Biology subjects

Koiwa, H.

Publications and source records attributed to Koiwa, H..

6 recordsLinked to original sources

bioRxiv2024

Reciprocal regulation of m6A modification and miRNA production machineries via phase separation-dependent and -independent mechanisms

Methyltransferase complex (MTC) deposits N6-adenosine (m6A) onto RNA, whereas microprocessor produces miRNA. Whether and how these two distinct complexes cross-regulate each other has been poorly studied. Here we report that the MTC subunit B (MTB) tends to form insoluble condensates with poor activity, with its level monitored by 20S proteasome. Conversely, the microprocessor component SERRATE (SE) forms liquid-like condensates, which in turn promotes solubility and stability of MTB, leading to increased MTC activity. Consistently, the hypomorphic lines expressing SE variants, defective in MTC interaction or liquid-like phase behavior, exhibit reduced m6A level. Reciprocally, MTC can recruit microprocessor to MIRNA loci, prompting co-transcriptional cleavage of primary miRNA (pri-miRNAs) substrates. Additionally, pri-miRNAs carrying m6A modifications at their single-stranded basal regions are enriched by m6A readers, which retain microprocessor in the nucleoplasm for continuing processing. This reveals an unappreciated mechanism of phase separation in RNA modification and processing through MTC and microprocessor coordination.

plant biology↗

bioRxiv2023

Roles of linker region flanked by transmembrane and peptidoglycan binding region of PomB in energy conversion of the Vibrio flagellar motor

The energy converting complex of the sodium-driven flagellar motor in bacteria comprises two proteins, PomA and PomB, whose transmembrane regions form ion conducting channels and is called the stator complex. The transmembrane protein PomB is attached to the cell wall by its periplasmic region and has a plug segment following the transmembrane helix to prevent ion flux. PomB ({Delta}41-120), which lacks the periplasmic region from E41 to K120 immediately following its transmembrane region shows similar motility as that of wild-type PomB. In this study, three deletion mutants after the plug region, PomB ({Delta}61-120), PomB ({Delta}61-140), and PomB ({Delta}71-150), were generated and those deletion mutants were examined for their functionality. PomB ({Delta}61-120) conferred similar motility as that of the wild-type protein, whereas the other two mutants showed almost no motility in soft agar plate; however, we observed some swimming cells with speed similar to that of the wild-type cells. To observe dominance of wild-type proteins, we introduced the two PomB mutants into a wild-type strain, and its ability to swim was not affected by the mutants. Then, we purified the mutant PomAB complexes to confirm the stator formation. When we introduced the PomB mutations in the plug region, the reduced motility by the deletion was rescued, suggesting that the stator was activated. Our results indicate that the deletion prevents stator from transformation to an active form; however, the linker and plug regions from E41 to S150 are not essential for the motor function of PomB but are important for its regulation. IMPORTANCEThe stator complex of flagella consists of PomA and PomB proteins and interacts with the rotor complex. PomB has a peptidoglycan binding (PGB) domain to fix the stator for generation of torque. PomB is attached to the cell wall only when the stator is activated by interaction between the cytoplasmic region of PomA and the rotor protein FliG. The flexible linker of PomB, which is a naturally unfolded region, is flanked by the peptidoglycan-binding (PGB) domain and transmembrane region. The plug region, which interacts with the periplasmic loops of PomA to prevent activation of the stator, is located next to its transmembrane region. In this study, we reveal the role of the flexible linker in activation of the stator complex.

microbiology↗

bioRxiv2023

Bacterial diversity and composition on the rinds of specific melon cultivars and hybrids from across different growing regions in the United States

The goal of this study was to characterize the bacterial diversity on different melon varieties grown in different regions of the US, and determine the influence that region, rind netting, and variety of melon has on the composition of the melon microbiome. Assessing the bacterial diversity of the microbiome on the melon rind can identify antagonistic and protagonistic bacteria for foodborne pathogens and spoilage organisms to improve melon safety, prolong shelf-life, and/or improve overall plant health. Bacterial community composition of melons (n = 603) grown in seven locations over a four-year period were used for 16S rRNA gene amplicon sequencing and analysis to identify bacterial diversity and constituents. Statistically significant differences in alpha diversity based on the rind netting and growing region (p < 0.01) were found among the melon samples. Principal Coordinate Analysis based on the Bray-Curtis dissimilarity distance matrix found that the melon bacterial communities clustered more by region rather than melon variety (R2 value: 0.09 & R2 value: 0.02 respectively). Taxonomic profiling among the growing regions found Enterobacteriaceae, Bacillaceae, Microbacteriaceae, and Pseudomonadaceae present on the different melon rinds at an abundance of [≥] 0.1%, but no specific core microbiome was found for netted melons. However, a core of Pseudomonadaceae, Bacillaceae, and Exiguobacteraceae were found for non-netted melons. The results of this study indicate that bacterial diversity is driven more by the region that the melons were grown in compared to rind netting or melon type. Establishing the foundation for regional differences could improve melon safety, shelf-life, and quality as well as the consumers health.

