An alternative miRISC targets a cancer-associated coding sequence mutation in FOXL2.

Recent evidence suggests that animal microRNAs (miRNAs) can target coding sequences (CDSs); however, the pathophysiological importance of such targeting remains unknown. Here, we show that a somatic heterozygous missense mutation (c.402C>G; p.C134W) in FOXL2, a feature shared by virtually all adult-type granulosa cell tumors (AGCTs), introduces a target site for miR-1236, which causes haploinsufficiency of the tumor-suppressor FOXL2. This miR-1236-mediated selective degradation of the variant FOXL2 mRNA is preferentially conducted by a distinct miRNA-loaded RNA-induced silencing complex (miRISC) directed by the Argonaute3 (AGO3) and DHX9 proteins. In both patients and a mouse model of AGCT, abundance of the inversely regulated variant FOXL2 with miR-1236 levels is highly correlated with malignant features of AGCT. Our study provides a molecular basis for understanding the conserved FOXL2 CDS mutation-mediated etiology of AGCT, revealing the existence of a previously unidentified mechanism of miRNA-targeting disease-associated mutations in the CDS by forming a non-canonical miRISC.

FOXL2 and FOXA1 cooperatively assemble on the TP53 promoter in alternative dimer configurations.

Although both the p53 and forkhead box (FOX) family proteins are key transcription factors associated with cancer progression, their direct relationship is unknown. Here, we found that FOX family proteins bind to the non-canonical homotypic cluster of the p53 promoter region (TP53). Analysis of crystal structures of FOX proteins (FOXL2 and FOXA1) bound to the p53 homotypic cluster indicated that they interact with a 2:1 stoichiometry accommodated by FOX-induced DNA allostery. In particular, FOX proteins exhibited distinct dimerization patterns in recognition of the same p53-DNA; dimer formation of FOXA1 involved protein-protein interaction, but FOXL2 did not. Biochemical and biological functional analyses confirmed the cooperative binding of FOX proteins to the TP53 promoter for the transcriptional activation of TP53. In addition, up-regulation of TP53 was necessary for FOX proteins to exhibit anti-proliferative activity in cancer cells. These analyses reveal the presence of a discrete characteristic within FOX family proteins in which FOX proteins regulate the transcription activity of the p53 tumor suppressor via cooperative binding to the TP53 promoter in alternative dimer configurations.

High-throughput RNA sequencing analysis of Mallotus japonicus revealed novel polerovirus and amalgavirus.

High-throughput RNA sequencing (RNA-seq) analysis of samples from Mallotus japonicus, a traditional medicinal plant, yielded two novel RNA viruses tentatively named Mallotus japonicus virus A (MjVA) and Mallotus japonicus virus B (MjVB). The MjVA and MjVB genomes encode proteins showing amino acid sequence similarities to those of poleroviruses (the genus Polerovirus, the family Solemoviridae) and amalgaviruses (the genus Amalgavirus, the family Amalgaviridae), respectively. The MjVA genome contains seven highly overlapping open reading frames, which are translated to seven proteins through various translational mechanisms, including -1 programmed ribosomal frameshifting (PRF) at the slippery motif GGGAAAC, non-AUG translational initiation, and stop codon readthrough. The MjVB genome encodes two proteins; one of which is translated by +1 PRF mechanism at the slippery motif UUUCGN. The abundance analysis of virus-derived RNA fragments revealed that MjVA is highly concentrated in plant parts with well-developed phloem tissues as previously demonstrated in other poleroviruses, which are transmitted by phloem feeders, such as aphids. MjVB, an amalgavirus generally transmitted by seeds, is distributed in all samples at low concentrations. Thus, this study demonstrates the effectiveness and usefulness of RNA-seq analysis of plant samples for the identification of novel RNA viruses and analysis of their tissue distribution. Keywords: Polerovirus; Amalgavirus; Mallotus japonicus; RNA virus; viral genome; programmed ribosomal frameshifting.

Identification of dicistro-like viruses in the transcriptome data of Striga asiatica and other plants.

