RNA structure-wide discovery of functional interactions with multiplexed RNA motif library

RNA構造のライブラリ化を通じてRNA 構造ごとにおけるRNA-タンパク質相互作用を大規模に解析するシステム「FOREST」の開発 --RNAを標的とする創薬に道--. 京都大学プレスリリース. 2020-12-09.Biochemical assays and computational analyses have discovered RNA structures throughout various transcripts. However, the roles of these structures are mostly unknown. Here we develop folded RNA element profiling with structure library (FOREST), a multiplexed affinity assay system to identify functional interactions from transcriptome-wide RNA structure datasets. We generate an RNA structure library by extracting validated or predicted RNA motifs from gene-annotated RNA regions. The RNA structure library with an affinity enrichment assay allows for the comprehensive identification of target-binding RNA sequences and structures in a high-throughput manner. As a proof-of-concept, FOREST discovers multiple RNA-protein interaction networks with quantitative scores, including translational regulatory elements that function in living cells. Moreover, FOREST reveals different binding landscapes of RNA G-quadruplex (rG4) structures-binding proteins and discovers rG4 structures in the terminal loops of precursor microRNAs. Overall, FOREST serves as a versatile platform to investigate RNA structure-function relationships on a large scale

Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires a Bicc1-Ccr4 RNA degradation complex

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3′ untranslated region (3′-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3′-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3′-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node

Surface structures of binary mixtures of imidazolium-based ionic liquids using high-resolution Rutherford backscattering spectroscopy and time of flight secondary ion mass spectroscopy.

Surface structures of binary mixtures of imidazolium-based ionic liquids having a common anion (bis(trifluoromethanesulfonyl)imide ([TFSI]), namely [C2MIM]1-x[C10MIM]x[TFSI] (x = 0.5 and 0.1), are studied using high-resolution Rutherford backscattering spectroscopy (HRBS) and time of flight secondary ion mass spectroscopy (TOF-SIMS). Although both measurements show surface segregation of [C10MIM] the degrees of the segregation are different. The surface fraction xsurf of [C10MIM] is estimated to be 0.6 ± 0.05 and 0.18 ± 0.02 by HRBS for x = 0.5 and 0.1, respectively. On the other hand, TOF-SIMS indicates much stronger surface segregation, namely xsurf = 0.83 ± 0.03 and 0.42 ± 0.04 for x = 0.5 and 0.1, respectively. The observed discrepancy can be attributed to the difference in the probing depth between HRBS and TOF-SIMS. The observed surface segregation can be roughly explained in terms of surface tension

Direct Numerical Simulation of the Neutrally Stratified Turbulent Ekman Boundary Layer

based on the geostrophic wind G, the kinematic viscosity ν and Coriolis parameter f. The simulations of Ref = 1140 and 1393 have been carried out on the Earth Simulator. In the cases of the higher Reynolds numbers, the mean velocity profiles show good agreement with the experimental data of Caldwell et al. (1972) and the DNS data of Coleman (1999). A low-speed large-scale structure is found in the upper region, whereas the well-known streaky structure appears in the vicinity of the wall. This large-scale structure is observed in a motion of a material line. The inclination of this structure does not coincide with the direction of the mean flow velocity. The reason is discussed based upon the motion of a material line whose initial position is horizontal in a vicinity of the wall. In addition, the eddy-viscosity model proposed by Blackadar (1962, 1965) is examined with respect to the obtained DNS data base. The improved model gives good agreement with the results by the present DNS

Electroextraction of Insoluble Proteins from the Organic Matrix of the Nacreous Layer of the Japanese Pearl Oyster, <i>Pinctada fucata</i>

The nacreous layer of shells and pearls is composed of aragonite crystals arranged in an organic matrix. The organic matrix contains chitin and several proteins that regulate the formation of the nacreous layer. Owing to their strong interactions in the organic matrix, the current method for extraction of insoluble proteins from the pre-powdered nacreous layer involves heating to high temperatures in the presence of a detergent (e.g., sodium dodecyl sulfate, SDS) and reductant (e.g., dithiothreitol, DTT), which is likely to induce protein degradation. Therefore, we have developed an electroextraction method to isolate proteins from the organic matrix of a nacreous organic sheet, that was obtained following the decalcification of shells in their original shape. Our electroextraction method employs milder conditions without heating or detergent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the electro-extracted proteins (EEPs) under non-reduced and reduced conditions revealed that this method yielded a greater number of different proteins compared with the conventional extraction method and the isolated EEPs retained their disulfide bonds. Our method is able to easily extract insoluble proteins from the nacreous layer under mild conditions and will undoubtedly aid future analyses into the functions of the nacreous layer proteins

A novel heterozygous ITGB3 p.T720del inducing spontaneous activation of integrin αIIbβ3 in autosomal dominant macrothrombocytopenia with aggregation dysfunction

We identified a novel heterozygous ITGB3 p.T720del mutation in a pedigree with macrothrombocytopenia exhibiting aggregation dysfunction. Platelet aggregation induced by ADP and collagen was significantly reduced, while ristocetin aggregation was normal. Integrin αIIbβ3 was partially activated in a resting status, but platelet expression of αIIbβ3 was downregulated. Functional analysis using a cell line showed spontaneous phosphorylation of FAK in αIIb/β3 (p.T720del)-transfected 293T cells in suspension conditions. Abnormal cytoplasmic protrusions, membrane ruffling, and cytoplasmic localization of αIIbβ3 were observed in αIIb/β3 (p.T720del)-transfected CHO cells. Such morphological changes were reversed by treatment with an FAK inhibitor. These findings imply spontaneous, but partial, activation of αIIbβ3 followed by phosphorylation of FAK as the initial mechanism of abnormal thrombopoiesis. Internalization and decreased surface expression of αIIbβ3 would contribute to aggregation dysfunction. We reviewed the literature of congenital macrothrombocytopenia associated with heterozygous ITGA2B or ITGB3 mutations. Reported mutations were highly clustered at the membrane proximal region of αIIbβ3, which affected the critical interaction between αIIb R995 and β3 D723, resulting in a constitutionally active form of the αIIbβ3 complex. Macrothrombocytopenia caused by a heterozygous activating mutation of ITGA2B or ITGB3 at the membrane proximal region forms a distinct entity of rare congenital thrombocytopenia

Prenatal PFAAs exposure and IGF2/H19 methylation

Prenatal exposure to perfluoroalkyl acids (PFAAs) influences fetal growth and long-term health. However, whether PFAAs affect offspring DNA methylation patterns to influence health outcomes is yet to be evaluated. Here, we assessed effect of prenatal PFAA exposure on cord blood insulin-like growth factor 2 (IGF2), H19, and long interspersed element 1 (LINE1) methylation and its associations with birth size. Mother-child pairs (N=177) from the Hokkaido Study on Environment and Children's Health were included in the study. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) levels in maternal serum were measured by liquid chromatography-tandem mass spectrometry. IGF2, H19, and LINE1 methylation in cord blood DNA was determined by pyrosequencing. After full adjustment in multiple linear regression models, IGF2 methylation showed a significant negative association with log-unit increase in PFOA (partial regression coefficient=-0.73; 95% confidence interval: -1.44 to -0.02). Mediation analysis suggested that reduced IGF2 methylation explained approximately 21% of the observed association between PFOA exposure and reduced ponderal index of the infant at birth. These results indicated that the effects of prenatal PFOA exposure could be mediated through DNA methylation. Further study will be required to determine the potential for long-term adverse health effects of reduced IGF2 methylation induced by PFOA exposure