Biology subjects

Kojima, S.

Publications and source records attributed to Kojima, S..

48 records · Page 3Linked to original sources

bioRxiv2020

Natural Antisense Transcript of Period2, Per2AS, regulates the amplitude of the mouse circadian clock

Circadian transcriptome studies identified a novel transcript at the Period2 (Per2) locus, which we named Per2AS. Per2AS is a long non-coding RNA transcribed from the antisense strand of Per2, and is expressed rhythmically and anti-phasic to Per2 mRNA. Previously, we mathematically tested the hypothesis that Per2AS and Per2 mutually inhibit each others expression by forming a double negative feedback loop, and found that Per2AS expands the oscillatory domain. In this study, we have experimentally tested this prediction by perturbing the expression of Per2AS in mouse fibroblasts. We found that Per2AS represses Per2 pre-transcriptionally in cis and regulates the amplitude of the circadian clock, but not period or phase. Unexpectedly, we also found that Per2 positively regulates Per2AS post-transcriptionally, indicating that Per2AS and Per2 form a single negative feedback loop. Because knock-down of Per2 does not recapitulate the phenotypes of Per2AS perturbation and Per2AS also activates Bmal1 in trans, we propose that Per2AS regulates the amplitude of the circadian clock without producing a protein by rewiring the molecular clock circuit.

molecular biology↗

bioRxiv2020

Multi-sample Full-length Transcriptome Analysis of 22 Breast Cancer Clinical Specimens with Long-Read Sequencing

Although transcriptome alteration is considered as one of the essential drivers of carcinogenesis, conventional short-read RNAseq technology has limited researchers from directly exploring full-length transcripts, only focusing on individual splice sites. We developed a pipeline for Multi-Sample long-read Transcriptome Assembly, MuSTA, and showed through simulations that it enables construction of transcriptome from the transcripts expressed in target samples and more accurate evaluation of transcript usage. We applied it to 22 breast cancer clinical specimens to successfully acquire cohort-wide full-length transcriptome from long-read RNAseq data. By comparing isoform existence and expression between estrogen receptor positive and triple-negative subtypes, we obtained a comprehensive set of subtype-specific isoforms and differentially used isoforms which consisted of both known and unannotated isoforms. We have also found that exon-intron structure of fusion transcripts tends to depend on their genomic regions, and have found three-piece fusion transcripts that were transcribed from complex structural rearrangements. For example, a three-piece fusion transcript resulted in aberrant expression of an endogenous retroviral gene, ERVFRD-1, which is normally expressed exclusively in placenta and supposed to protect fetus from maternal rejection, and expression of which were increased in several TCGA samples with ERVFRD-1 fusions. Our analyses of real clinical specimens and simulated data provide direct evidence that full-length transcript sequencing in multiple samples can add to our understanding of cancer biology and genomics in general.

genomics↗

bioRxiv2020

Genome-wide correlation analysis reveals Rorc as potential amplitude regulator of circadian transcriptome output

Cell-autonomous circadian system, consisting of core clock genes, generates near 24-hour rhythms and regulates the downstream rhythmic gene expression. While it has become clear that the percentage of rhythmic genes varies among mouse tissues, it remains unclear how this variation can be generated, particularly when the clock machinery is nearly identical in all tissues. In this study, we sought to characterize circadian transcriptome datasets that are publicly available and identify the critical component(s) involved in creating this variation. We found that the relative amplitude of 13 genes and the average level of 197 genes correlated with the percentage of cycling genes. Of those, the correlation of Rorc in both relative amplitude and the average level was one of the strongest. In addition, the level of Per2AS, a novel non-coding transcript that is expressed at the Period 2 locus, was also linearly correlated, although with a much lesser degree compared to Rorc. Overall, our study provides insight into how the variation in the percentage of clock-controlled genes can be generated in mouse tissues and suggests that Rorc and potentially Per2AS are involved in regulating the amplitude of circadian transcriptome output.

genomics↗

bioRxiv2020

Genome analysis of the fatal tapeworm Sparganum proliferum unravels the cryptic lifecycle and mechanisms underlying the aberrant larval proliferation

BackgroundThe cryptic parasite Sparganum proliferum proliferates in humans and invades tissues and organs. Only scattered cases have been reported, but S. proliferum infection is always fatal. However, the S. proliferum phylogeny and lifecycle are still an enigma. ResultsTo investigate the phylogenetic relationships between S. proliferum and other cestode species, and to examine the underlying mechanisms of pathogenicity, we sequenced the entire S. proliferum genome. Additionally, S. proliferum plerocercoid larvae transcriptome analyses were performed to identify genes involved in asexual reproduction in the host. The genome sequences confirmed that the S. proliferum genetic sequence is distinct from that of the closely related Spirometra erinaceieuropaei. Moreover, nonordinal extracellular matrix coordination allows for asexual reproduction in the host and loss of sexual maturity in S. proliferum is related to its fatal pathogenicity in humans. ConclusionsThe high-quality reference genome sequences generated should prove valuable for future studies of pseudophyllidean tapeworm biology and parasitism.

