Biology subjects

Nomura, Y.

Publications and source records attributed to Nomura, Y..

23 records · Page 2Linked to original sources

bioRxiv2020

DeLTa-Seq: direct-lysate targeted RNA-Seq from crude tissue lysate.

Using current mRNA quantification methods such as RT-qPCR and RNA-Seq, it is very difficult to examine thousands of tissue samples due to cost and labor of RNA extraction and quantification steps. Here, we developed Direct-RT buffer in which homogenization of tissue samples and direct-lysate reverse transcription can be conducted without RNA purification. We showed that appreciate concentration of DTT prevented RNA degradation but not RT in the lysates of several plants tissues, yeast, and zebrafish larvae. Using the buffer, direct reverse transcription on the lysates could produce comparable amount of cDNA with that synthesized from purified RNA. Furthermore, we established DeLTa-Seq (Direct-Lysate reverse transcription and Targeted RNA-Seq) method. DeLTa-Seq is a cost-effective, high-throughput and highly-precise quantification method for the expressions of hundreds of genes. It enables us to conduct large-scale studies using thousands of samples such as chemical screening, field experiments and studies focusing on individual variability.

molecular biology↗

bioRxiv2020

Cryo-EM structure of the MgtE Mg2+ channel pore domain in Mg2+-free conditions reveals cytoplasmic pore opening

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed state and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. However, due to the lack of a structure of the MgtE channel, including its transmembrane domain in Mg2+-free conditions, the pore-opening mechanism of MgtE has remained unclear. Here, we determined the cryoelectron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE transmembrane domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

biophysics↗

bioRxiv2020

Drastic shift in flowering phenology, an instant reproductive isolation mechanism, explains the population structure of Imperata cylindrica in Japan

Reproductive isolation plays an important role in population differentiation and speciation, thus enhancing biodiversity in wild plants. Hybridisation sometimes involves rapid reproductive isolation between parents and their hybrids through the novel traits of hybrids derived from a new combination of genomes. Here, we report how a hybrids new phenotype contributes to rapid reproductive isolation between two ecotypes of Imperata cylindrica. The two ecotypes differ in their flowering phenology and habitats. An analysis with genetic markers revealed that hybrid populations consisted of only F1 individuals. Both parental ecotypes flowered in spring, but F1s flowered in fall. This drastic shift in flowering phenology prevented backcrossing parental ecotypes to F1. F1s flowered in fall and dispersed seeds in winter. The germination percentage of seeds set on F1 was extremely low in their habitats, and seedlings did not survive due to the low temperatures in winter, resulting in the absence of a F2 generation. In conclusion, flowering phenology mismatch promotes reproductive isolation between parents and F1, resulting in a hybrid population consisting of only F1s.

evolutionary biology↗

bioRxiv2020

Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes

Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1-7 complex initiates mRNA decay, while the nuclear Lsm2-8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2-8 bound to RNA identify the unique 2',3' cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1-7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1-7 loads onto RNA from the 3' end and removal of the Lsm1 C-terminal region allows Lsm1-7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites. These data reveal the molecular basis for RNA binding by Lsm proteins, a fundamental step in the formation of molecular assemblies that are central to eukaryotic mRNA metabolism.

biochemistry↗

bioRxiv2019

MIR3607 regulates cerebral cortex development via activation of Wnt/βCat signaling

The evolutionary expansion of the mammalian cerebral cortex is recapitulated during embryonic development in large mammals, but the underlying genetic mechanisms remain mostly unknown. Previous transcriptomic analyses of the developing ferret cortex identify candidate genes related to the expansion of germinal layers and cortex size. Here we focused on MIR3607, a microRNA differentially expressed between germinal layers of the large human and ferret cortex, not expressed in the small mouse cortex. Expression of MIR3607 in mouse embryos at E14.5 leads to increased progenitor cell proliferation. This is reflected in transcriptomic changes, which also reveal increased Wnt/{beta}Catenin signaling. Expression of MIR3607 at E12.5, when progenitor cells expand, causes amplification and severe delamination of apical progenitors, leading to rosette formation. This is rescued by co-expressing Adenomatous Polyposis Coli, inhibitor of canonical Wnt signaling. A similar phenotype is produced in human cerebral organoids. Our findings demonstrate that MIR3607 expands and delaminates apical progenitor cells via activating Wnt/{beta}Catenin, and suggest that a secondary loss of expression in mouse may underlie their reduction in cortex size during recent evolution.

developmental biology↗