PRMT5-mediated arginine methylation of FXR1 is essential for RNA binding in cancer cells.

Emerging evidence indicates that arginine methylation promotes the stability of arginine-glycine-rich (RGG) motif-containing RNA-binding proteins (RBPs) and regulates gene expression. Here, we report that post-translational modification of FXR1 enhances the binding with mRNAs and is involved in cancer cell growth and proliferation. Independent point mutations in arginine residues of FXR1's nuclear export signal (R386 and R388) and RGG (R453, R455 and R459) domains prevent it from binding to RNAs that form G-quadruplex (G4) RNA structures. Disruption of G4-RNA structures by lithium chloride failed to bind with FXR1, indicating its preference for G4-RNA structure containing mRNAs. Furthermore, loss-of-function of PRMT5 inhibited FXR1 methylation both in vivo and in vitro, affecting FXR1 protein stability, inhibiting RNA-binding activity and cancer cell growth and proliferation. Finally, the enhanced crosslinking and immunoprecipitation (eCLIP) analyses reveal that FXR1 binds with the G4-enriched mRNA targets such as AHNAK, MAP1B, AHNAK2, HUWE1, DYNC1H1 and UBR4 and controls its mRNA expression in cancer cells. Our findings suggest that PRMT5-mediated FXR1 methylation is required for RNA/G4-RNA binding, which promotes gene expression in cancer cells. Thus, FXR1's structural characteristics and affinity for RNAs preferentially G4 regions provide new insights into the molecular mechanism of FXR1 in oral cancer cells.

RNA-binding protein HuR reprograms immune T cells and promotes oral squamous cell carcinoma.

Hu Antigen R, also known as ELAVL1 (HuR), is a key posttranscriptional regulator in eukaryotic cells. HuR overexpression promotes several malignancies, including head and neck squamous cell carcinoma (HNSCC). However, its immune dysfunction-associated tumorigenesis pathways remain unknown. We examined HuR's effects on oral malignancies and immune cell function in vitro and in vivo using oral carcinoma cells and transgenic HuR knockout (KO) mice. CRISPR/Cas9-mediated HuR deletion in mice syngeneic oral cancer cells eliminated colony formation and tumor development. HuR-KO tumors had a lower tumor volume, fewer CD4+CD25+FoxP3+ regulatory T cells, and more CD8+ T cells, suggesting that HuR may suppress the immune response during oral cancer progression. In contrast, HuR KO oral epithelial tissues are resistant to 4NQO-induced oral malignancies compared to control tumor-bearing mice. HuR KO mice showed fewer Tregs and greater IFN levels than WT tumor-bearing mice, suggesting anticancer activity. Finally, the HuR inhibitor pyrvinium pamoate lowers tumor burden by enhancing CD8+ infiltration at the expense of CD4+, suggesting anticancer benefits. Thus, HuR-dependent oral neoplasia relies on immunological dysfunction, suggesting that decreasing HuR may boost antitumor potential and offer a novel HNSCC therapy.

Leaf lipidome and transcriptome profiling of Portulaca oleracea: characterization of lysophosphatidylcholine acyltransferase.

MAIN CONCLUSION: Portulaca leaves serve as an alternative bioresource for edible PUFAs. Transcriptome data provide information to explore Portulaca as a model system for galactolipids, leaf lipid metabolism, and PUFA-rich designer lipids. Poly-unsaturated fatty acids (PUFAs) are gaining importance due to their innumerable health benefits, and hence, understanding their biosynthesis in plants has attained prominence in recent years. The most common source of PUFAs is of marine origin. Although reports have identified Portulaca oleracea (purslane) as a leaf source of omega-3 fatty acids in the form of alpha-linolenic acid (ALA), the mechanism of ALA accumulation and its distribution into various lipids has not been elucidated. Here, we present the lipid profiles of leaves and seeds of several accessions of P. oleracea. Among the nineteen distinct accessions, the RR04 accession has the highest amount of ALA and is primarily associated with galactolipids. In addition, we report the transcriptome of RR04, and we have mapped the potential genes involved in lipid metabolism. Phosphatidylcholine (PC) is the major site of acyl editing, which is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT), an integral membrane protein that plays a major role in supplying oleate to the PC pool for further unsaturation. Our investigations using mass spectrometric analysis of leaf microsomal fractions identified LPCAT as part of a membrane protein complex. Both native and recombinant LPCAT showed strong acyltransferase activity with various acyl-CoA substrates. Altogether, the results suggest that ALA-rich glycerolipid biosynthetic machinery is highly active in nutritionally important Portulaca leaves. Furthermore, lipidome, transcriptome, and mass spectrometric analyses of RR04 provide novel information for exploring Portulaca as a potential resource and a model system for studying leaf lipid metabolism.

Emerging themes in heterotrimeric G-protein signaling in plants.

