Three-dimensional architecture of ESCRT-III flat spirals on the membrane.

The endosomal sorting complexes required for transport (ESCRTs) are responsible for membrane remodeling in many cellular processes, such as multivesicular body biogenesis, viral budding, and cytokinetic abscission. ESCRT-III, the most abundant ESCRT subunit, assembles into flat spirals as the primed state, essential to initiate membrane invagination. However, the three-dimensional architecture of ESCRT-III flat spirals remained vague for decades due to highly curved filaments with a small diameter and a single preferred orientation on the membrane. Here, we unveiled that yeast Snf7, a component of ESCRT-III, forms flat spirals on the lipid monolayers using cryogenic electron microscopy. We developed a geometry-constrained Euler angle-assigned reconstruction strategy and obtained moderate-resolution structures of Snf7 flat spirals with varying curvatures. Our analyses showed that Snf7 subunits recline on the membrane with N-terminal motifs α0 as anchors, adopt an open state with fused α2/3 helices, and bend α2/3 gradually from the outer to inner parts of flat spirals. In all, we provide the orientation and conformations of ESCRT-III flat spirals on the membrane and unveil the underlying assembly mechanism, which will serve as the initial step in understanding how ESCRTs drive membrane abscission.

Transcriptomic and free monoterpene analyses of aroma reveal that isopentenyl diphosphate isomerase inhibits monoterpene biosynthesis in grape (Vitis vinifera L.).

BACKGROUND: Monoterpenes are among the most important volatile aromatic compounds contributing to the flavor and aroma of grapes and wine. However, the molecular basis of monoterpene biosynthesis has not yet been fully elucidated. RESULTS: In our study, transcriptomics and gas chromatography-mass spectrometry (GC-MS) were used to mine candidate genes and transcription factors involved in monoterpene biosynthesis between high-monoterpene and zero-monoterpene table grape cultivars. We found that monoterpene biosynthesis was positively correlated by the expression of five genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (VvDXSs), one encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (VvHDR), three hydroxy-3-methylglutaryl-CoA synthases (VvHMGSs) and one mevalonate kinase (VvMVK), whereas the expression of one isopentenyl diphosphate isomerase (VvIDI) and one 3-hydroxy-3-methylglutaryl-CoA reductase (VvHMGR) negatively correlated monoterpene biosynthesis. Of these genes, VvIDI was selected to validate its function in monoterpene accumulation through a transient overexpression experiment, and was shown to inhibit the biosynthesis of grape linalool and α-terpineol. Meanwhile, we found that a 64-amino acid extension sequence at the N-terminus can guide the VvIDI protein to target the chloroplast. CONCLUSIONS: The findings of this study should help to guide future functional analysis of key genes as well as mining the potential regulatory mechanism of monoterpene biosynthesis in grapes and grape products.

LncRNA RP11-301G19.1 is required for the maintenance of vascular smooth muscle cell contractile phenotype via sponging miR-17-5P/ATOH8 axis.

Long noncoding RNAs (LncRNAs) play essential roles in regulating gene expression in various biological processes. However, the function of lncRNAs in vascular smooth muscle cell (VSMC) transformation remains to be explained. In this work, we discover that a new bone marrow protein (BMP) signaling target, lncRNA RP11-301G19.1, is significantly induced in BMP7-treated VSMCs through lncRNA microarray analysis. Addition of BMP signaling inhibitor LDN-193189 attenuates the expression of ACTA2 and SM-22α, as well as the mRNA level of RP11-301G19.1. Furthermore, lncRNA RP11-301G19.1 is critical to the VSMC differentiation and is directly activated by SMAD1/9. Mechanistically, knocking down of RP11-301G19.1 leads to the decrease of ATOH8, another BMP target, while the forced expression of RP11-301G19.1 reactivates ATOH8. In addition, miR-17-5p, a miRNA negatively regulated by BMP-7, contains predicted binding sites for lncRNA RP11-301G19.1 and ATOH8 3'UTR. Accordingly, overexpression of miR-17-5p decreases the levels of them. Together, our results revealed the role of lncRNA RP11-301G19.1 as a miRNA sponge to upregulate ATOH8 in VSMC phenotype transformation.

Target of rapamycin (TOR) plays a role in regulating ROS-induced chloroplast damage during cucumber (Cucumis sativus) leaf senescence.

