the rice blast fungus, Magnaporthe oryzae. Four dsRNAs, dsRNA 1 (3554 bp), dsRNA 2 (3250 bp), dsRNA 3 (307 bp) and dsRNA 4 (3043 bp), were detected in isolate S-0412-II 1a of M. oryzae. By picking up single conidia of S-0412-II 1a, cured strains of the fungus were isolated that had completely lost the mycovirus. The cured strains had normal mycelial growth and pigmentation, suggesting that this mycovirus modulates host traits. The buoyant densities of isometric virus particles (∼35 nm diameter) containing these dsRNAs in CsCl ranged from 1.37 to 1.40 g cm⁻³. The single ORF (3384 nt) of dsRNA 1 encoded a gene product highly homologous to the viral RNA-dependent RNA polymerase of members of the family Chrysoviridae. It is noteworthy that mycovirus S-0412-II 1a was detected not only in host cells but also in culture supernatant. Furthermore, abnormal aggregation of mycelia was observed after adding the mycovirus-containing culture supernatant to an uninfected strain of M. oryzae and mycoviral dsRNAs were detectable from the aggregated mycelia. This novel dsRNA mycovirus was named Magnaporthe oryzae chrysovirus 1.

Specific requirement of DRB4, a dsRNA-binding protein, for the in vitro dsRNA-cleaving activity of Arabidopsis Dicer-like 4

Arabidopsis thaliana Dicer-like 4 (DCL4) produces 21-nt small interfering RNAs from both endogenous and exogenous double-stranded RNAs (dsRNAs), and it interacts with DRB4, a dsRNA-binding protein, in vivo and in vitro. However, the role of DRB4 in DCL4 activity remains unclear because the dsRNA-cleaving activity of DCL4 has not been characterized biochemically. In this study, we biochemically characterize DCL4s Dicer activity and establish that DRB4 is required for this activity in vitro. Crude extracts from Arabidopsis seedlings cleave long dsRNAs into 21-nt small RNAs in a DCL4/DRB4-dependent manner. Immunoaffinity-purified DCL4 complexes produce 21-nt small RNAs from long dsRNA, and these complexes have biochemical properties similar to those of known Dicer family proteins. The DCL4 complexes purified from drb4-1 do not cleave dsRNA, and the addition of recombinant DRB4 to drb4-1 complexes specifically recovers the 21-nt small RNA generation. These results reveal that DCL4 requires DRB4 to cleave long dsRNA into 21-nt small RNAs in vitro. Amino acid substitutions in conserved dsRNA-binding domains (dsRBDs) of DRB4 impair three activities: binding to dsRNA, interacting with DCL4, and facilitating DCL4 activity. These observations indicate that the dsRBDs are critical for DRB4 function. Our biochemical approach and observations clearly show that DRB4 is specifically required for DCL4 activity in vitro.

The wide distribution of endornaviruses, large double-stranded RNA replicons with plasmid-like properties.

Summary.The International Committee on Taxonomy of Viruses (ICTV) recently accepted Endornavirus as a new genus of plant dsRNA virus. We have determined the partial nucleotide sequences of the RNA-dependent RNA polymerase regions from the large dsRNAs (about 14 kbp) isolated from barley (Hordeum vulgare), kidney bean (Phaseolus vulgaris), melon (Cucumis melo), bottle gourd (Lagenaria siceraria), Malabar spinach (Basella alba), seagrass (Zostera marina), and the fungus Helicobasidium mompa. Phylogenetic analyses of these seven dsRNAs indicate that these dsRNAs are new members of the genus Endornavirus that are widely distributed over the plant and fungal kingdoms.

Prions of Yeast and Fungi PROTEINS AS GENETIC MATERIAL

The genetic properties of [URE3] and [PSI], two non-chromosomal genetic elements of Saccharomyces cerevisiae, indicated that they were infectious proteins (prions) (1). Subsequent studies have supported this proposal, and the genetic criteria we proposed have been used in the discovery of another new prion, [Het-s], in the filamentous fungus Podospora anserina (2). The prion hypothesis has long been an intriguing explanation of the transmissible spongiform encephalopathies, such as scrapie, Creutzfeldt-Jakob disease, and “mad cow disease” (3–5) (reviewed in Refs. 6 and 7). Studies using Saccharomyces and Podospora have provided evidence of a type not available from studies of scrapie that there can be such a thing as an infectious protein. This work also revealed that prions can be the basis for inherited traits and initiated the use of the powerful yeast system to study this phenomenon. Here we review the basis for the proposal that [URE3], [PSI], and [Hets] are prions of the chromosomally encoded Ure2p, Sup35p, and Het-s protein, respectively. We also review the properties of [URE3] and [Het-s]. Further studies of [PSI] are reviewed by Liebman and Derkatch in the following minireview (8), and other reviews of these subjects have appeared (9–12).

