Evidence for widespread existence of functional novel and non-canonical human transcripts.

BACKGROUND: Fraction of functional sequence in the human genome remains a key unresolved question in Biology and the subject of vigorous debate. While a plethora of studies have connected a significant fraction of human DNA to various biochemical processes, the classical definition of function requires evidence of effects on cellular or organismal fitness that such studies do not provide. Although multiple high-throughput reverse genetics screens have been developed to address this issue, they are limited to annotated genomic elements and suffer from non-specific effects, arguing for a strong need to develop additional functional genomics approaches. RESULTS: In this work, we established a high-throughput lentivirus-based insertional mutagenesis strategy as a forward genetics screen tool in aneuploid cells. Application of this approach to human cell lines in multiple phenotypic screens suggested the presence of many yet uncharacterized functional elements in the human genome, represented at least in part by novel exons of known and novel genes. The novel transcripts containing these exons can be massively, up to thousands-fold, induced by specific stresses, and at least some can represent bi-cistronic protein-coding mRNAs. CONCLUSIONS: Altogether, these results argue that many unannotated and non-canonical human transcripts, including those that appear as aberrant splice products, have biological relevance under specific biological conditions.

A versatile 5' RACE-Seq methodology for the accurate identification of the 5' termini of mRNAs.

BACKGROUND: Technological advancements in the era of massive parallel sequencing have enabled the functional dissection of the human transcriptome. However, 5' ends of mRNAs are significantly underrepresented in these datasets, hindering the efficient analysis of the complex human transcriptome. The implementation of the template-switching mechanism at the reverse transcription stage along with 5' rapid amplification of cDNA ends (RACE) constitutes the most prominent and efficient strategy to specify the actual 5' ends of cDNAs. In the current study, we developed a 5' RACE-seq method by coupling a custom template-switching and 5' RACE assay with targeted nanopore sequencing, to accurately unveil 5' termini of mRNA targets. RESULTS: The optimization of the described 5' RACE-seq method was accomplished using the human BCL2L12 as control gene. We unveiled that the selection of hybrid DNA/RNA template-switching oligonucleotides as well as the complete separation of the cDNA extension incubation from the template-switching process, significantly increase the overall efficiency of the downstream 5' RACE. Collectively, our results support the existence of two distinct 5' termini for BCL2L12, being in complete accordance with the results derived from both direct RNA and PCR-cDNA sequencing approaches from Oxford Nanopore Technologies. As proof of concept, we implemented the described 5' RACE-seq methodology to investigate the 5' UTRs of several kallikrein-related peptidases (KLKs) gene family members. Our results confirmed the existence of multiple annotated 5' UTRs of the human KLK gene family members, but also identified novel, previously uncharacterized ones. CONCLUSIONS: In this work we present an in-house developed 5' RACE-seq method, based on the template-switching mechanism and targeted nanopore sequencing. This approach enables the broad and in-depth study of 5' UTRs of any mRNA of interest, by offering a tremendous sequencing depth, while significantly reducing the cost-per reaction compared to commercially available kits.

Molecular cloning and 3D model of first cytochrome P450 from CYP3A subfamily in saltwater crocodile (Crocodylus porosus).

Cytochrome P450s (CYPs) play critical role in oxidative metabolism of numerous xenobiotics and endogenous compounds. The first CYP3A subfamily member in saltwater crocodile has been cloned and modelled for three-dimensional (3D) structure. The full-length cDNA was obtained employing reverse transcription polymerase chain reaction (RT-PCR) strategy and rapid amplification of cDNA ends (RACE). The cDNA sequence of 1659 nucleotides includes 132 nucleotides from 5' untranslated region (UTR), an open reading frame of 1527 nucleotides encoding 509 amino acids designated as CYP3A163. The alignment of CYP3A163 sequence with CYP3A subfamily across the lineages exhibit the loss of 1 residue in birds and 7 residues in mammals in comparison to reptiles suggesting the adaptation processes during evolution. The amino acid identity of CYP3A163 with Alligator mississippiensis CYP3A77 and Homo sapiens CYP3A4 is 91% and 62% respectively. The 3D structure of CYP3A163 modelled using human CYP3A4 structure as a template with Phyre2 software, represents high similarity with its functionally important motifs and catalytic domain. Both sequence and structure of CYP3A163 display the common and conserved features of CYP3A subfamily. Overall, this study provides primary molecular and structural data of CYP3A163 required to investigate the xenobiotic metabolism in saltwater crocodiles.