Dicistroviruses (the family Dicistroviridae) are positive-sense single-stranded RNA viruses of the order Picornavirales, which is a rapidly growing viral group. They have been detected in a wide range of animals, predominantly in insects and crustaceans. In this study, we identified the genome sequences of 14 dicistro-like viruses in the transcriptome data from 12 plant species, including Striga asiatica dicistro-like virus 1 and 2 identified in the transcriptome data of Striga asiatica. Sequence comparison and phylogenetic analysis indicated that these 14 plant-associated dicistro-like viruses were novel members of the family Dicistroviridae, five of which are placed within the genera Aparavirus and Cripavirus, which mainly consist of viruses infecting animals, including insects. The other nine plant dicistro-like viruses formed clades with unclassified dicistroviruses. Our study implies that a wide range of plant species may serve as hosts for dicistroviruses or reservoirs for their transmission. Keywords: dicistrovirus; Dicistroviridae; plant; transcriptome; Striga asiatica.

Artemisia capillaris nucleorhabdovirus 1, a novel member of the genus Alphanucleorhabdovirus, identified in the Artemisia capillaris transcriptome.

A novel, negative-sense, single-stranded RNA virus, Artemisia capillaris nucleorhabdovirus 1 (AcNRV1), was identified in the transcriptome data of Artemisia capillaris (commonly known as capillary wormwood) root tissue. The AcNRV1 genome contains six open reading frames encoding a nucleocapsid (N), phosphoprotein, movement protein P3, matrix protein, glycoprotein, and polymerase (L). Sequence comparison and phylogenetic analysis using L and N protein sequences revealed that AcNRV1 is a novel member of the genus Alphanucleorhabdovirus, one of the six plant-infecting rhabdovirus genera of the family Rhabdoviridae. Wheat yellow striate virus and rice yellow stunt virus were identified as the closest known rhabdoviruses of AcNRV1. The conserved regulatory sequences involved in transcription termination/polyadenylation (TTP) and transcription initiation (TI) of individual genes were identified in the AcNRV1 genome with the consensus sequence 3'-(A/U)UUAUUUUU-GGG-UUG-5' (in the negative-sense genome), whereby dashes separate the TTP, untranscribed intergenic spacer, and TI elements. The AcNRV1 genome sequence will contribute to further understanding the genome structural evolution of plant rhabdoviruses. Keywords: Artemisia capillaris nucleorhabdovirus 1; plant virus; Alphanucleorhabdovirus; Rhabdoviridae.

A novel RNA virus, Thesium chinense closterovirus 1, identified by high-throughput RNA-sequencing of the parasitic plant Thesium chinense.

The genome sequence of a closterovirus (genus Closterovirus, family Closteroviridae), tentatively named Thesium chinense closterovirus 1 (TcCV1), was identified by performing high-throughput RNA-sequencing of the haustoria and root tissues of Thesium chinense, a parasitic plant. The TcCV1 genome was predicted to encode nine proteins, eight of which have orthologs in previously identified closteroviruses. The TcCV1 RNA-dependent RNA polymerase (RdRp) and heat shock protein 70 homolog (Hsp70h) showed 27.8-68.2% and 23.8-55.1% amino acid identity, respectively, to orthologous proteins of known closteroviruses. The putative +1 ribosomal frameshifting site required for producing RdRp was identified as GUUUAGC with UAG stop codon and the skipped nucleotide U. Phylogenetic trees based on RdRp and Hsp70h show that TcCV1 is a novel member of the genus Closterovirus, forming a subclade with a group of known closteroviruses, including mint virus 1 and carnation necrotic fleck virus. The genome sequence of TcCV1 may be useful for studying the genome evolution of closteroviruses. Keywords: Thesium chinense closterovirus 1; Closterovirus; Closteroviridae; Thesium chinense.

Rapid protein sequence evolution via compensatory frameshift is widespread in RNA virus genomes.

BACKGROUND: RNA viruses possess remarkable evolutionary versatility driven by the high mutability of their genomes. Frameshifting nucleotide insertions or deletions (indels), which cause the premature termination of proteins, are frequently observed in the coding sequences of various viral genomes. When a secondary indel occurs near the primary indel site, the open reading frame can be restored to produce functional proteins, a phenomenon known as the compensatory frameshift. RESULTS: In this study, we systematically analyzed publicly available viral genome sequences and identified compensatory frameshift events in hundreds of viral protein-coding sequences. Compensatory frameshift events resulted in large-scale amino acid differences between the compensatory frameshift form and the wild type even though their nucleotide sequences were almost identical. Phylogenetic analyses revealed that the evolutionary distance between proteins with and without a compensatory frameshift were significantly overestimated because amino acid mismatches caused by compensatory frameshifts were counted as substitutions. Further, this could cause compensatory frameshift forms to branch in different locations in the protein and nucleotide trees, which may obscure the correct interpretation of phylogenetic relationships between variant viruses. CONCLUSIONS: Our results imply that the compensatory frameshift is one of the mechanisms driving the rapid protein evolution of RNA viruses and potentially assisting their host-range expansion and adaptation.