In cucumber production, delaying leaf senescence is crucial for improving cucumber yield and quality. Target of rapamycin (TOR) is a highly conserved serine/threonine protein kinase in eukaryotes, which can integrate exogenous and endogenous signals (such as cell energy state levels) to stimulate cell growth, proliferation, and differentiation. However, no studies have yet examined the regulatory role of TOR signalling in cucumber leaf senescence. In this study, the effects of TOR signalling on dark-induced cucumber leaf senescence were investigated using the TOR activator MHY1485 and inhibitor AZD8055 combined with transient transformation techniques. The results indicate that TOR responds to dark-induced leaf senescence, and alterations in TOR activity/expression influence cucumber leaf resistance to dark-induced senescence. Specifically, in plants with elevated TOR activity/expression, we observed reduced expression of senescence-related genes, less membrane lipid damage, decreased cell apoptosis, lower levels of reactive oxygen species production, and less damage to the photosynthetic system compared to the control. In contrast, in plants with reduced TOR activity/expression, we observed higher expression of senescence-related genes, increased membrane lipid damage, enhanced cell apoptosis, elevated levels of reactive oxygen species production, and more damage to the photosynthetic system. These comprehensive results underscore the critical role of TOR in regulating dark-induced cucumber leaf senescence. These findings provide a foundation for controlling premature leaf senescence in cucumber production and offer insights for further exploration of leaf senescence mechanisms and the development of more effective control methods.

Knockdown of DNMT1 and DNMT3a Promotes the Angiogenesis of Human Mesenchymal Stem Cells Leading to Arterial Specific Differentiation.

Human mesenchymal stem cells (hMSCs) possess the potential to differentiate into endothelial cells (EC). DNA methylation plays an important role in cell differentiation during development. However, the role of the DNA methyltransferases Dnmt1 and Dnmt3a in specific arterial differentiation of hMSCs is not clear. Here, we show that the CpG islands in the promoter regions of the EC specification and arterial marker genes were highly methylated in hMSCs based on bisulfite genomic sequencing. Treatment with the DNMT inhibitor 5-aza-dc induced the reactivation of EC specification and arterial marker genes by promoting demethylation of these genes as well as stimulating tube-like structure formation. The hMSCs with stable knockdown of Dnmt1/Dnmt3a were highly angiogenic and expressed several arterial specific transcription factors and marker genes. A Matrigel plug assay confirmed that Dnmt1/Dnmt3a stable knockdown hMSCs enhanced blood vessel formation compared with WT MSCs. We also identified that the transcription factor E2F1 could upregulate the transcription of arterial marker genes by binding to the promoters of arterial genes, suggesting its critical role for arterial specification. Moreover, miRNA gain/loss-of-function analyses revealed that miR152 and miR30a were involved in endothelial differentiation of hMSCs by targeting Dnmt1 and Dnmt3a, respectively. Taken together, these data suggest that Dnmt1 and Dnmt3a are critical regulators for epigenetic silencing of EC marker genes and that E2F1 plays an important role in promoting arterial cell determination. Stem Cells 2016;34:1273-1283.

miR-219a-5p inhibits breast cancer cell migration and epithelial-mesenchymal transition by targeting myocardin-related transcription factor A.

Although many miRNAs are reported to be involved in tumor formation and progression, the effect of miR-219a-5p on breast cancer metastasis is not well-known. The aim of this study is to investigate the effect of miR-219a-5p on the migratory ability and epithelial-mesenchymal transition (EMT) of breast cancer cells. First, miR-219a-5p was found to be highly expressed in low-invasive breast cancer MCF-7 cells, but lowly expressed in high-invasive breast cancer MDA-MB-231 cells. Wound scratch assay and transwell assay showed that miR-219a-5p inhibited the migratory ability of MDA-MB-231 cells. miR-219a-5p also suppressed the cellular EMT, confirmed by suppressing the expression of mesenchymal markers vimentin and N-cadherin and increasing the expression of epithelial marker E-cadherin. Using the epithelial-mesenchymal-epithelial model in MCF-7 cells, we confirmed that the level of miR-219a-5p was highly expressed in epithelial-type cells and lowly expressed in mesenchymal-type cells. Importantly, we identified myocardin-related transcription factor A (MRTF-A) as a novel potential target gene of miR-219a-5p. Overexpression of miR-219a-5p in MDA-MB-231 cells could inhibit the expression of MRTF-A as revealed by real-time PCR and western blot analysis. miR-219a-5p inhibited the transcription of MRTF-A by targeting the 3'UTR of MRTF-A, which was confirmed by wild-type or mutant MRTF-A 3'UTR luciferase reporter system. Furthermore, knockdown of MRTF-A using siRNA for MRTF-A could depress breast cell migration. In conclusion, our present study revealed the tumor suppressive role of miR-219a-5p in regulating breast cancer migration by targeting MRTF-A, suggesting that miR-219a-5p might be a therapeutic target in breast cancer through regulating EMT.

