Double-stranded RNA Elements Research Articles

Metatranscriptomic Sequencing of Sheath Blight-Associated Isolates of Rhizoctonia solani Revealed Multi-Infection by Diverse Groups of RNA Viruses.

Rice sheath blight, caused by the soil-borne fungus Rhizoctonia solani (teleomorph: Thanatephorus cucumeris, Basidiomycota), is one of the most devastating phytopathogenic fungal diseases and causes yield loss. Here, we report on a very high prevalence (100%) of potential virus-associated double-stranded RNA (dsRNA) elements for a collection of 39 fungal strains of R. solani from the rice sheath blight samples from at least four major rice-growing areas in the Philippines and a reference isolate from the International Rice Research Institute, showing different colony phenotypes. Their dsRNA profiles suggested the presence of multiple viral infections among these Philippine R. solani populations. Using next-generation sequencing, the viral sequences of the three representative R. solani strains (Ilo-Rs-6, Tar-Rs-3, and Tar-Rs-5) from different rice-growing areas revealed the presence of at least 36 viruses or virus-like agents, with the Tar-Rs-3 strain harboring the largest number of viruses (at least 20 in total). These mycoviruses or their candidates are believed to have single-stranded RNA or dsRNA genomes and they belong to or are associated with the orders Martellivirales, Hepelivirales, Durnavirales, Cryppavirales, Ourlivirales, and Ghabrivirales based on their coding-complete RNA-dependent RNA polymerase sequences. The complete genome sequences of two novel RNA viruses belonging to the proposed family Phlegiviridae and family Mitoviridae were determined.

Incidence of putative RNA mycoviruses in entomopathogenic fungi in Korea

Entomopathogenic fungi have potential as biocontrol agents against insect pests, and mycovirus-mediated hypervirulence may enhance their efficacy. Before initiating research on hypervirulence, the presence or absence of double-stranded (ds) RNA elements was determined in 94 Korean entomopathogenic fungi. dsRNA elements varying in size from ca. 0.8 to 7 kbp were found in 14.9% (14/94) of the strains examined, including Beauveria bassiana, Metarhizium pemphigi, M. pinghaense, M. rileyi, and Cordyceps fumosorosea. This study provides information on the incidence and electrophoretic banding patterns of dsRNA elements and is the first report of mycoviruses entomopathogenic fungi in Korea.

PBRM1 bromodomains associate with RNA to facilitate chromatin association.

PBRM1 is a subunit of the PBAF chromatin remodeling complex, which is mutated in 40-50% of clear cell renal cell carcinoma patients. It is thought to largely function as a chromatin binding subunit of the PBAF complex, but the molecular mechanism underlying this activity is not fully known. PBRM1 contains six tandem bromodomains which are known to cooperate in binding of nucleosomes acetylated at histone H3 lysine 14 (H3K14ac). Here, we demonstrate that the second and fourth bromodomains from PBRM1 also bind nucleic acids, selectively associating with double stranded RNA elements. Disruption of the RNA binding pocket is found to compromise PBRM1 chromatin binding and inhibit PBRM1-mediated cellular growth effects.

Abstract P2055: Secondary Structure And Antisense Oligonucleotides Regulate Cardiac Messenger RNA Translation And Modulates Cardiomyocyte Hypertrophy