genomics↗

bioRxiv2020

The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching

The bacterial flagellar motor is an intricate nanomachine that switches rotational directions between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants (CCW-biased fliG-G214S and CW-locked fliG-G215A) in Vibrio alginolyticus. Sub-tomogram averaging was utilized to resolve two distinct conformations of the C-ring. Comparison of the C-ring structures in two rotational senses provide direct evidence that the C-ring undergoes major structural remodeling during rotational switch. Specifically, FliG conformational changes elicit a large rearrangement of the C-ring that coincides with rotational switching, whereas FliM and FliN form a spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.

microbiology↗

bioRxiv2020

Outer membrane-deprived cyanobacteria liberate periplasmic and thylakoid luminal components that support the growth of heterotrophs

Chloroplasts originate from endosymbiosis of a cyanobacterium within a heterotrophic host cell. Establishing endosymbiosis requires the translocation across its envelope of photosynthetic products generated inside the once free-living cyanobacterium to be exploited by host metabolism. However, the nature of this translocation event is unknown. We previously found that most cyanobacterial outer membrane components were eliminated during the primitive stage of chloroplast evolution, suggesting the importance of evolutionary changes of the outer membrane. Here, we removed the outer membrane from Synechocystis sp. PCC 6803 by disrupting the physical interaction with peptidoglycan, and characterized the effects on cell function. Outer membrane-deprived cells liberated diverse substances into the environment without significantly compromising photoautotrophic growth. The amount of liberated proteins increased to ~0.35 g/L within five days of culture. Proteomic analysis showed that most liberated proteins were periplasmic and thylakoid luminal components. Connectivity between the thylakoid lumen-extracellular space was confirmed by findings that an exogenous hydrophilic oxidant was reduced by photosynthetic electron transport chain on the thylakoid membrane. Metabolomic analysis detected the release of nucleotide-related metabolites at concentrations around 1 M. The liberated materials supported the proliferation of heterotrophic bacteria. These findings show that breaching the outer membrane, without any manipulations to the cytoplasmic membrane, converts a cyanobacterium to a chloroplast-like organism that conducts photosynthesis and releases its biogenic materials. This conversion not only represents a potential explanation why the outer membrane markedly changed during the earliest stage of chloroplast evolution, but also provides the opportunity to harness cyanobacterial photosynthesis for biomanufacturing processes. SIGNIFICANCE STATEMENTAlthough it is well accepted that chloroplasts stem from endosymbiosis of a cyanobacterium within a heterotrophic host cell, the issue of how photosynthetic products generated inside a formerly free-living cyanobacterium are translocated across its envelope and exploited by host metabolism has been little addressed. Here we show that breaching the cyanobacterial outer membrane barrier converts a cyanobacterium to a chloroplast-like organism that conducts photosynthesis and releases its diverse biogenic materials into its external environment, which sustains the growth of heterotrophic organisms. This conversion represents a possible example of metabolic exploitation of cyanobacterial photosynthesis. Further, this "quasi-chloroplast" provides a potential opportunity for industrial application such as producing feedstock for biomanufacturing processes that harnesses heterotrophic bacteria.

microbiology↗

bioRxiv2019

Dynamic production and loss of flagellar filaments during the bacterial life cycle

Bacterial flagella are large extracellular protein organelles that drive bacteria motility and taxis in response to environmental changes. Previous research has focused mostly on describing the flagellar assembly, its rotation speed and power output. However, whether flagella are permanent cell structures and, if not, the circumstances and timing of their production and loss during the bacterial life cycle remain poorly understood. Here we used the single polar flagellum of Vibrio alginolyticus as our model and, using in vivo fluorescence imaging, revealed that the percentage of flagellated bacteria (PFB) in a population varies substantially across different bacterial growth phases. In the early-exponential phase, the PFB increases rapidly in respect to incubation time, mostly through widespread flagella production. In the mid-exponential phase, the PFB peaks at around 76% and the partitioning of flagella between the daughter cells is 1:1 and strictly at the old poles. After entering the stationary phase, the PFB starts to decline, mainly because daughter cells stop making new flagella after cell division. Interestingly, we discovered that bacteria can actively abandon flagella after prolonged stationary culturing, though cell division has long been suspended. Lack of glucose was found to be a major factor promoting flagellar disassembly. We also revealed that the active loss of flagella was initiated by breakage in the rod connecting the extracellular filament to the basal body formed by MS- and C-rings. Our results highlight the dynamic production and loss of flagellar filaments during the bacterial life cycle.