Two novel poty-like viruses identified from the transcriptome data of purple witchweed (Striga hermonthica).

Potyvirids (the family Potyviridae) are the largest family of plant RNA viruses. Two novel potyvirid viruses, Striga-associated poty-like virus 1 (SaPlV1) and Striga-associated poty-like virus 2 (SaPlV2), were identified from the transcriptome data of purple witchweed (Striga hermonthica). SaPlV1 was most closely related to bellflower veinal mottle virus (BVMoV), the only member of the genus Bevemovirus, and then to macluraviruses (the genus Macluravirus). The SaPlV1 genome encodes a 2462-amino acid (aa) polyprotein that may be cleaved into nine mature peptides. The cleavage sites of SaPlV1, BVMoV, and macluravirus polyproteins shared strong sequence similarities. SaPlV2 was most closely related to celery latent virus, the sole species of the genus Celavirus, which is the most divergent potyvirid genus. The SaPlV2 polyprotein contained 3329 aa and it may be cleaved into at least seven or eight mature peptides. Phylogenetic analysis suggested that SaPlV1 and SaPlV2 may be novel species of the genera Bevemovirus and Celavirus, respectively. The genome sequences of SaPlV1 and SaPlV2 are useful resources for studying the genome evolution of potyvirids. Keywords: Striga-associated poty-like virus 1; Striga-associated poty-like virus 2; Potyviridae; Beve- movirus; Celavirus; purple witchweed; Striga hermonthica.

Identification of the genome sequence of Zostera associated varicosavirus 1, a novel negative-sense RNA virus, in the common eelgrass (Zostera marina) transcriptome.

Varicosaviruses (the genus Varicosavirus) are bipartite, negative-sense, single-stranded RNA viruses that infect plants. We analyzed a transcriptome dataset isolated from the common eelgrass (Zostera marina) and identified a novel varicosavirus named Zostera associated varicosavirus 1 (ZaVV1). The ZaVV1 genome consists of two genomic segments: RNA1 (6,632-nt) has an open reading frame (ORF) encoding a large multi-functional polymerase protein (L), while RNA2 (4,304-nt) has four ORFs: one for a nucleocapsid protein and three for proteins with unknown functions (P2, P3, and P4). Sequence comparison and phylogenetic analysis using L proteins showed that ZaVV1 is a novel member of the genus Varicosavirus of the family Rhabdoviridae. The conserved regulatory elements involved in transcription termination/polyadenylation and transcription initiation were identified in the ZaVV1 gene-junction regions with the consensus sequence 3'-UAUUAUUCUUUUUGCUCU-5' (in the negative-sense genome). The ZaVV1 genome sequence may be useful for studying the phylogenetic relationships of varicosaviruses and genome evolution of rhabdoviruses. Keywords: Zostera associated varicosavirus 1; Varicosavirus; Rhabdoviridae; common eelgrass; Zostera marina.

A novel tepovirus, Agave virus T, identified by the analysis of the transcriptome data of blue agave (Agave tequilana).

The genome sequence of a novel RNA virus was identified by analyzing transcriptome data obtained from the stem sample of a blue agave (Agave tequilana) plant. Sequence comparison and phylogenetic analysis showed that the RNA virus, Agave virus T (AgVT), was a new member of the genus Tepovirus in the family Betaflexiviridae. AgVT genome had three open reading frames: a 1605-amino acid (aa) replicase (REP), 355-aa movement protein (MP), and 220-aa coat protein (CP). Phylogenetic analyses based on the REP, MP, and CP sequences of AgVT, previously reported tepoviruses, and other Betaflexiviridae viruses revealed that tepoviruses could be classified into two subclades: "potato virus T (PVT)-clade" and "Prunus virus T (PrVT)-clade." PVT, the type species and founding member of the genus Tepovirus, belong to "PVT-clade" along with AgVT, while the other five tepoviruses belong to "PrVT-clade." The genome sequence of AgVT may be useful for studying the phylogenetic relationships between tepoviruses and other closely related viruses. Keywords: Agave virus T; Tepovirus; Betaflexiviridae; blue agave; Agave tequilana.

Two novel closteroviruses, fig virus A and fig virus B, identified by the analysis of the high-throughput RNA-sequencing data of fig (Ficus carica) latex.

