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Potyviruses encode a large polyprotein that undergoes proteolytic processing, producing 10 mature proteins: P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb-RdRp, and CP. While P1/HC-Pro and HC-Pro/P3 junctions are cleaved by P1 and HC-Pro, respectively, the remaining seven are processed by NIa-Pro. In this study, we analyzed 135 polyprotein sequences from approved potyvirus species and deduced the consensus amino acid residues at five positions (from −4 to +1, where a protease cleaves between −1 and +1) in each of nine cleavage sites. In general, the newly deduced consensus sequences were consistent with the previous ones. However, seven NIa-Pro cleavage sites showed distinct amino acid preferences despite being processed by the same protease. At position −2, histidine was the dominant amino acid residue in most cleavage sites (57.8–60.7% of analyzed sequences), except for the NIa-Pro/NIb-RdRp junction where it was absent. At position −1, glutamine was highly dominant in most sites (88.2–97.8%), except for the VPg/NIa-Pro junction where glutamic acid was found in all the analyzed proteins (100%). At position +1, serine was the most abundant residue (47.4–86.7%) in five out of seven sites, while alanine (52.6%) and glycine (82.2%) were the most abundant in the P3/6K1 and 6K2/VPg junctions, respectively. These findings suggest that each NIa-Pro cleavage site is finely tuned for differential characteristics of proteolytic reactions. The newly deduced consensus sequences may be useful resources for the development of models and methods to accurately predict potyvirus polyprotein processing sites.

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.

Quinoa (Chenopodium quinoa) is an important crop that is used as a model host for studying plant viruses. Genome sequences of five partitiviruses (two novel betapartitiviruses, two novel deltapartitiviruses, and one known gammapartitivirus) were identified from 65 quinoa transcriptome datasets. All quinoa transcriptome datasets analyzed in this study contained at least one of these partitivirus genome segments. These partitivirus sequences may affect the biology of quinoa plants or artificially infected viruses, and therefore could interfere with the accurate interpretation of the experimental results obtained from using quinoa as the host plants.

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.

Transfer RNA halves (tRHs) have various biological functions. However, the biogenesis of specific 5′-tRHs under certain conditions remains unknown. Here, we report that inositol-requiring enzyme 1α (IRE1α) cleaves the anticodon stem-loop region of tRNA Gly(GCC) to produce 5′-tRHs (5′-tRH-Gly GCC ) with highly selective target discrimination upon endoplasmic reticulum (ER) stress. Levels of 5′-tRH-Gly GCC positively affect cancer cell proliferation and modulate mRNA isoform biogenesis both in vitro and in vivo; these effects require co-expression of two nuclear ribonucleoproteins, HNRNPM and HNRNPH2, which we identify as binding proteins of 5′-tRH-Gly GCC . In addition, under ER stress in vivo, we observe simultaneous induction of IRE1α and 5′-tRH-Gly GCC expression in mouse organs and a distantly related organism, Cryptococcus neoformans . Thus, collectively, our findings indicate an evolutionarily conserved function for IRE1α-generated 5′-tRH-Gly GCC in cellular adaptation upon ER stress. RNA fragments derived from cleaved transfer RNAs play roles in gene regulation and cell proliferation. Here, the authors report that upon ER stress, an ER ribonuclease produces a specific tRNA fragment that promotes cancer cell proliferation and modulates mRNA alternative splicing.

Background Cohesin is a chromosome-associated SMC–kleisin complex that mediates sister chromatid cohesion, recombination, and most chromosomal processes during mitosis and meiosis. However, it remains unclear whether meiosis-specific cohesin complexes are functionally active in mitotic chromosomes. Results Through high-resolution 3D-structured illumination microscopy (3D-SIM) and functional analyses, we report multiple biological processes associated with the meiosis-specific cohesin components, α-kleisin REC8 and STAG3, and the distinct loss of function of meiotic cohesin during the cell cycle of embryonic stem cells (ESCs). First, we show that STAG3 is required for the efficient localization of REC8 to the nucleus by interacting with REC8. REC8-STAG3-containing cohesin regulates topological properties of chromosomes and maintains sister chromatid cohesion. Second, REC8-cohesin has additional sister chromatid cohesion roles in concert with mitotic RAD21-cohesin on ESC chromosomes. SIM imaging of REC8 and RAD21 co-staining revealed that the two types of α-kleisin subunits exhibited distinct loading patterns along ESC chromosomes. Third, knockdown of REC8 or RAD21-cohesin not only leads to higher rates of premature sister chromatid separation and delayed replication fork progression, which can cause proliferation and developmental defects, but also enhances chromosome compaction by hyperloading of retinoblastoma protein–condensin complexes from the prophase onward. Conclusions Our findings indicate that the delicate balance between mitotic and meiotic cohesins may regulate ESC-specific chromosomal organization and the mitotic program.