microbiology↗

bioRxiv2019

Critical role of rhythmic poly(A) tail regulation in circadian gene expression

The mammalian circadian clock is deeply rooted in rhythmic regulation of gene expression. Rhythmic transcriptional control mediated by the circadian transcription factors is thought to be the main driver of mammalian circadian gene expression. However, mounting evidence has demonstrated the importance of rhythmic post-transcriptional controls, and it remains unclear how the transcriptional and post-transcriptional mechanisms collectively control rhythmic gene expression. A recent study discovered rhythmicity in poly(A) tail length in mouse liver and its strong correlation with protein expression rhythms. To understand the role of rhythmic poly(A) regulation in circadian gene expression, we constructed a parsimonious model that depicts rhythmic control imposed upon basic mRNA expression and poly(A) regulation processes, including transcription, deadenylation, polyadenylation, and degradation. The model results reveal the rhythmicity in deadenylation as the strongest contributor to the rhythmicity in poly(A) tail length and the rhythmicity in the abundance of the mRNA subpopulation with long poly(A) tails (a rough proxy for mRNA translatability). In line with this finding, the model further shows that the experimentally observed distinct peak phases in the expression of deadenylases, regardless of other rhythmic controls, can robustly group the rhythmic mRNAs by their peak phases in poly(A) tail length and in abundance of the subpopulation with long poly(A) tails. This provides a potential mechanism to synchronize the phases of target gene expression regulated by the same deadenylases. Our findings highlight the critical role of rhythmic deadenylation in regulating poly(A) rhythms and circadian gene expression. Author SummaryThe biological circadian clock regulates various bodily functions such that they anticipate and respond to the day-and-night cycle. To achieve this, the circadian clock controls rhythmic gene expression, and these genes ultimately drive the rhythmicity of downstream biological processes. As a mechanism of driving circadian gene expression, rhythmic transcriptional control has attracted the central focus. However, mounting evidence has also demonstrated the importance of rhythmic post-transcriptional controls. Here we use mathematical modeling to investigate how transcriptional and post-transcriptional rhythms coordinately control rhythmic gene expression. We have particularly focused on rhythmic regulation of the length of poly(A) tail, a nearly universal feature of mRNAs that controls mRNA stability and translation. Our model reveals that the rhythmicity of deadenylation, the process that shortens the poly(A) tail, is the dominant contributor to the rhythmicity in poly(A) tail length and mRNA translatability. Particularly, the phase of deadenylation nearly overrides the other rhythmic processes in controlling the phases of poly(A) tail length and mRNA translatability. Our finding highlights the critical role of rhythmic deadenylation in circadian gene expression control.

systems biology↗

bioRxiv2019

ESUCA: a pipeline for genome-wide identification of upstream open reading frames with evolutionarily conserved sequences and determination of the taxonomic range of their conservation

BackgroundUpstream open reading frames (uORFs) in the 5'-untranslated regions (5'-UTRs) of certain eukaryotic mRNAs encode evolutionarily conserved functional peptides, such as cis-acting regulatory peptides that control translation of downstream main ORFs (mORFs). For genome-wide searches for uORFs with conserved peptide sequences (CPuORFs), comparative genomic studies have been conducted, in which uORF sequences were compared between selected species. To increase chances of identifying CPuORFs, we previously developed an approach in which uORF sequences were compared using BLAST between Arabidopsis and any other plant species with available transcript sequence databases. If this approach is applied to multiple plant species belonging to phylogenetically distant clades, it is expected to further comprehensively identify CPuORFs conserved in various plant lineages, including those conserved among relatively small taxonomic groups.\n\nResultsTo efficiently compare uORF sequences among many species and efficiently identify CPuORFs conserved in various taxonomic lineages, we developed a novel pipeline, ESUCA. We applied ESUCA to the genomes of five angiosperm species, which belong to phylogenetically distant clades, and selected CPuORFs conserved among at least three different orders. Through these analyses, we identified 88 novel CPuORF families. As expected, ESUCA analysis of each of the five angiosperm genomes identified many CPuORFs that were not identified from ESUCA analyses of the other four species. However, unexpectedly, these CPuORFs include those conserved in wide taxonomic ranges, indicating that the approach used here is useful not only for comprehensive identification of narrowly conserved CPuORFs but also for that of widely conserved CPuORFs. Examination of the effects of 11 selected CPuORFs on mORF translation revealed that CPuORFs conserved only in relatively narrow taxonomic ranges can have sequence-dependent regulatory effects, suggesting that most of the identified CPuORFs are conserved because of functional constraints of their encoded peptides.\n\nConclusionsThis study demonstrates that ESUCA is capable of efficiently identifying CPuORFs likely to be conserved because of the functional importance of their encoded peptides. Furthermore, our data show that the approach in which uORF sequences from multiple species are compared with those of many other species, using ESUCA, is highly effective in comprehensively identifying CPuORFs conserved in various taxonomic ranges.