ZEA exerts toxic effects on reproduction and development by mediating Dio3os in mouse endometrial stromal cells.

Zearalenone (ZEA) and imprinted long noncoding RNAs (lncRNAs) are both closely related to reproduction and development. However, whether they have connections in regulating reproduction and development is not clear yet. The aim of this research is to investigate their relationship. lncRNA microarray was performed to analyze differentially expressed genes, and real-time quantitative polymerase chain reaction (PCR) was used to verify the accuracy of microarray analysis. Meanwhile, the technologies of rapid amplification of cDNA ends, RNA fluorescence in situ hybridization and bioinformatics were adopted to characterize the selected lncRNA. Analysis of lncRNA microarray showed lncRNAs and messenger RNAs related to reproduction and development were significantly differently expressed, and Dio3os was probably the target lncRNA. Then, the experiment of real-time quantitative PCR verified the accuracy of microarray data. Characterization of Dio3os showed Dio3os, an antisense lncRNA with 2312 bp and 15 open reading frames, was enriched in the cytoplasm. Our findings suggest ZEA probably exerts toxic effects on reproduction and development by mediating Dio3os.

Unique GH18 chitinase from Euglena gracilis: full-length cDNA cloning and characterization of its catalytic domain.

A cDNA of putative chitinase from Euglena gracilis, designated EgChiA, encoded 960 amino acid residues, which is arranged from N-terminus in the order of signal peptide, glycoside hydrolase family 18 (GH18) domain, carbohydrate binding module family 18 (CBM18) domain, GH18 domain, CBM18 domain, and transmembrane helix. It is likely that EgChiA is anchored on the cell surface. The recombinant second GH18 domain of EgChiA, designated as CatD2, displayed optimal catalytic activity at pH 3.0 and 50 °C. The lower the polymerization degree of the chitin oligosaccharides [(GlcNAc)4-6] used as the substrates, the higher was the rate of degradation by CatD2. CatD2 degraded chitin nanofibers as an insoluble substrate, and it produced only (GlcNAc)2 and GlcNAc. Therefore, we speculated that EgChiA localizes to the cell surface of E. gracilis and is involved in degradation of chitin polymers into (GlcNAc)2 or GlcNAc, which are easily taken up by the cells.

Cloning of a novel lectin from Artocarpus lingnanensis that induces apoptosis in human B-lymphoma cells.

We isolated a novel lectin (Artocarpus nitidus subsp. lingnanensis lectin, ALL) from Artocarpus nitidus subsp. lingnanensis and showed its mitogenic activities. In this study, we determined the amino acid sequence of ALL by cDNA sequencing. ALL cDNA (933 bp) contains a 657-bp open reading frame (ORF), which encodes a protein with 218 amino acids. ALL shares high sequence similarities with Jacalin and Morniga G and belongs to jacalin-related lectin family. We also examined the antitumor activity of ALL using Raji, a human B-lymphoma cell line. ALL exhibits a strong binding affinity to cell membrane, which can be effectively inhibited by N-acetyl-D-galactosamine (GalNAc). ALL inhibits Raji cell proliferation in a time- and dose-dependent manner through apoptosis, evidenced by morphological changes, phosphatidylserine externalization, poly ADP-ribose polymerase (PARP) cleavage, Bcl-2 down-regulation, and caspase-3 activation. We further showed that the activation of p38 mitogen-activated protein kinase (MAPK) signaling pathways is required for the pro-apoptotic activity of ALL.

An ectotherm homologue of human predicted gene NAT16 encodes histidine N-acetyltransferase responsible for Nα-acetylhistidine synthesis.

BACKGROUND: Nα-Acetylhistidine (NAH) is present in very high concentrations exclusively in the brain and lens of ectothermic vertebrates, including ray-finned fishes, amphibians and reptiles, and not in those of endothermic birds and mammals. Although NAH is known to be synthesized from l-His and acetyl-CoA by histidine N-acetyltransferase (HISAT; EC 2.3.1.33), the gene encoding HISAT has remained unknown for any organism. METHODS: HISAT was purified from the blue mackerel brain, and its partial amino acid sequences were analyzed using mass spectrometry and Edman degradation. Using the sequence information, the corresponding gene was cloned and sequenced. Recombinant proteins encoded by the fish gene and its human homologue were expressed in a cell-free translation system. RESULTS: HISAT was identified to be a protein encoded by a fish homologue of the human predicted gene NAT16 (N-acetyltransferase 16). HISAT is an unstable enzyme that is rapidly and irreversibly inactivated during preincubation at 37°C in the absence of acetyl-CoA. In fish brain, the HISAT gene is expressed as two splice variants containing an identical ORF but differing lengths of 5'-UTR. Both variants are expressed exclusively in the fish brain and lens. Interestingly, the recombinant human NAT16 protein, unlike the recombinant fish HISAT, has only trace enzyme activity for NAH synthesis. CONCLUSIONS: These results propose that the function of mammalian NAT16 has been altered from l-His acetylation (NAH synthesis) to another different biological role. GENERAL SIGNIFICANCE: The molecular identification of HISAT will allow progress in the understanding of the physiological function of NAH in ectothermic vertebrates.