Heterotrimeric G-proteins are key signaling components involved during the regulation of a multitude of growth and developmental pathways in all eukaryotes. Although the core proteins (Gα, Gß, Gγ subunits) and their basic biochemistries are conserved between plants and non-plant systems, seemingly different inherent properties of specific components, altered wirings of G-protein network architectures, and the presence of novel receptors and effector proteins make plant G-protein signaling mechanisms somewhat distinct from the well-established animal paradigm. G-protein research in plants is getting a lot of attention recently due to the emerging roles of these proteins in controlling many agronomically important traits. New findings on both canonical and novel G-protein components and their conserved and unique signaling mechanisms are expected to improve our understanding of this important module in affecting critical plant growth and development pathways and eventually their utilization to produce plants for the future needs. In this review, we briefly summarize what is currently known in plant G-protein research, describe new findings and how they are changing our perceptions of the field, and discuss important issues that still need to be addressed.

Arabidopsis Type III Gγ Protein AGG3 Is a Positive Regulator of Yield and Stress Responses in the Model Monocot Setaria viridis.

Heterotrimeric G-proteins are key regulators of a multitude of growth and development pathways in eukaryotes. Along with the conserved G-protein components found in all organisms, plants have certain novel variants with unique architecture, which may be involved in the regulation of plant-specific traits. The higher plant-specific type III (or Class C) Gγ protein, which possesses a large C terminal extension, represented by AGG3 in Arabidopsis, is one such variant of canonical Gγ proteins. The type III Gγ proteins are involved in regulation of many agronomically important traits in plants, including seed yield, organ size regulation, abscisic acid (ABA)-dependent signaling and stress responses, and nitrogen use efficiency. However, the extant data, especially in the monocots, present a relatively complex and sometimes contradictory picture of the regulatory role of these proteins. It remains unclear if the positive traits observed in certain naturally occurring populations are due to the presence of specific allelic variants of the proteins or due to the altered expression of the gene itself. To address these possibilities, we have overexpressed the Arabidopsis AGG3 gene in the model monocot Setaria viridis and systematically evaluated its role in conferring agriculturally relevant phenotypes. Our data show that AtAGG3 is indeed functional in Setaria and suggest that a subset of the traits affected by the type III Gγ proteins are indeed positively correlated with the gene expression level, while others might have more complex, allele specific regulation.

Arabidopsis serine/threonine/tyrosine protein kinase phosphorylates oil body proteins that regulate oil content in the seeds.

Protein phosphorylation is an important post-translational modification that can regulate the protein function. The current knowledge on the phosphorylation status of plant oil body (OB) proteins is inadequate. This present study identifies the distinct physiological substrates of Arabidopsis serine/threonine/tyrosine protein kinase (STYK) and its role in seed oil accumulation; the role of Arabidopsis OLE1, a major seed OB protein has also been elucidated. In vitro kinase assay followed by mass spectrometry identifies residue that are phosphorylated by STYK. Further, co-expression of OLE1 and STYK in yeast cells increases the cellular lipid levels and reduces the total lipid when OLE1 was replaced with OLE1T166A. Moreover, in vivo experiments with OB isolated from wild-type and styk knock-out lines show the ability of STYK to phosphorylate distinct OB proteins. OLE1T166A mutant and Arabidopsis styk mutant demonstrate the significant reduction of its substrate phosphorylation. styk mutant line significantly reduces the amount of total seed oil as compared to wild-type seeds. Together, our results provide the evidences that Arabidopsis At2G24360 (STYK) is phosphorylating oil body proteins and the phosphorylation regulates the oil content in Arabidopsis seeds.

Optimization of Phenotyping Assays for the Model Monocot Setaria viridis.

Setaria viridis (green foxtail) is an important model plant for the study of C4 photosynthesis in panicoid grasses, and is fast emerging as a system of choice for the study of plant development, domestication, abiotic stress responses and evolution. Basic research findings in Setaria are expected to advance research not only in this species and its close relative S. italica (foxtail millet), but also in other panicoid grasses, many of which are important food or bioenergy crops. Here we report on the standardization of multiple growth and development assays for S. viridis under controlled conditions, and in response to several phytohormones and abiotic stresses. We optimized these assays at three different stages of the plant's life: seed germination and post-germination growth using agar plate-based assays, early seedling growth and development using germination pouch-based assays, and adult plant growth and development under environmentally controlled growth chambers and greenhouses. These assays will be useful for the community to perform large scale phenotyping analyses, mutant screens, comparative physiological analysis, and functional characterization of novel genes of Setaria or other related agricultural crops. Precise description of various growth conditions, effective treatment conditions and description of the resultant phenotypes will help expand the use of S. viridis as an effective model system.

The Arabidopsis ABHD11 Mutant Accumulates Polar Lipids in Leaves as a Consequence of Absent Acylhydrolase Activity.