Identification of fruit related microRNAs in cucumber (Cucumis sativus L.) using high-throughput sequencing technology.

MicroRNAs (miRNAs) are approximately 21 nt noncoding RNAs that influence the phenotypes of different species through the post-transcriptional regulation of gene expression. Although many miRNAs have been identified in a few model plants, less is known about miRNAs specific to cucumber (Cucumis sativus L.). In this study, two libraries of cucumber RNA, one based on fruit samples and another based on mixed samples from leaves, stems, and roots, were prepared for deep-sequencing. A total of 110 sequences were matched to known miRNAs in 47 families, while 56 sequences in 46 families are newly identified in cucumber. Of these, 77 known and 44 new miRNAs were differentially expressed, with a fold-change of at least 2 and p-value < 0.05. In addition, we predicted the potential targets of known and new miRNAs. The identification and characterization of known and new miRNAs will enable us to better understand the role of these miRNAs in the formation of cucumber fruit.

Structure and transport mechanism of the human calcium pump SPCA1.

Secretory-pathway Ca2+-ATPases (SPCAs) play critical roles in maintaining Ca2+ homeostasis, but the exact mechanism of SPCAs-mediated Ca2+ transport remains unclear. Here, we determined six cryo-electron microscopy (cryo-EM) structures of human SPCA1 (hSPCA1) in a series of intermediate states, revealing a near-complete conformational cycle. With the aid of molecular dynamics simulations, these structures offer a clear structural basis for Ca2+ entry and release in hSPCA1. We found that hSPCA1 undergoes unique conformational changes during ATP binding and phosphorylation compared to other well-studied P-type II ATPases. In addition, we observed a conformational distortion of the Ca2+-binding site induced by the separation of transmembrane helices 4L and 6, unveiling a distinct Ca2+ release mechanism. Particularly, we determined a structure of the long-sought CaE2P state of P-type IIA ATPases, providing valuable insights into the Ca2+ transport cycle. Together, these findings enhance our understanding of Ca2+ transport by hSPCA1 and broaden our knowledge of P-type ATPases.

Transcriptome Sequence Analysis of the Defense Responses of Resistant and Susceptible Cucumber Strains to Podosphaera xanthii.

Powdery mildew (PM) caused by Podosphaera xanthii poses a continuous threat to the performance and yield of the cucumber (Cucumis sativus L.). Control in the initial stages of infection is particularly important. Here, we studied the differential physiological and transcriptomic changes between PM-resistant strain B21-a-2-1-2 and PM-susceptible strain B21-a-2-2-2 at the early stage of P. xanthii attack. When challenged with P. xanthii, the tolerant line can postpone the formation of the pathogen primary germ. Comparative transcriptomic analysis suggested that DEGs related to the cell wall and to pathogen and hormone responses were similar enriched in both cucumber lines under P. xanthii infection. Notably, the number of DEGs triggered by P. xanthii in B21-a-2-1-2 was quintuple that in B21-a-2-2-2, revealing that the success of defense of resistant cucumber is due to rapidly mobilizing multiple responses. The unique responses detected were genes related to SA signaling, MAPK signaling, and Dof and WRKY transcription factors. Furthermore, 5 P. xanthii -inducible hub genes were identified, including GLPK, ILK1, EIN2, BCDHß1, and RGGA, which are considered to be key candidate genes for disease control. This study combined multiple analytical approaches to capture potential molecular players and will provide key resources for developing cucumber cultivars resistant to pathogen stress.

Transcriptome analysis reveals the effects of grafting on sugar and α-linolenic acid metabolisms in fruits of cucumber with two different rootstocks.