Translation attenuation via 3' terminal codon usage in bovine csn1s2 is responsible for the difference in αs2- and ß-casein profile in milk.

The rate of secretion of αs2-casein into bovine milk is approximately 25% of that of ß-casein, yet mammary expression of their respective mRNA transcripts (csn1s2 and csn2) is not different. Our objective was to identify molecular mechanisms that explain the difference in translation efficiency between csn1s2 and csn2. Cell-free translational efficiency of csn2 was 5 times that of csn1s2. Transcripts of csn1s2 distributed into heavier polysomes than csn2 transcripts, indicating an attenuation of elongation and/or termination. Stimulatory and inhibitory effects of the 5' and 3' UTRs on translational efficiency were different with luciferase and casein sequences in the coding regions. Substituting the 5' and 3' UTRs from csn2 into csn1s2 did not improve csn1s2 translation, implicating the coding region itself in the translation difference. Deletion of a 28-codon fragment from the 3' terminus of the csn1s2 coding region, which displays codons with low correlations to cell fitness, increased translation to a par with csn2. We conclude that the usage of the last 28 codons of csn1s2 is the main regulatory element that attenuates its expression and is responsible for the differential translational expression of csn1s2 and csn2.

DDX6 post-transcriptionally down-regulates miR-143/145 expression through host gene NCR143/145 in cancer cells.

In various human malignancies, widespread dysregulation of microRNA (miRNA) expression is reported to occur and affects various cell growth programs. Recent studies suggest that the expression levels of miRNAs that act as tumor suppressors are frequently reduced in cancers because of chromosome deletions, epigenetical changes, aberrant transcription, and disturbances in miRNA processing. MiR-143 and -145 are well-recognized miRNAs that are highly expressed in several tissues, but down-regulated in most types of cancers. However, the mechanism of this down-regulation has not been investigated in detail. Here, we show that DEAD-box RNA helicase 6, DDX6 (p54/RCK), post-transcriptionally down-regulated miR-143/145 expression by prompting the degradation of its host gene product, NCR143/145 RNA. In human gastric cancer cell line MKN45, DDX6 protein was abundantly expressed and accumulated in processing bodies (P-bodies). DDX6 preferentially increased the instability of non-coding RNA, NCR143/145, which encompasses the miR-143/145 cluster, and down-regulated the expression of mature miR-143/145. In human monocytic cell line THP-1, lipopolysaccharide treatment promoted the assembly of P-bodies and down-regulated the expression of NCR143/145 and its miR-143/145 rapidly. In these cells, cycloheximide treatment led to a loss of P-bodies and to an increase in NCR143/145 RNA stability, thus resulting in up-regulation of miR-143/145 expression. These data demonstrate that DDX6 contributed to the control of NCR143/145 RNA stability in P-bodies and post-transcriptionally regulated miR-143/145 expression in cancer cells.

Evaluation of hypothalamic murine and human melanocortin 3 receptor transcript structure.

The melanocortin 3 receptor (MC3R) is involved in regulation of energy homeostasis. However, its transcript structure is not well understood. We therefore studied initiation and termination sites for hypothalamic murine Mc3r and human MC3R transcripts. Rapid Amplification of cDNA Ends (RACE) was performed for the 5' and 3' ends of murine and human hypothalamic RNA. 5' RACE experiments using hypothalamic murine RNA indicated mouse hypothalamus expresses two major Mc3r transcription start sites: one with a 5' UTR approximately 368 bases in length and another previously unknown transcript with a 5' UTR approximately 440 bases in length. 5' RACE experiments using human hypothalamic RNA identified a 5' UTR beginning 533 bases upstream of the start codon with a 248 base splice. 3' RACE experiments using hypothalamic murine RNA indicated the 3' UTR terminates approximately 1286 bases after the translational stop codon, with a previously unknown 787 base splice between consensus splice donor and acceptor sites. 3' RACE experiments using human MC3R transcript indicated the 3' UTR terminates approximately 115-160 bases after the translational stop codon. These data provide insight into melanocortin 3 receptor transcript structure.