Translation of upstream open reading frames (uORFs) typically abrogates translation for the main ORF (mORF) and regulates protein expression. uORFs are present in nearly 50% of messenger RNA (mRNA) transcripts, but their impact and regulation on biological processes are understudied. Mining of ribosome profiling data from next-generation sequencing analysis of human and mouse hearts reveals that most translated uORFs reside within mRNAs of transcription factors, which play essential roles in cardiomyocyte (CM) development and growth. We show that the uORF start codon synergizes with an immediate downstream double-stranded RNA (dsRNA) element to activate uORF and inhibit mORF translation. Biochemical and genetic evidence support that this mechanism is utilized by mRNAs encoding multiple transcription factors such as GATA4 (GATA binding protein 4), encoding a master transcription regulator of CM differentiation and pathological hypertrophy. Intriguingly, a trans-acting regulatory factor, DEAD-box RNA helicase DDX3X, is involved in unwinding the dsRNA structure and promoting mORF translation. We develop two types of antisense oligonucleotides (ASOs) that mimic the unwinding or stabilizing of the dsRNA secondary structure, thereby suppressing or enhancing the translation of this uORF, respectively. Genetic or chemical inactivation of this GATA4 uORF using CRISPR-Cas9 or ASOs in human embryonic stem cells lead to enhanced differentiation into CMs and cellular hypertrophy. At the organismal level, short-term treatment of isoproterenol and surgery-induced cardiac hypertrophy mouse models with uORF-enhancing ASOs reduces GATA4 mRNA translation and antagonizes cardiac hypertrophy and remodeling. As a summary, this uORF-dsRNA element is a new tunable regulator of cardiac transcription factor mRNA translation and can be targeted to alter cellular and organismal phenotypes. Moreover, mechanism-based translation-manipulating ASOs can serve as promising biotechnological tools to regulate gene expression in vitro and in vivo and can be adapted to create novel RNA-based therapeutics.

De novo Sequencing of Novel Mycoviruses From Fusarium sambucinum: An Attempt on Direct RNA Sequencing of Viral dsRNAs.

An increasing number of viruses are continuously being found in a wide range of organisms, including fungi. Recent studies have revealed a wide viral diversity in microbes and a potential importance of these viruses in the natural environment. Although virus exploration has been accelerated by short-read, high-throughput sequencing (HTS), and viral de novo sequencing is still challenging because of several biological/molecular features such as micro-diversity and secondary structure of RNA genomes. This study conducted de novo sequencing of multiple double-stranded (ds) RNA (dsRNA) elements that were obtained from fungal viruses infecting two Fusarium sambucinum strains, FA1837 and FA2242, using conventional HTS and long-read direct RNA sequencing (DRS). De novo assembly of the read data from both technologies generated near-entire genomic sequence of the viruses, and the sequence homology search and phylogenetic analysis suggested that these represented novel species of the Hypoviridae, Totiviridae, and Mitoviridae families. However, the DRS-based consensus sequences contained numerous indel errors that differed from the HTS consensus sequences, and these errors hampered accurate open reading frame (ORF) prediction. Although with its present performance, the use of DRS is premature to determine viral genome sequences, the DRS-mediated sequencing shows great potential as a user-friendly platform for a one-shot, whole-genome sequencing of RNA viruses due to its long-reading ability and relative structure-tolerant nature.

Molecular characterization of a novel partitivirus isolated from the phytopathogenic fungus Aplosporella javeedii.

Aplosporella javeedii is a pathogenic fungus that causes canker and dieback of jujube in China. In this study, we report a new mycovirus, Aplosporella javeedii partitivirus 1 (AjPV1), isolated from A. javeedii strain NX55-3. The AjPV1 genome contains two double-stranded RNA elements (dsRNA1 and dsRNA2). The size of dsRNA1 is 2,360 bp, and it encodes a putative RNA-dependent RNA polymerase (RdRp), while dsRNA2 is 2,301 bp in length and encodes a putative capsid protein (CP). The sequences of RdRp and CP have significant similarity to those of members of the family Partitiviridae. Sequence alignment and phylogenetic analysis showed that AjPV1 is a new member of the family Partitiviridae that is related to members of the genus Betapartitivirus. To our knowledge, AjPV1 is the first mycovirus reported from A. javeedii.

Completion of the sequence of the Aspergillus fumigatus partitivirus 1 genome

A Portuguese isolate of Aspergillus fumigatus was found to contain three double-stranded (ds) RNA elements ranging in size from 1.1 to 1.8 kbp and comprising the genome of a strain of Aspergillus fumigatus partitivirus 1 (AfuPV-1) previously thought to contain only the two largest dsRNA elements. The sequence of the smallest dsRNA element is described here, completing the sequence of the AfuPV-1 genome. Sequence analysis of the element revealed an open reading frame encoding a protein of unknown function similar in size and distantly related to elements previously identified in other members of the family Partitiviridae.