Flavor quality in cucumber is affected by different rootstocks, but the molecular mechanism is largely unclean. To clarify the differences of sugar and aromatic compounds, cucumber (cucumis sativus) fruits from plants of self-grafted (SG) or grafted onto figleaf gourd (Cucurbita ficifolia; G1) or 'Weisheng No.1' rootstock (Cucurbita moschata ⅹCucurbita moschata hybrids; G2) were performed the transcriptome analysis. We obtained 1013 and 920 differentially expressed genes (DEGs) from G1 and G2 compared to SG respectively, in which 453 genes were co-expressed. Functional annotations showed many DEGs were involved in glycolysis/gluconeogenesis metabolism, fructose metabolism and α-Linolenic acid metabolisms, 20 DEGs were selected from the 3 pathways to validate sequencing accuracy by quantitative real-time PCR. The gene relative expression levels were concurrent with RNA-seq results and sugar and aromatic compounds content phenotypes. Moreover, some vital transcript factors and transport proteins were analyzed. These findings indicate that different rootstocks could induce significantly changes in the physiological profiling and transcripts of sugar- and aromatic flavor-related genes. This study provides a novel insight into the molecular mechanisms of fruit quality regulated by candidate genes.

The Two Translationally Controlled Tumor Protein Genes, CsTCTP1 and CsTCTP2, Are Negative Modulators in the Cucumis sativus Defense Response to Sphaerotheca fuliginea.

Pathogen stress often significantly decreases cucumber production. However, knowledge regarding the molecular mechanism and signals of cucumber disease resistance is far from complete. Here, we report two translationally controlled tumor protein genes, CsTCTP1 and CsTCTP2, that are both negative modulators in the Cucumis sativus defense response to Sphaerotheca fuliginea. Subcellular localization analysis showed that CsTCTP1 and CsTCTP2 were both localized in the cytoplasm. Expression analysis indicated that the transcript levels of CsTCTP1 and CsTCTP2 were linked to the degree of cucumber resistance to S. fuliginea. Transient overexpression of either CsTCTP1 or CsTCTP2 in cucumber cotyledons impaired resistance to S. fuliginea, whereas silencing of either CsTCTP1 or CsTCTP2 enhanced cucumber resistance to S. fuliginea. The relationship of several defense-related genes and ABA and target of rapamycin (TOR) signaling pathway-related genes to the overexpressing and silencing of CsTCTP1/CsTCTP2 in non-infested cucumber plants was investigated. The results indicated that CsTCTP1 participates in the defense response to S. fuliginea by regulating the expression of certain defense-associated genes and/or ABA signaling pathway-associated genes, and CsTCTP2 participates through regulating the expression of TOR signaling pathway-associated genes. Our findings will guide enhancing the resistance of cucumber to powdery mildew.

LncRNA MALAT1 regulates smooth muscle cell phenotype switch via activation of autophagy.

Vascular smooth muscle cells (VSMCs), switching from a differentiated to a proliferative phenotype, contribute to various vascular diseases. However, the role of long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 MALAT1 in the phenotype switching of VSMCs remains unclear. Here, we report that the knockdown of MALAT1 promotes the transformation of smooth muscle cells from a proliferative phenotype to a differentiated phenotype. MALAT1 knockdown inhibited cellular proliferation and migration, leading to significant cell cycle arrest in the G2 phase. MALAT1 was downregulated in bone morphogenetic protein-7 (BMP-7)-induced cellular differentiation, while MALAT1 was upregulated in platelet-derived growth factor-BB (PDGF-BB)-induced cellular proliferation. PDGF induced the transformation of smooth muscle cells into a proliferative phenotype accompanied by an increase in autophagy. The downregulation of MALAT1 attenuated PDGF-BB-induced proliferation and migration by inhibiting autophagy. MALAT1 could act as a competing endogenous RNA (ceRNA) to regulate autophagy-related 7 (ATG7) gene expression by sponging miR142-3p. The present study reveals a novel mechanism by which MALAT1 promotes the transformation of smooth muscle cells from contraction to synthetic phenotypes.

MicroRNA-29a-3p attenuates ET-1-induced hypertrophic responses in H9c2 cardiomyocytes.

Transcription factor nuclear factor of activated T cells c4 (NFATc4) is the best-characterized target for the development of cardiac hypertrophy. Aberrant microRNA-29 (miR-29) expression is involved in the development of cardiac fibrosis and congestive heart failure. However, whether miR-29 regulates hypertrophic processes is still not clear. In this study, we investigated the potential functions of miR-29a-3p in endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy. We showed that miR-29a-3p was down-regulated in ET-1-treated H9c2 cardiomyocytes. Overexpression of miR-29a-3p significantly reduced ET-1-induced hypertrophic responses in H9c2 cardiomyocytes, which was accompanied by a decrease in NFATc4 expression. miR-29a-3p targeted directly to the 3'-UTR of NFATc4 mRNA and silenced NFATc4 expression. Our results indicate that miR-29a-3p inhibits ET-1-induced cardiomyocyte hypertrophy via inhibiting NFATc4 expression.