Closteroviruses (the genus Closterovirus, the family Closteroviridae) are RNA viruses that infect and cause viral diseases in many economically important plants. Genome sequences of two novel closteroviruses named fig virus A (FiVA) and fig virus B (FiVB) were identified in high-throughput sequencing data obtained from a fig latex sample. FiVA and FiVB genomes, whose lengths are 19,333 bp and 18,741 bp, respectively, were predicted to have 14 shared open reading frames, nine of which had homologs in other closteroviruses. Phylogenetic analysis confirmed that FiVA and FiVB are novel closteroviruses forming a strong subclade with fig mild mottle-associated virus within the genus Closterovirus. FiVA and FiVB genome sequences identified in this study are useful resources for investigating the evolution of closterovirus genome organization. Keywords: fig virus A; fig virus B; Closterovirus; common fig; Ficus carica.

Diet-Related Alterations of Gut Bile Salt Hydrolases Determined Using a Metagenomic Analysis of the Human Microbiome.

The metabolism of bile acid by the gut microbiota is associated with host health. Bile salt hydrolases (BSHs) play a crucial role in controlling microbial bile acid metabolism. Herein, we conducted a comparative study to investigate the alterations in the abundance of BSHs using data from three human studies involving dietary interventions, which included a ketogenetic diet (KD) versus baseline diet (BD), overfeeding diet (OFD) versus underfeeding diet, and low-carbohydrate diet (LCD) versus BD. The KD increased BSH abundance compared to the BD, while the OFD and LCD did not change the total abundance of BSHs in the human gut. BSHs can be classified into seven clusters; Clusters 1 to 4 are relatively abundant in the gut. In the KD cohort, the levels of BSHs from Clusters 1, 3, and 4 increased significantly, whereas there was no notable change in the levels of BSHs from the clusters in the OFD and LCD cohorts. Taxonomic studies showed that members of the phyla Bacteroidetes, Firmicutes, and Actinobacteria predominantly produced BSHs. The KD altered the community structure of BSH-active bacteria, causing an increase in the abundance of Bacteroidetes and decrease in Actinobacteria. In contrast, the abundance of BSH-active Bacteroidetes decreased in the OFD cohort, and no significant change was observed in the LCD cohort. These results highlight that dietary patterns are associated with the abundance of BSHs and community structure of BSH-active bacteria and demonstrate the possibility of manipulating the composition of BSHs in the gut through dietary interventions to impact human health.

Functional relevance of synonymous alleles reflected in allele rareness in the population.

We provide theoretical evidence supporting the non-neutrality of synonymous alleles by investigating the rareness of synonymous alleles in the population. We find a significantly greater number of synonymous rare alleles than conventional neutral alleles derived from noncoding regions. A permutation experiment shows that the rareness of synonymous alleles is not a byproduct of random statistical noise. We then compare the frequencies of synonymous rare alleles and common alleles in various functional contexts in which synonymous alleles are known to be involved. Subsequently, we perform logistic regression analysis to elucidate the effect size of each independent factor contributing to the rareness of synonymous alleles. Additionally, we show that changes in optimality caused by synonymous mutations resulting in rare SNPs in the population tend to be biased toward optimality loss. We think that our study will contribute to the development of novel strategies for identifying functional synonymous mutations.

Gene Tree Discordance Can Generate Patterns of Diminishing Convergence over Time.

Phenotypic convergence is an exciting outcome of adaptive evolution, occurring when different species find similar solutions to the same problem. Unraveling the molecular basis of convergence provides a way to link genotype to adaptive phenotypes, but can also shed light on the extent to which molecular evolution is repeatable and predictable. Many recent genome-wide studies have uncovered a striking pattern of diminishing convergence over time, ascribing this pattern to the presence of intramolecular epistatic interactions. Here, we consider gene tree discordance as an alternative cause of changes in convergence levels over time in a primate dataset. We demonstrate that gene tree discordance can produce patterns of diminishing convergence by itself, and that controlling for discordance as a cause of apparent convergence makes the pattern disappear. We also show that synonymous substitutions, where neither selection nor epistasis should be prevalent, have the same diminishing pattern of molecular convergence in primates. Finally, we demonstrate that even in situations where biological discordance is not possible, discordance due to errors in species tree inference can drive similar patterns. Though intramolecular epistasis could in principle create a pattern of declining convergence over time, our results suggest a possible alternative explanation for this widespread pattern. These results contribute to a growing appreciation not just of the presence of gene tree discordance, but of the unpredictable effects this discordance can have on analyses of molecular evolution.