Phylogenetic analysis of some large double-stranded RNA replicons from plants suggests they evolved from a defective single-stranded RNA virus.

Sequences were recently obtained from four double-stranded (ds) RNAs from different plant species. These dsRNAs are not associated with particles and as they appeared not to be horizontally transmitted, they were thought to be a kind of RNA plasmid. Here we report that the RNA-dependent RNA polymerase (RdRp) and helicase domains encoded by these dsRNAs are related to those of viruses of the alpha-like virus supergroup. Recent work on the RdRp sequences of alpha-like viruses raised doubts about their relatedness, but our analyses confirm that almost all the viruses previously assigned to the supergroup are related. Alpha-like viruses have single-stranded (ss) RNA genomes and produce particles, and they are much more diverse than the dsRNAs. This difference in diversity suggests the ssRNA alpha-like virus form is older, and we speculate that the transformation to a dsRNA form began when an ancestral ssRNA virus lost its virion protein gene. The phylogeny of the dsRNAs indicates this transformation was not recent and features of the dsRNA genome structure and translation strategy suggest it is now irreversible. Our analyses also show some dsRNAs from distantly related plants are closely related, indicating they have not strictly co-speciated with their hosts. In view of the affinities of the dsRNAs, we believe they should be classified as viruses and we suggest they be recognized as members of a new virus genus (Endornavirus) and family (Endoviridae).

Double-stranded RNA in rice: A novel RNA replicon in plants

The entire sequence of 13952 nucleotides of a plasmid-like, double-stranded RNA (dsRNA) from rice was assembled from more than 50 independent cDNA clones. The 5′ non-coding region of the coding (sense) strand spans over 166 nucleotides, followed by one long open reading frame (ORF) of 13716 nucleotides that encodes a large putative polyprotein of 4572 amino acid residues, and by a 70-nucleotide 3′ noncoding region. This ORF is apparently the longest reported to date in the plant kingdom. Amino acid sequence comparisons revealed that the large putative polyprotein includes an RNA helicase-like domain and an RNA-dependent RNA polymerase (replicase)-like domain. Comparisons of the amino acid sequences of these two domains and of the entire genetic organization of the rice dsRNA with those found in potyviruses and the CHV1-713 dsRNA of chestnut blight fungus suggest that the rice dsRNA is located evolutionarily between potyviruses and the CHV1-713 dsRNA. This plasmid-like dsRNA in rice seems to constitute a novel RNA replicon in plants.

[URE3] prion propagation is abolished by a mutation of the primary cytosolic Hsp70 of budding yeast

[URE3] and [PSI+] are infectious protein forms of the Saccharomyces cerevisiae Ure2p and Sup35p, respectively. We isolated an allele of SSA2, the primary cytosolic Hsp70, in a screen for mutants unable to maintain [URE3]. Designated ssa2‐10, the mutation results in a leucine substitution for proline 395, a conserved residue of the peptide‐binding domain. This allele also unexpectedly destabilizes [URE3] in newly formed heterozygotes: [URE3] is either absent in heterozygotes formed by crossing wild‐type [URE3] cells with ssa2‐10 mutants, or present and fully stable. SSA2 deletion mutants are weakly capable of maintaining [URE3]. The ssa2‐10 allele is compatible with propagation of [PSI+]. However, in combination with a deletion of SSA1, ssa2‐10 eliminates the nonsense‐suppression phenotype of [PSI+] cells. Copyright

Bell pepper endornavirus: molecular and biological properties, and occurrence in the genus Capsicum

Bell peppers (Capsicum annuum) harbour a large dsRNA virus. The linear genome (14.7 kbp) of two isolates from Japanese and USA bell pepper cultivars were completely sequenced and compared. They shared extensive sequence identity and contained a single, long ORF encoding a 4815 aa protein. This polyprotein contained conserved motifs of putative viral methyltransferase (MTR), helicase 1 (Hel-1), UDP-glycosyltransferase and RNA-dependent RNA polymerase. This unique arrangement of conserved domains has not been reported in any of the known endornaviruses. Hence this virus, for which the name Bell pepper endornavirus (BPEV) is proposed, is a distinct species in the genus Endornavirus (family Endornaviridae). The BPEV-encoded polyprotein contains a cysteine-rich region between the MTR and Hel-1 domains, with conserved CXCC motifs shared among several endornaviruses, suggesting an additional functional domain. In agreement with general endornavirus features, BPEV contains a nick in the positive-strand RNA molecule. The virus was detected in all bell pepper cultivars tested and transmitted through seed but not by graft inoculations. Analysis of dsRNA patterns and RT-PCR using degenerate primers revealed putative variants of BPEV, or closely related species, infecting other C. annuum genotypes and three other Capsicum species (C. baccatum, C. chinense and C. frutescens).