Endogenous double-stranded Alu RNA elements stimulate IFN-responses in relapsing remitting multiple sclerosis

Various sensors that detect double-stranded RNA, presumably of viral origin, exist in eukaryotic cells and induce IFN-responses. Ongoing IFN-responses have also been documented in a variety of human autoimmune diseases including relapsing-remitting multiple sclerosis (RRMS) but their origins remain obscure. We find increased IFN-responses in leukocytes in relapsing-remitting multiple sclerosis at distinct stages of disease. Moreover, endogenous RNAs isolated from blood cells of these same patients recapitulate this IFN-response if transfected into naïve cells. These endogenous RNAs are double-stranded RNAs, contain Alu and Line elements and are transcribed from leukocyte transcriptional enhancers. Thus, transcribed endogenous retrotransposon elements can co-opt pattern recognition sensors to induce IFN-responses in RRMS.

A novel monopartite dsRNA virus isolated from the entomopathogenic and nematophagous fungus Purpureocillium lilacinum.

Purpureocillium lilacinum is a ubiquitous saprophytic fungus commonly isolated from soils and widely known as a biological control agent against phytopathogenic nematodes and pest insects. Mycoviruses infect a wide number of fungal species, but the study of viruses infecting entomopathogenic fungi is still quite recent. In this study, a total of 86 P. lilacinum isolates collected from soil in natural and cultivated habitats throughout the Czech Republic were analyzed; 22% of the isolates harbored double-stranded RNA (dsRNA) elements with viral characteristics. These results suggest that mycoviruses are common in P. lilacinum. One of the most common dsRNA elements detected in the survey was completely sequenced and corresponded to the 2,864-bp genome of a previously undescribed mycovirus, designated Purpureocillium lilacinum nonsegmented virus 1 (PlNV-1). Phylogenetic analysis of the RNA-dependent RNA polymerase of PlNV-1 indicated that this virus might belong to a new taxon related to the family Partitiviridae.

Genome sequence of a novel mitovirus identified in the phytopathogenic fungus Alternaria arborescens.

The phytopathogenic fungus Alternaria spp. contains a variety of double-stranded RNA (dsRNA) elements of different sizes. Detailed analysis of next-generation sequencing data obtained using dsRNA purified from Alternaria arborescens, from which we had previously found Alternaria arborescens victorivirus 1, revealed the presence of another mycoviral-like dsRNA of approximately 2.5kbp in length. When using the fungal mitochondrial genetic code, this dsRNA has a single open reading frame that potentially encodes an RNA-dependent RNA polymerase (RdRp) with significant to sequence similarity to those of viruses of the genus Mitovirus. Moreover, both the 5'- and 3'-untranslated regions have the potential to fold into stable stem-loop structures, which is characteristic of mitoviruses. Pairwise comparisons and phylogenetic analysis of the deduced amino acid sequences of RdRp indicated that the virus we identified in A. arborescens is a distinct member of the genus Mitovirus in the family Narnaviridae, designated as "Alternaria arborescens mitovirus 1" (AaMV1).

Genome sequence of a novel victorivirus identified in the phytopathogenic fungus Alternaria arborescens.

Strains of the phytopathogenic fungus Alternaria spp. have been found to contain a variety of double-stranded RNA (dsRNA) elements indicative of mycovirus infection. Here, we report the molecular characterization of a novel dsRNA mycovirus, Alternaria arborescens victorivirus 1 (AaVV1), from A. arborescens, the tomato pathotype of A. alternata. Using next-generation sequencing of dsRNA purified from an A. arborescens strain from the United States of America, we found that the AaVV1 genome is 5203bp in length and contains two open reading frames (ORF1 and 2) that overlap at the tetranucleotide AUGA. Proteins encoded by ORF1 and ORF2 showed significant similarities to the coat protein (CP) and the RNA-dependent RNA polymerase (RdRp), respectively, of dsRNA mycoviruses of the genus Victorivirus. Pairwise comparisons and phylogenetic analysis of the deduced amino acid sequences of both CP and RdRp indicated that AaVV1 is a member of a distinct species of the genus Victorivirus in the family Totiviridae.