microbiology↗

bioRxiv2023

Bidirectional feedforward regulatory loop of Dicer-like 4 and flavonoids causes floral bicolor patterning in petunia and dahlia

Floral bicolor pigmentation is caused by naturally occurring RNA interference (RNAi) in some cultivars of petunia and dahlia. In both plants, the chalcone synthase gene is highly expressed only in the pigmented region of bicolor petals. However, it remains unknown why RNAi is induced only in the unpigmented region. To elucidate the mechanism of this clear bicolor pattern formation, we examined the dicing activity of Dicer-like 4 (DCL4), which produces small interfering RNAs essential for the induction of RNAi. We showed that the crude extract in the pigmented petal region inhibits dicing activity of DCL4, but not when flavonoids were depleted from the extract. Moreover, we showed the inhibitory activity was associated with flavonoid aglycons. The in vivo dicing activities were detected in the intact protoplasts prepared from the unpigmented region but not from the pigmented region. These results suggest that in the unpigmented region, flavonoids that inhibit DCL4 are not synthesized, and RNAi is maintained, whereas in the pigmented region, DCL4 is inhibited by flavonoids, RNAi is not induced, and anthocyanin biosynthesis is maintained, which ensures RNAi inhibition. Therefore, a clear bicolor pattern is generated by the bidirectional feedforward mechanism of antagonizing DCL4 and flavonoids.

plant biology↗

bioRxiv2021

Function of the Vibrio PomB plug region based on the stator rotation model for bacterial flagellar motor

Bacterial flagella are the only real rotational motor organs in the biological world. The spiral-shaped flagellar filaments that extend from the cell surface rotate like a screw to create a propulsive force. The base of the flagellar filament has a protein motor consisting of a stator and a rotor embedded in the membrane. The motor part has stators composed of two types of membrane subunits, PomA(MotA) and PomB(MotB), which are energy converters coupled to the ion flow that assemble around the rotor. Recently, structures of the stator, in which two molecules of MotB stuck in the center of the MotA ring made of five molecules, were reported and a model in which the MotA ring rotates with respect to MotB, which is coupled to the influx of ions, was proposed. We focused on the Vibrio PomB plug region, which has been reported to control the activation of flagellar motors. We searched for the plug region, which is the interacting region, through site-directed photo-cross-linking and disulfide cross-linking experiments. Our results demonstrated that it interacts with the extracellular short loop region of PomA, which is between transmembrane 3 and 4. Although the motor halted following cross-linking, its function was recovered with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion inflow by stopping the rotation of the rotor. ImportanceThe flagellar biological motor resembles a mechanical motor, which is composed of stator and rotor and where the rotational force is transmitted by gear-like movements. We hypothesized that the flagellar the rotation of stator that the pentamer of A subunits revolves around the axis of the B subunit dimer with ion flow. The plug region of the B subunit has been shown to regulate the ion flow. Herein, we demonstrated that the ion flow was terminated by the crosslinking between the plug region and the A subunit. These finding support the rotation hypothesis and explain the role of the plug region in terminating the rotation.

microbiology↗

bioRxiv2017

Coding sequence of firefly luciferase reporter gene affects specific hyper expression in cpl1 mutant.

Forward genetic screening of mutants using firefly luciferase (LUC) reporter gene became a standard practice in plant research. Such screenings frequently identified alleles of CPL1 (Carboxyl-terminal Phosphatase-Like 1) regardless of promoters or pathways studied. Expression of the corresponding endogenous genes often shows the minimal difference between wild type and cpl1. Here we show that the LUC coding sequence is responsible for the high expression in cpl1, using a classical RD29a-LUC. Deletion of the LUC 3-UTR did not change hyperactivation of LUC in cpl1. However, a codon-modified LUC (LUC2) produced similar expression levels both in wild type and in cpl1. These results indicate that the coding region of LUC is responsible for the cpl1-specific LUC overexpression uncoupled with the expression of the endogenous counterpart.

plant biology↗