Identifying and characterizing Hfq-RNA interactions.

To regulate stress responses and virulence, bacteria use small regulatory RNAs (sRNAs). These RNAs can up or down regulate target mRNAs through base pairing by influencing ribosomal access and RNA decay. A large class of these sRNAs, called trans-encoded sRNAs, requires the RNA binding protein Hfq to facilitate base pairing between the regulatory RNA and its target mRNA. The resulting network of regulation is best characterized in Escherichia coli and Salmonella typhimurium, but the importance of Hfq dependent sRNA regulation is recognized in a diverse population of bacteria. In this review we present the approaches and methods used to discover Hfq binding RNAs, characterize their interactions and elucidate their functions.

Discovery of a disused desaturase gene from the pheromone gland of the moth Ascotis selenaria, which secretes an epoxyalkenyl sex pheromone.

Female Ascotis selenaria (Geometridae) moths use 3,4-epoxy-(Z,Z)-6,9-nonadecadiene, which is synthesized from linolenic acid, as the main component of their sex pheromone. While the use of dietary linolenic or linoleic fatty acid derivatives as sex pheromone components has been observed in moth species belonging to a few families including Geometridae, the majority of moths use derivatives of a common saturated fatty acid, palmitic acid, as their sex pheromone components. We attempted to gain insight into the differentiation of pheromone biosynthetic pathways in geometrids by analyzing the desaturase genes expressed in the pheromone gland of A. selenaria. We demonstrated that a Δ11-desaturase-like gene (Asdesat1) was specifically expressed in the pheromone gland of A. selenaria in spite of the absence of a desaturation step in the pheromone biosynthetic pathway in this species. Further analysis revealed that the presumed transmembrane domains were degenerated in Asdesat1. Phylogenetic analysis demonstrated that Asdesat1 anciently diverged from the lineage of Δ11-desaturases, which are currently widely used in the biosynthesis of sex pheromones by moths. These results suggest that an ancestral Δ11-desaturase became dysfunctional in A. selenaria after a shift in pheromone biosynthetic pathways.

Phosphatidic acid and phosphoinositides facilitate liposome association of Yas3p and potentiate derepression of ARE1 (alkane-responsive element one)-mediated transcription control.

In the n-alkane assimilating yeast Yarrowia lipolytica, the expression of ALK1, encoding a cytochrome P450 that catalyzes terminal mono-oxygenation of n-alkanes, is induced by n-alkanes. The transcription of ALK1 is regulated by a heterocomplex that comprises the basic helix-loop-helix transcription activators, Yas1p and Yas2p, and binds to alkane-responsive element 1 (ARE1) in the ALK1 promoter. An Opi1 family transcription repressor, Yas3p, represses transcription by binding to Yas2p. Yas3p localizes in the nucleus when Y. lipolytica is grown on glucose but localizes to the endoplasmic reticulum (ER) upon the addition of n-alkanes. In this study, we showed that recombinant Yas3p binds to the acidic phospholipids, phosphatidic acid (PA) and phosphoinositides (PIPs), in vitro. The ARE1-mediated transcription was enhanced in vivo in mutants defective in an ortholog of the Saccharomyces cerevisiae gene PAH1, encoding PA phosphatase, and in an ortholog of SAC1, encoding PIP phosphatase in the ER. Truncation mutation analyses for Yas3p revealed two regions that bound to PA and PIPs. These results suggest that the interaction with acidic phospholipids is important for the n-alkane-induced association of Yas3p with the ER membrane.

Molecular cloning of the alpha subunit of complement component C8 (CpC8α) of whitespotted bamboo shark (Chiloscyllium plagiosum).