In this study, we identified a key 7α-dehydroxylating bacterial group predicted to be largely responsible for converting primary bile acids (BAs) to secondary BAs in the human gut through sequence similarity network, genome neighborhood network, and gene abundance analyses using human gut metagenomes. The key bacterial group was phylogenetically quite different from known 7α-dehydroxylating bacteria, and their abundance was highly correlated with the occurrence of diverse diseases associated with bile acid 7α-dehydroxylation. The metabolism of bile acids (BAs) by gut bacteria plays an important role in human health. This study identified and characterized 7α-dehydroxylating bacteria, which are majorly responsible for converting primary BAs to secondary BAs, in the human gut and investigated their association with human disease. Six 7α-dehydratase (BaiE) clusters were identified from human gut metagenomes through sequence similarity network and genome neighborhood network analyses. Abundance analyses of gut metagenomes and metatranscriptomes identified a cluster of bacteria (cluster 1) harboring baiE genes that may be key 7α-dehydroxylating bacteria in the human gut. The baiE gene abundance of cluster 1 was significantly and positively correlated with the ratio of secondary BAs to primary BAs. Furthermore, the baiE gene abundances of cluster 1 were significantly negatively correlated with inflammatory bowel disease, including Crohn’s disease and ulcerative colitis, as well as advanced nonalcoholic fatty liver disease, liver cirrhosis, and ankylosing spondylitis. Phylogenetic and metagenome-assembled genome analyses showed that the 7α-dehydroxylating bacterial clade of cluster 1 was affiliated with the family Oscillospiraceae and may demonstrate efficient BA dehydroxylation ability by harboring both a complete bai operon, for proteins which produce secondary BAs from primary BAs, and a gene for bile salt hydrolase, which deconjugates BAs, in the human gut. IMPORTANCE In this study, we identified a key 7α-dehydroxylating bacterial group predicted to be largely responsible for converting primary bile acids (BAs) to secondary BAs in the human gut through sequence similarity network, genome neighborhood network, and gene abundance analyses using human gut metagenomes. The key bacterial group was phylogenetically quite different from known 7α-dehydroxylating bacteria, and their abundance was highly correlated with the occurrence of diverse diseases associated with bile acid 7α-dehydroxylation. In addition, we characterized the metabolic features of the key bacterial group using their metagenome-assembled genomes. This approach is useful to identify and characterize key gut bacteria highly associated with human health and diseases.

Alteromonas species are globally distributed copiotrophic bacteria in marine habitats. Among these, sea-tidal flats are distinctive: undergoing seasonal temperature and oxygen-tension changes, plus periodic exposure to petroleum hydrocarbons. Strain SN2 of the genus Alteromonas was isolated from hydrocarbon-contaminated sea-tidal flat sediment and has been shown to metabolize aromatic hydrocarbons there. Strain SN2's genomic features were analyzed bioinformatically and compared to those of Alteromonas macleodii ecotypes: AltDE and ATCC 27126. Strain SN2's genome differs from that of the other two strains in: size, average nucleotide identity value, tRNA genes, noncoding RNAs, dioxygenase gene content, signal transduction genes, and the degree to which genes collected during the Global Ocean Sampling project are represented. Patterns in genetic characteristics (e.g., GC content, GC skew, Karlin signature, CRISPR gene homology) indicate that strain SN2's genome architecture has been altered via horizontal gene transfer (HGT). Experiments proved that strain SN2 was far more cold tolerant, especially at 5°C, than the other two strains. Consistent with the HGT hypothesis, a total of 15 genomic islands in strain SN2 likely confer ecological fitness traits (especially membrane transport, aromatic hydrocarbon metabolism, and fatty acid biosynthesis) specific to the adaptation of strain SN2 to its seasonally cold sea-tidal flat habitat.

Most contemporary methods for the quantification of DNA methylation employ bisulfite conversion and PCR amplification. However, many reports have indicated that bisulfite-mediated PCR methodologies can result in inaccurate measurements of DNA methylation owing to amplification biases. To calibrate analytical biases in quantification of gene methylation, especially those that arise during PCR, we utilized reference materials that represent exact bisulfite-converted sequences with 0% and 100% methylation status of specific genes. After determining relative quantities using qPCR, pairs of plasmids were gravimetrically mixed to generate working standards with predefined DNA methylation levels at 10% intervals in terms of mole fractions. The working standards were used as controls to optimize the experimental conditions and also as calibration standards in melting-based and sequencing-based analyses of DNA methylation. Use of the reference materials enabled precise characterization and proper calibration of various biases during PCR and subsequent methylation measurement processes, resulting in accurate measurements.

Background Post-translational modification of lysine residues of specific proteins by ubiquitin modulates the degradation, localization, and activity of these target proteins. Here, we identified gains of ubiquitylation sites in highly conserved regions of human proteins that occurred during human evolution. Results We analyzed human ubiquitylation site data and multiple alignments of orthologous mammalian proteins including those from humans, primates, other placental mammals, opossum, and platypus. In our analysis, we identified 281 ubiquitylation sites in 252 proteins that first appeared along the human lineage during primate evolution: one protein had four novel sites; four proteins had three sites each; 18 proteins had two sites each; and the remaining 229 proteins had one site each. PML, which is involved in neurodevelopment and neurodegeneration, acquired three sites, two of which have been reported to be involved in the degradation of PML. Thirteen human proteins, including ERCC2 (also known as XPD) and NBR1, gained human-specific ubiquitylated lysines after the human-chimpanzee divergence. ERCC2 has a Lys/Gln polymorphism, the derived (major) allele of which confers enhanced DNA repair capacity and reduced cancer risk compared with the ancestral (minor) allele. NBR1 and eight other proteins that are involved in the human autophagy protein interaction network gained a novel ubiquitylation site. Conclusions The gain of novel ubiquitylation sites could be involved in the evolution of protein degradation and other regulatory networks. Although gains of ubiquitylation sites do not necessarily equate to adaptive evolution, they are useful candidates for molecular functional analyses to identify novel advantageous genetic modifications and innovative phenotypes acquired during human evolution.