Novel mitoviruses in Rhizoctonia solani AG-3PT infecting potato

Double-stranded RNA (dsRNA) elements are ubiquitous in Rhizoctonia solani. Total dsRNA was randomly amplified from a R. solani isolate (RS002) belonging to anastomosis group-3PT (AG-3PT), associated with black scurf in potato. Assembly of resulting cDNA sequences identified a nearly complete genome of a novel virus related to the genus Mitovirus (family Narnaviridae), herein named Rhizoctonia mitovirus 1 RS002 (RMV-1-RS002). The 2797 nucleotide partial genome of RMV-1-RS002 is A-U rich (59.06 %), and can be folded into stable stem-loop structures at 5′ and 3′ ends. Universal and mold mitochondrial codon usages revealed a large open reading frame in the genome, putatively encoding an 826 amino acid polypeptide, which has conserved motifs for mitoviral RNA-dependent RNA polymerase. The full length putative polypeptide shared 25.6 % sequence identity with the corresponding region of Tuber excavatum mitovirus (TeMV). The partial genome of a second mitovirus (proposed name Rhizoctonia mitovirus 2 RS002 (RMV-2-RS002)) was also amplified from RS002. A nearly identical copy of RMV-1-RS002 was detected in two additional AG-3PT isolates. These data indicate that multiple mitoviruses can exist in a single isolate of R. solani AG-3PT, and that mitoviruses such as RMV-1-RS002 are probably widespread in this pathogen. The roles of mitoviruses in the biology of R. solani AG-3PT remain unknown.

Identification and sequence determination of a novel double-stranded RNA mycovirus from the entomopathogenic fungus Beauveria bassiana.

An isolate of the entomopathogenic fungus Beauveria bassiana was found to contain five double-stranded (ds) RNA elements ranging from 1.5 to more than 3 kbp. The complete sequence of the largest dsRNA element is described here. Analysis of the RdRp nucleotide sequence reveals its similarity to unclassified dsRNA elements, such as Alternaria longipes dsRNA virus 1, and its distant relationship to the RNA-dependent RNA polymerases of members of the family Partitiviridae.

Characterization of a novel dsRNA endornavirus in the plant pathogenic fungus Thielaviopsis basicola

Double-stranded (ds) RNA elements are commonly present in strains of the plant pathogenic fungus Thielaviopsis basicola which infects a wide range of plant species. To characterize a novel 12 kb dsRNA in strain NC1527 of this fungus, reverse transcription-polymerase chain reaction (RT-PCR) was used to obtain an 11,602 bp cDNA sequence from overlapping cDNA clones. Northern blot analysis confirmed that 15 individual cDNA clones that covered the entire length of the cDNA sequence all hybridized to the 12 kb dsRNA. An open reading frame (ORF) search revealed that the 5′non-coding region of this sequence spans 27 nucleotides, followed by one large ORF of 11,575 bp nucleotides, which potentially encodes a large putative polyprotein of 3858 amino acid residues. Specialized Basic Local Alignment Search Tool (BLAST) searches of conserved domains indicated that the putative polyprotein contained a viral RNA helicase1 (Hel), glycosyl transferase (GT) and RNA-dependent RNA polymerase (RdRp) domain regions. BLASTp searches in the protein database using translated nucleotides showed that the cloned dsRNA had homology to endornaviruses, which are present in a few fungi as well as some plant species. The amino acid homologies ranged from 28% to 34% in the RdRp domain region, 23–32% in the helicase domain region and 29–30% in GT region. We designate this dsRNA element as a new endornavirus, Chalara elegans endornavirus 1 (CeEV1). Phylogenetic comparison of the sequence of RdRP with other endornavirus indicated that CeEV1 was relatively distant and may be an ancestral form.

