Biology subjects

Kurokawa, K.

Publications and source records attributed to Kurokawa, K..

23 records · Page 2Linked to original sources

bioRxiv2020

De novo virus inference and host prediction from metagenome using CRISPR spacers

Viruses are the most numerous biological entity, existing in all environments and infecting all cellular organisms. Compared with cellular life, the evolution and origin of viruses are poorly understood; viruses are enormously diverse and most lack sequence similarity to cellular genes. To uncover viral sequences without relying on either reference viral sequences from databases or marker genes known to characterize specific viral taxa, we developed an analysis pipeline for virus inference based on clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR is a prokaryotic nucleic acid restriction system that stores memory of previous exposure. Our protocol can infer viral sequences targeted by CRISPR and predict their hosts using unassembled short-read metagenomic sequencing data. Analysing human gut metagenomic data, we extracted 11,391 terminally redundant CRISPR-targeted sequences which are likely complete circular genomes of viruses or plasmids. The sequences include 257 complete crAssphage family genomes, 11 genomes larger than 200 kilobases, 766 genomes of Microviridae species, 114 genomes of Inoviridae species and many entirely novel genomes of unknown taxa. We predicted the host(s) of approximately 70% of discovered genomes by linking protospacers to taxonomically assigned CRISPR direct repeats. These results support that our protocol is efficient for de novo inference of viral genomes and host prediction. In addition, we investigated the origin of the diversity-generating retroelement (DGR) locus of the crAssphage family. Phylogenetic analysis and gene locus comparisons indicate that DGR is orthologous in human gut crAssphages and shares a common ancestor with baboon-derived crAssphage; however, the locus has likely been lost in multiple lineages recently.

genomics↗

bioRxiv2020

Rapid-Eye-Movement Sleep Behavior Disorder Exhibits Similar but Distinct Gut Dysbiosis Compared to Parkinson's Disease

Gut dysbiosis has been reported repeatedly in Parkinsons disease (PD), but once in rapid-eye-movement sleep behavior disorder (RBD) from Germany. Abnormal aggregation of -synuclein fibrils causing PD possibly starts from the intestine. RBD patients frequently develop PD. Early-stage gut dysbiosis that is causally associated with PD is thus expected to be observed in RBD. We analyzed gut microbiota in 26 RBD patients and 137 controls by 16S rRNA-seq. Our RBD dataset was meta-analyzed with the German RBD dataset, and was compared with gut microbiota in 223 PD patients. Unsupervised clustering of gut microbiota by LIGER, a topic model-based tool for single-cell RNA-seq analysis, revealed four enterotypes in controls, RBD, and PD. Short-chain fatty acid (SCFA)-producing bacteria were conserved in an enterotype observed in controls and RBD, whereas they were less in enterotypes observed in PD. Genus Akkermansia and family Akkermansiaceae were consistently increased in both RBD in two countries and PD in five countries. No short-chain fatty acid (SCFA)-producing bacteria were significantly changed in RBD in two counties. In contrast, we previously reported that recognized and putative SCFA-producing genera Faecalibacterium, Roseburia, and Lachnospiraceae ND3007 group were consistently decreased in PD in five countries. Increased mucin-layer-degrading genus Akkermansia possibly accounts for the development of RBD, and an additional decrease of SCFA-producing genera is likely to be associated with the transition from RBD to PD. ImportanceNineteen studies have been reported on gut microbiota in PD, whereas only one study has been reported in RBD from Germany. RBD has the highest likelihood ratio to develop PD. Our meta-analysis of RBD in Japan and Germany revealed increased mucin-layer-degrading genus Akkermansia in RBD. Genus Akkermansia may increase the intestinal permeability, as we previously observed in PD patients, and make the intestinal neural plexus exposed to oxidative stress, which can lead to abnormal aggregation of prion-like -synuclein fibrils in the intestine. In contrast to PD, SCFA-producing bacteria were not decreased in RBD. As SCFA induces Treg cells, a decrease of SCFA-producing bacteria may be a prerequisite for the development of PD. We propose that prebiotic and/or probiotic therapeutic strategies to increase the intestinal mucin layer and to increase intestinal SCFA potentially retard the development of RBD and PD.