Complement-mediated cytolysis is the important effect of immune response, which results from the assembly of terminal complement components (C5b-9). Among them, α subunit of C8 (C8α) is the first protein that traverses the lipid bilayer, and then initiates the recruitment of C9 molecules to form pore on target membranes. In this article, a full-length cDNA of C8α (CpC8α) is identified from the whitespotted bamboo shark (Chiloscyllium plagiosum) by RACE. The CpC8α cDNA is 2183 bp in length, encoding a protein of 591 amino acids. The deduced CpC8α exhibits 89%, 49% and 44% identity with nurse shark, frog and human orthologs, respectively. Sequence alignment indicates that the C8α is well conserved during the evolution process from sharks to mammals, with the same modular architecture as well as the identical cysteine composition in the mature protein. Phylogenetic analysis places CpC8α and nurse shark C8α in cartilaginous fish clade, in parallel with the teleost taxa, to form the C8α cluster with higher vertebrates. Hydrophobicity analysis also indicates a similar hydrophobicity of CpC8α to mammals. Finally, expression analysis revealed CpC8α transcripts were constitutively highly expressed in shark liver, with much less expression in other tissues. The well conserved structure and properties suggests an analogous function of CpC8α to mammalian C8α, though it remains to be confirmed by further study.

Molecular characterization and expression analysis of complement component C9 gene in the whitespotted bambooshark, Chiloscyllium plagiosum.

Complement system is known as highly sophisticated immune defense mechanism for antigen recognition as well as effector functions. Activation of the terminal pathway of the complement system leads to the assembly of terminal complement complexes (C5b-9), which induces the characteristic complement-mediated cytolysis. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. In this article, a full-length cDNA of C9 (CpC9) is identified from cartilaginous species, the whitespotted bambooshark, Chiloscyllium plagiosum by RACE. The CpC9 cDNA is 2263 bp in length, encoding a protein of 603 amino acids, which shares 42% and 43% identity with human and Xenopus C9 respectively. Through sequence alignment and comparative analysis, the CpC9 protein was found well conserved, with the typical modular architecture in TCCs and nearly unanimous cysteine composition from fish to mammal. Phylogenetic analysis places it in a clade with C9 orthologs in higher vertebrate and as a sister taxa to the Xenopus. Expression analysis revealed that CpC9 is constitutively highly expressed in shark liver, with much less or even undetectable expression in other tissues; demonstrating liver is the primary tissue for C9synthesis. To sum up, the structural conservation and distinctive phylogenetics might indicate the potentially vital role of CpC9 in shark immune response, though it remains to be confirmed by further study.

Multiple structurally distinct ERα mRNA variants in zebrafish are differentially expressed by tissue type, stage of development and estrogen exposure.

It is well established that estrogen-like environmental chemicals interact with the ligand-binding site of estrogen receptors (ERs) to disrupt transcriptional control of estrogen responsive targets. Here we investigate the possibility that estrogens also impact splicing decisions on estrogen responsive genes, such as that encoding ERα itself. Targeted PCR cloning was applied to identify six ERα mRNA variants in zebrafish. Sequencing revealed alternate use of transcription and translation start sites, multiple exon deletions, intron retention and alternate polyadenylation. As determined by quantitative (q)PCR, N-terminal mRNA variants predicting long (ERαA(L)) and short (ERα(S)) isoforms were differentially expressed by tissue-type, sex, stage of development and estrogen exposure. Whereas ERα(L) mRNA was diffusely distributed in liver, brain, heart, eye, and gonads, ERα(S) mRNA was preferentially expressed in liver (female>male) and ovary. Neither ERα(L) nor ERα(S) transcripts varied significantly during development, but 17ß-estradiol selectively increased accumulation of ERα(S) mRNA (∼170-fold by 120 hpf), an effect mimicked by bisphenol-A and diethylstilbestrol. Significantly, a C-truncated variant (ERα(S)-Cx) lacking most of the ligand binding and AF-2 domains was transcribed exclusively from the short isoform promoter and was similar to ERα(S) in its tissue-, stage- and estrogen inducible expression. These results support the idea that promoter choice and alternative splicing of the esr1 gene of zebrafish are part of the autoregulatory mechanism by which estrogen modulates subsequent ERα expression, and further suggest that environmental estrogens could exert some of their toxic effects by altering the relative abundance of structurally and functionally distinct ERα isoforms.

Molecular cloning of cDNA of gonadotropin-releasing hormones in the Chinese sturgeon (Acipenser sinensis) and the effect of 17ß-estradiol on gene expression.