bioinformatics↗

bioRxiv2019

Sensory feedback independent pre-song vocalizations correlate with current state of motor preparation

Many self-initiated, learned, motor sequences begin by repeating a simple movement, like ball-bouncing before a tennis serve, and this repetition is thought to represent motor preparation. Do these simple movements provide real-time sensory feedback used by the brain for getting ready or do they simply reflect internal neural preparatory processes? Here, we addressed this question by examining the introductory notes (INs) that zebra finches repeat before starting their learned song sequence. INs progress from a variable initial state to a stereotyped final state before each song and are thought to represent motor preparation before song. Here, we found that the mean number of INs before song and the progression of INs to song were not affected by removal of two sensory feedback pathways (auditory and proprioceptive). In both feedback-intact and feedback-deprived birds, the presence of calls (other non-song vocalizations), just before the first IN, was correlated with fewer INs before song and an initial state closer to song. Finally, the initial IN state correlated with the time to song initiation. Overall, these results show that INs do not provide real-time sensory feedback for preparing the motor system. Rather, repetition of INs, and possibly, other such simple movements, may reflect the \"current\" state of internal neural preparatory processes involved in getting the brain ready to initiate a learned movement sequence.\n\nSUMMARY STATEMENTThe number and progression of introductory notes to song in the zebra finch are not affected by removal of sensory feedback.

neuroscience↗

bioRxiv2018

Transitioning between preparatory and precisely sequenced neuronal activity in production of a skilled behavior

Precise neural sequences are associated with the production of well-learned skilled behaviors. Yet, how neural sequences arise in the brain remains unclear. In songbirds, premotor projection neurons in the cortical song nucleus HVC are necessary for producing learned song and exhibit precise sequential activity during singing. Using cell-type specific calcium imaging we identify populations of HVC premotor neurons associated with the beginning and ending of singing-related neural sequences. We discovered neurons that bookend singing-related sequences and neuronal populations that transition from sparse preparatory activity prior to song to precise neural sequences during singing. Recordings from downstream premotor neurons or the respiratory system suggest that pre-song activity may be involved in motor preparation to sing. These findings reveal population mechanisms associated with moving from non-vocal to vocal behavioral states and suggest that precise neural sequences begin and end as part of orchestrated activity across functionally diverse populations of cortical premotor neurons.

neuroscience↗

bioRxiv2018

The Vibrio H-ring facilitates the outer membrane penetration of polar-sheathed flagellum

The bacterial flagellum has evolved as one of the most remarkable nanomachines in nature. It provides swimming and swarming motilities that are often essential for the bacterial life cycle and for pathogenesis. Many bacteria such as Salmonella and Vibrio species use flagella as an external propeller to move to favorable environments, while spirochetes utilize internal periplasmic flagella to drive a serpentine movement of the cell bodies through tissues. Here we use cryo-electron tomography to visualize the polar-sheathed flagellum of Vibrio alginolyticus with particular focus on a Vibrio specific feature, the H-ring. We characterized the H-ring by identifying its two components FlgT and FlgO. Surprisingly, we discovered that the majority of flagella are located within the periplasmic space in the absence of the H-ring, which are dramatically different from external flagella in wild-type cells. Our results indicate the H-ring has a novel function in facilitating the penetration of the outer membrane and the assembly of the external sheathed flagella. This unexpected finding is however consistent with the notion that the flagella have evolved to adapt highly diverse needs by receiving or removing accessary genes.\n\nSignificance StatementFlagellum is the major organelle for motility in many bacterial species. While most bacteria possess external flagella such as the multiple peritrichous flagella found in Escherichia coli and Salmonella enterica or the single polar-sheathed flagellum in Vibrio spp., spirochetes uniquely assemble periplasmic flagella, which are embedded between their inner and outer membranes. Here, we show for the first time that the external flagella in Vibrio alginolyticus can be changed as periplasmic flagella by deleting two flagellar genes. The discovery here may provide a new paradigm to understand the molecular basis underlying flagella assembly, diversity, and evolution.

microbiology↗