Mycoviruses ofBotrytis cinereaisolates from different hosts

Botrytis cinerea (teleomorph Botryotinia fuckeliana) is a necrotrophic plant pathogenic fungus that causes grey mould and enormous economic losses worldwide in different crops. Control of B. cinerea is difficult due to the appearance of fungicide-resistant isolates, and the diversity in virulence due to genetic variability and, perhaps, the infection with mycoviruses or fungal viruses. The discovery of mycoviruses and their possible application as biocontrol agents, as well as their use as tools to study the plant–pathogen interaction, has encouraged their study in B. cinerea. Herein, we have analysed the occurrence of mycoviruses in Spanish B. cinerea isolates to approach a better understanding of the interactions among viruses, fungi and plants in this pathosystem. Fifty-five percent of the B. cinerea isolates analysed contained double-stranded RNA (dsRNA) elements, and the number of dsRNA elements, their relative concentration and size were variable among isolates. Some of these dsRNAs were related to the presence of virus like rod or isometric particles, and to cellular degeneration and malformed mitochondria. We have also demonstrated that a 3 kb dsRNA present in 55% of the isolates having dsRNA elements was a mycovirus genome. Partial sequence of that mycovirus presented high identity in nucleotide and amino acid sequence with Botrytis cinerea mitovirus 1 (BcMV1). Analysis of the genetic distance within Spanish BcMV1 sequences showed the existence of different isolates of this mitovirus inside the Spanish B. cinerea population analysed. This is the first report of the variability of dsRNA elements and the partial genome sequence of a mitovirus associated with Spanish B. cinerea isolates and the genetic diversity within Spanish isolates of BcMV1.

A novel virus in the family Hypoviridae from the plant pathogenic fungus Fusarium graminearum

A double-stranded (ds) RNA element, sized at approximately 13kb pairs, was purified from a field isolate, HN10, of Fusarium graminearum. The coding strand of the dsRNA was 13,023 nucleotides (nt) long (excluding the 3′ poly(A) tail) and was predicted to contain two discontiguous open reading frames (ORF A and ORF B). The 5′ proximal ORF A of 531nt encoded a protein of 176 amino acids (aa), and a BLAST search showed it to be similar to the putative papain-like protease domains encoded by Valsa ceratosperma hypovirus 1 (35% identity) and Cryphonectria hypovirus 4 (CHV4) (31% identity). The 3′ proximal ORF B of 11,118nt encoded a large polyprotein with three conserved domains, including papain-like protease, RNA-dependent RNA polymerase and RNA helicase domains. The polyprotein shared significant aa identities with CHV1 (32%) and CHV2 (32%). Both the genome organization and phylogenetic analysis suggested that the characterized RNA represented a novel hypovirus, designated “Fusarium graminearum hypovirus 1 (FgHV1)”, which was closely related to CHV1 and CHV2 in the Hypoviridae family. Elimination of the virus resulted in no dramatic phenotypic alteration of the fungus.

Isolation and characterization of a mycovirus in Lentinula edodes

A mycovirus was isolated from an edible mushroom, Lentinula edodes, that was suffering from a severe epidemic. Fractionation of the diseased cell extract by isopycnic centrifugation with 50% CsCl revealed that the diseased mushroom was infected by Lentinula edodes spherical virus (LeSV), a new spherical virus with a diameter of 55 nm. The particle of LeSV encapsidated the 12 kb RNA genome by a 120 kDa coat protein. BLAST analysis of the partially sequenced LeSV genome showed 95% sequence identity with a putative RNA-dependent RNA polymerase (RdRp) gene of the mycovirus HKB, which was previously reported as being a double-stranded RNA (dsRNA) element. In contrast to HKB, the RNA genome in LeSV is encapsidated by the 120 kDa coat protein. To confirm that the LeSV coat protein is encoded by the viral genome, the N-terminal amino acid sequence of the coat protein was determined. The resulting N-terminal amino acid sequence, N-SALDVAPVVPELYFXXLEV-C, was found to be located in the middle of the HKB ORF1, suggesting that the LeSV coat protein was indeed encoded by the virus. To detect LeSV in L. edodes, a primer set targeting the RdRp gene was designed based on the partial sequence of the LeSV genome. RT-PCR analysis showed that 56 of the 84 commercially available dikaryotic cultivars carry LeSV. The transmission pattern of the virus was determined by analysing basidiospores from LeSV-infected and LeSV-free fruiting bodies. Nine out of 10 basidiospores from the LeSV-infected cultivars contained the virus while the spores from the LeSV-free parent were free of LeSV, suggesting that vertical transmission is the primary mode of LeSV propagation.