H3K9 methyltransferase G9a negatively regulates UHRF1 transcription during leukemia cell differentiation.

Histone H3K9 methyltransferase (HMTase) G9a-mediated transcriptional repression is a major epigenetic silencing mechanism. UHRF1 (ubiquitin-like with PHD and ring finger domains 1) binds to hemimethylated DNA and plays an essential role in the maintenance of DNA methylation. Here, we provide evidence that UHRF1 is transcriptionally downregulated by H3K9 HMTase G9a. We found that increased expression of G9a along with transcription factor YY1 specifically represses UHRF1 transcription during TPA-mediated leukemia cell differentiation. Using ChIP analysis, we found that UHRF1 was among the transcriptionally silenced genes during leukemia cell differentiation. Using a DNA methylation profiling array, we discovered that the UHRF1 promoter was hypomethylated in samples from leukemia patients, further supporting its overexpression and oncogenic activity. Finally, we showed that G9a regulates UHRF1-mediated H3K23 ubiquitination and proper DNA replication maintenance. Therefore, we propose that H3K9 HMTase G9a is a specific epigenetic regulator of UHRF1.

The acquisition of novel N-glycosylation sites in conserved proteins during human evolution.

BACKGROUND: N-linked protein glycosylation plays an important role in various biological processes, including protein folding and trafficking, and cell adhesion and signaling. The acquisition of a novel N-glycosylation site may have significant effect on protein structure and function, and therefore, on the phenotype. RESULTS: We analyzed the human glycoproteome data set (2,534 N-glycosylation sites in 1,027 proteins) and identified 112 novel N-glycosylation sites in 91 proteins that arose in the human lineage since the last common ancestor of Euarchonta (primates and treeshrews). Three of them, Asn-196 in adipocyte plasma membrane-associated protein (APMAP), Asn-91 in cluster of differentiation 166 (CD166/ALCAM), and Asn-76 in thyroglobulin, are human-specific. Molecular evolutionary analysis suggested that these sites were under positive selection during human evolution. Notably, the Asn-76 of thyroglobulin might be involved in the increased production of thyroid hormones in humans, especially thyroxine (T4), because the removal of the glycan moiety from this site was reported to result in a significant decrease in T4 production. CONCLUSIONS: We propose that the novel N-glycosylation sites described in this study may be useful candidates for functional analyses to identify innovative genetic modifications for beneficial phenotypes acquired in the human lineage.

The RNA polymerase II C-terminal domain-interacting domain of yeast Nrd1 contributes to the choice of termination pathway and couples to RNA processing by the nuclear exosome.

The RNA polymerase II (RNApII) C-terminal domain (CTD)-interacting domain (CID) proteins are involved in two distinct RNApII termination pathways and recognize different phosphorylated forms of CTD. To investigate the role of differential CTD-CID interactions in the choice of termination pathway, we altered the CTD-binding specificity of Nrd1 by domain swapping. Nrd1 with the CID from Rtt103 (Nrd1(CID(Rtt103))) causes read-through transcription at many genes, but can also trigger termination where multiple Nrd1/Nab3-binding sites and the Ser(P)-2 CTD co-exist. Therefore, CTD-CID interactions target specific termination complexes to help choose an RNApII termination pathway. Interactions of Nrd1 with both CTD and nascent transcripts contribute to efficient termination by the Nrd1 complex. Surprisingly, replacing the Nrd1 CID with that from Rtt103 reduces binding to Rrp6/Trf4, and RNA transcripts terminated by Nrd1(CID(Rtt103)) are predominantly processed by core exosome. Thus, the Nrd1 CID couples Ser(P)-5 CTD not only to termination, but also to RNA processing by the nuclear exosome.

Retroduplication and loss of parental genes is a mechanism for the generation of intronless genes in Ciona intestinalis and Ciona savignyi.

Tunicates, the sister clade of vertebrates, have miniature genomes and numerous intronless genes compared to other animals. It is still unclear how the tunicates acquired such a large number of intronless genes. Here, we analyzed sequences and intron-exon organizations of homologous genes from two closely related tunicates, Ciona intestinalis and Ciona savignyi. We found seven cases in which ancestral introns of a gene were completely lost in a species after their divergence. In four cases, both the intronless copy and the intron-containing copy were present in the genome, indicating that the intronless copy was generated by retroduplication. In the other three cases, the intron-containing copy was absent, implying it was lost after retroduplication. This result suggests that retroduplication and loss of parental genes is a major mechanism for the accumulation of intronless genes in tunicates.