The Chinese sturgeon, Acipenser sinensis, is a rare and large-sized anadromous bony fish and understanding of its reproductive regulation is a precondition for controlled reproduction. In this study, two gonadotropin-releasing hormone (GnRH) precursor cDNAs, AsGnRH1 (mammalian type) and AsGnRH2 (chicken type 2), were sequenced in A. sinensis. The precursor cDNAs of the AsGnRH1 and AsGnRH2 are 381 and 649 base pairs (bp), encoding signal peptide plus precursors of 92 and 86 amino acids, respectively. Multiple sequence alignment suggests that AsGnRH1 and AsGnRH2 decapeptides are highly conserved among vertebrates. Besides, AsGnRH1 had closer evolutionary relationship with tetrapods, while AsGnRH2 was conservatively grouped with teleosts in the phylogenetic analysis. Tissue distribution analysis shows that AsGnRH2 is exclusively transcribed in the brain, whereas AsGnRH1 exhibits more extensive tissue distribution including brain, liver, spleen and gonad. Furthermore, Chinese sturgeons were subcutaneously implanted with 17ß-estradiol (E2) and the effect of E2 on brain GnRH mRNA levels was evaluated by real-time PCR. A significant increase in AsGnRH1 and AsGnRH2 mRNA levels is detected in fish receiving E2 implantation compared to controls after one month (P<0.05). These results indicate that E2 exerts positive feedback effects on the transcription of the two GnRHs in immature Chinese sturgeon.

Sixteen Novel Mycoviruses Containing Positive Single-Stranded RNA, Double-Stranded RNA, and Negative Single-Stranded RNA Genomes Co-Infect a Single Strain of Rhizoctonia zeae.

In the present study, sixteen novel RNA mycoviruses co-infecting a single strain of Rhizoctonia zeae (strain D40) were identified and molecularly characterized using metatranscriptome sequencing combined with a method for rapid amplification of cDNA ends. The fungal strain was isolated from diseased seedlings of sugar beet with damping-off symptoms. Based on genome analysis and phylogenetic analysis of amino acid sequences of RNA-dependent RNA polymerase, the sixteen mycoviruses associated with strain D40 contained three genome types with nine distinct lineages, including positive single-stranded RNA (Hypoviridae, Yadokariviridae, Botourmiaviridae, and Gammaflexiviridae), double-stranded RNA (Phlegiviridae, Megabirnaviridae, Megatotiviridae, and Yadonushiviridae), and negative single-stranded RNA (Tulasviridae), suggesting a complex composition of a mycoviral community in this single strain of R. zeae (strain D40). Full genome sequences of six novel mycoviruses and the nearly full-length sequences of the remaining ten novel mycoviruses were obtained. Furthermore, seven of these sixteen mycoviruses were confirmed to assemble virus particles present in the R. zeae strain D40. To the best of our knowledge, this is the first detailed study of mycoviruses infecting R. zeae.

Rapid whole genome sequencing methods for RNA viruses.

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.

Six Novel Mycoviruses Containing Positive Single-Stranded RNA and Double-Stranded RNA Genomes Co-Infect a Single Strain of the Rhizoctoniasolani AG-3 PT.

Six novel mycoviruses that collectively represent the mycovirome of Rhizoctonia solani anastomosis group (AG)-3 PT strain ZJ-2H, which causes potato black scurf, were identified through metatranscriptome sequencing and putatively designated as Rhizoctonia solani fusarivirus 4 [RsFV4, positive single-stranded RNA (+ssRNA)], Rhizoctonia solani fusarivirus 5 (RsFV5, +ssRNA), Rhizoctonia solani mitovirus 40 (RsMV40, +ssRNA), Rhizoctonia solani partitivirus 10 [RsPV10, double-stranded RNA (dsRNA)], Rhizoctonia solani partitivirus 11 (RsPV11, dsRNA), and Rhizoctonia solani RNA virus 11 (RsRV11, dsRNA). Whole genome sequences of RsFV4, RsMV40, RsPV10, RsPV11, and RsRV11, as well as a partial genome sequence of RsFV5, were obtained. The 3'- and 5'- untranslated regions of the five mycoviruses with complete genome sequences were folded into stable stem-loop or panhandle secondary structures. RsFV4 and RsFV5 are most closely related to Rhizoctonia solani fusarivirus 1 (RsFV1), however, the first open reading frame (ORF) of RsFV4 and RsFV5 encode a hypothetical protein that differs from the first ORF of RsFV1, which encodes a helicase. We confirmed that RsPV10 and RsPV11 assemble into the spherical virus particles (approximately 30 nm in diameter) that were extracted from strain ZJ-2H. This is the first report that +ssRNA and dsRNA viruses co-infect a single strain of R. solani AG-3 PT.