Appearance of mycovirus-like double-stranded RNAs in the white root rot fungus,Rosellinia necatrix, in an apple orchard

In general, mycoviruses are transmitted through hyphal anastomosis between vegetatively compatible strains of the same fungi, and their entire intracellular life cycle within host fungi limits transmission to separate species and even to incompatible strains belonging to the same species. Based on field observations of the white root rot fungus, Rosellinia necatrix, we found two interesting phenomena concerning mycovirus epidemiology. Specifically, apple trees in an orchard were inoculated with one or two R. necatrix strains that belonged to different mycelial compatibility groups (MCGs), strains W563 (virus-free, MCG139) and NW10 (carrying a mycovirus-like double-stranded (ds) RNA element (N10), MCG442). Forty-two sub-isolates of R. necatrix, which were retrieved 2-3 years later, were all genetically identical to W563 or NW10: however, 22 of the sub-isolates contained novel dsRNAs. Six novel dsRNAs (S1-S6) were isolated: S1 was a new victorivirus; S2, S3, and S4 were new partitiviruses; and S5 and S6 were novel viruses that could not be assigned to any known mycovirus family. N10 dsRNA was detected in three W563 sub-isolates. These findings indicated that novel mycoviruses, from an unknown source, were infecting strains W563 and NW10 of R. necatrix in the soil, and that N10 dsRNA was being transmitted between incompatible strains, NW10 to W563.

5'-UTR RNA G-quadruplexes: translation regulation and targeting

RNA structures in the untranslated regions (UTRs) of mRNAs influence post-transcriptional regulation of gene expression. Much of the knowledge in this area depends on canonical double-stranded RNA elements. There has been considerable recent advancement of our understanding of guanine(G)-rich nucleic acids sequences that form four-stranded structures, called G-quadruplexes. While much of the research has been focused on DNA G-quadruplexes, there has recently been a rapid emergence of interest in RNA G-quadruplexes, particularly in the 5′-UTRs of mRNAs. Collectively, these studies suggest that RNA G-quadruplexes exist in the 5′-UTRs of many genes, including genes of clinical interest, and that such structural elements can influence translation. This review features the progresses in the study of 5′-UTR RNA G-quadruplex-mediated translational control. It covers computational analysis, cell-free, cell-based and chemical biology studies that have sought to elucidate the roles of RNA G-quadruplexes in both cap-dependent and -independent regulation of mRNA translation. We also discuss protein trans-acting factors that have been implicated and the evidence that such RNA motifs have potential as small molecule target. Finally, we close the review with a perspective on the future challenges in the field of 5′-UTR RNA G-quadruplex-mediated translation regulation.

Mycoviruses infecting the endophytic and entomopathogenic fungus Tolypocladium cylindrosporum

A mixed virus infection in a strain of the endophytic and entomopathogenic fungus Tolypocladium cylindrosporum was deduced from a study of the transmission to conidia of several double-stranded RNA (dsRNA) elements. The transmission rates of each dsRNA were different, and monosporic isolates harbouring different combinations of the original set of six dsRNAs were obtained. A 5196bp dsRNA element was sequenced and represents the genome of T. cylindrosporum virus 1 (TcV1), a new member of the genus Victorivirus in the Totiviridae family. This virus was transmitted to 81.4% of the conidia; in contrast, four dsRNAs of 3.1–3.7kbp were transmitted only to 4.7% of the monosporic isolates obtained from the infected parental strain. These four dsRNAs did not show segregation during transmission, and one of them was shown by sequence analysis to encode an RdRp, suggesting that the four molecules might represent the whole genome of a quadripartite chrysovirus. A third possible virus with a genome of approximately 4.2kbp was transmitted to 79.1% of the monosporic isolates produced by the infected strain. Ribavirin was used to cure T. cylindrosporum from viruses, and TcV1 was sensitive to this drug. All monosporic cultures derived from the infected strain treated with 80 and 100μM concentrations of the drug were free of TcV1.