microbiology↗

bioRxiv2020

A functional RNA structure in the influenza A virus ribonucleoprotein complex for segment bundling

The influenza A virus genome is segmented into eight viral RNAs (vRNA). Secondary structures on vRNA are thought to be involved in the viral proliferation process, such as intersegment interactions that are necessary for segment bundling. However, the functional RNA structure on vRNA is not well known because the secondary structure of vRNA in virion was partially unwound by binding viral non-specific RNA binding proteins in a sequence-independent manner. Here, we establish the global map of the vRNA secondary structure in virion using the combination of dimethyl sulfate (DMS)-seq and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE)-seq. By integrating DMS-seq and SHAPE-seq analyses with robust statistical analysis, we inferred quite a few bases paired regions including a pseudoknot structure on segment 5. Notably, when cells were infected with the recombinant virus which had mutations in the pseudoknot structure, the impairment of replication and packaging was observed on the other specific segment. Moreover, we analyzed the comprehensive intersegment RNA interactions in virion by ligation of interacting RNA followed by high-throughput sequencing (LIGR-seq). Our LIGR-seq analysis revealed that the intersegment interactions of the specific segment became less frequent and rearranged in the recombinant virus in concordance with the strength of genome packaging impairment. Our data provide evidence that the functional RNA structure motif on the influenza A virus genome can affect the efficiency of replication and segment bundling through the segment interactions.

microbiology↗

bioRxiv2019

Retrograde Transport of Golgi Enzymes by GOLPH3 Across Maturing Cisternae Regulates Glycan Assembly on Sphingolipids and CellGrowth

Glycans are ubiquitous sugar polymers with major biological functions that are assembled by glyco-enzymes onto cargo molecules during their transport through the Golgi complex. How the Golgi determines glycan assembly is poorly understood. By relying on the Golgi cisternal maturation model and using the glyco-enzyme adaptor and oncoprotein GOLPH3 as a molecular tool, we define the first example of how the Golgi controls glycosylation and associated cell functions. GOLPH3, acting as a component of the cisternal maturation mechanism, selectively binds and recycles a subset of glyco-enzymes of the glycosphingolipid synthetic pathway, hinders their escape to the lysosomes and hence increases their levels through a novel lysosomal degradation-regulated mechanism. This enhances the production of specific growth-inducing glycosphingolipids and reprograms the glycosphingolipid pathway to potentiate mitogenic signaling and cell proliferation. These findings unravel unforeseen organizing principles of Golgi-dependent glycosylation and delineate a paradigm for glycan assembly by the Golgi transport mechanisms. Moreover, they indicate a new role of cisternal maturation as a regulator of glycosylation, and outline a novel mechanism of action for GOLPH3-induced proliferation.

cell biology↗

bioRxiv2019

A non-canonical unfolded protein response pathway and mitochondrial dynamics control the number of ER-mitochondria contact sites

Mitochondria maintain their morphology and functions through the optimized balance between the mitochondrial fusion and division. Here we report a novel role of mitochondrial dynamics in controlling the number of ER-mitochondria encounter structure (ERMES) clusters in a yeast cell. Loss of mitochondrial fusion or division caused the increased or decreased number, respectively, of ERMES foci observed in cells. ERMES complexes, therefore, appear to cluster with each other and mitochondrial division may inhibit undesired ERMES hyper-clustering. Furthermore, our microscopic analyses suggest that ER stress induces dissociation of ERMES clusters, increasing the number of ERMES foci even in the absence of Ire1 and Hac1, which are essential factors for the UPR response. Interestingly, we found that ER stress leads to expansion of both the ER and mitochondrial membranes in an ERMES function-dependent manner. These findings imply that a cell is equipped with two independent regulatory mechanisms controlling the number of ER-mitochondria contact sites to meet the cellular as well as environmental demands.

cell biology↗