Alpha/beta hydrolase domain (ABHD)-containing proteins are structurally related with diverse catalytic activities. In various species, some ABHD proteins have been characterized and shown to play roles in lipid homeostasis. However, little is known about ABHD proteins in plants. Here, we characterized AT4G10030 (AtABHD11), an Arabidopsis (Arabidopsis thaliana) homolog of a human ABHD11 gene. In silico analyses of AtABHD11 revealed homology with other plant species with a conserved GXSXG lipid motif. Interestingly, Arabidopsis abhd11 mutant plants exhibited an enhanced growth rate compared with wild-type plants. Quantitative analyses of the total lipids showed that the mutant abhd11 has a high amount of phospholipid and galactolipid in Arabidopsis leaves. The overexpression of AtABHD11 in Escherichia coli led to a reduction in phospholipid levels. The bacterially expressed recombinant AtABHD11 hydrolyzed lyso(phospho)lipid and monoacylglycerol. Furthermore, using whole-genome microarray and real-time PCR analyses of abhd11 and wild-type plants, we noted the up-regulation of MGD1, -2, and -3 and DGD1. Together, these findings suggested that AtABHD11 is a lyso(phospho)lipase. The disruption of AtABHD11 caused the accumulation of the polar lipids in leaves, which in turn promoted a higher growth rate compared with wild-type plants.

A bifunctional enzyme that has both monoacylglycerol acyltransferase and acyl hydrolase activities.

Monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, the precursor of triacylglycerol biosynthesis and an important signaling molecule. Here, we describe the isolation and characterization of the peanut (Arachis hypogaea) MGAT gene. The soluble enzyme utilizes invariant histidine-62 and aspartate-67 residues of the acyltransferase motif for its MGAT activity. A sequence analysis revealed the presence of a hydrolase (GXSXG) motif, and enzyme assays revealed the presence of monoacylglycerol (MAG) and lysophosphatidylcholine (LPC) hydrolytic activities, indicating the bifunctional nature of the enzyme. The overexpression of the MGAT gene in yeast (Saccharomyces cerevisiae) caused an increase in triacylglycerol accumulation. Similar to the peanut MGAT, the Arabidopsis (Arabidopsis thaliana) homolog (At1g52760) also exhibited both acyltransferase and hydrolase activities. Interestingly, the yeast homolog lacks the conserved HX(4)D motif, and it is deficient in the acyltransferase function but exhibits MAG and LPC hydrolase activities. This study demonstrates the presence of a soluble MGAT/hydrolase in plants. The predicted three-dimensional homology modeling and substrate docking suggested the presence of two separate substrate (MAG and LPC)-binding sites in a single polypeptide. Our study describes a soluble bifunctional enzyme that has both MGAT and hydrolase functions.

Serine/threonine/tyrosine protein kinase phosphorylates oleosin, a regulator of lipid metabolic functions.

Plant oils are stored in oleosomes or oil bodies, which are surrounded by a monolayer of phospholipids embedded with oleosin proteins that stabilize the structure. Recently, a structural protein, Oleosin3 (OLE3), was shown to exhibit both monoacylglycerol acyltransferase and phospholipase A(2) activities. The regulation of these distinct dual activities in a single protein is unclear. Here, we report that a serine/threonine/tyrosine protein kinase phosphorylates oleosin. Using bimolecular fluorescence complementation analysis, we demonstrate that this kinase interacts with OLE3 and that the fluorescence was associated with chloroplasts. Oleosin-green fluorescent protein fusion protein was exclusively associated with the chloroplasts. Phosphorylated OLE3 exhibited reduced monoacylglycerol acyltransferase and increased phospholipase A(2) activities. Moreover, phosphatidylcholine and diacylglycerol activated oleosin phosphorylation, whereas lysophosphatidylcholine, oleic acid, and Ca(2+) inhibited phosphorylation. In addition, recombinant peanut (Arachis hypogaea) kinase was determined to predominantly phosphorylate serine residues, specifically serine-18 in OLE3. Phosphorylation levels of OLE3 during seed germination were determined to be higher than in developing peanut seeds. These findings provide direct evidence for the in vivo substrate selectivity of the dual-specificity kinase and demonstrate that the bifunctional activities of oleosin are regulated by phosphorylation.

Effects of three adhesion boosters on the shear bond strength of new and rebonded brackets--an in vitro study.

AIM: To evaluate the effects of three adhesion boosters--All-Bond 2, Enhance LC, and Ortho Solo--on the shear bond strength of new and rebonded (previously debonded) brackets. METHODS: One hundred new and 100 sandblasted debonded brackets were bonded to 200 extracted human premolars and divided into eight groups. RESULTS: The new brackets/Ortho Solo group yielded the highest bond strength, followed by the new brackets/All-Bond 2 and the new brackets/Enhance LC groups. During rebonding, Ortho Solo improved the bond strength significantly; however, All-Bond 2 and Enhance LC did not. CONCLUSION: (1) Bond strength is significantly improved when new brackets are bonded with an adhesion booster; (2) without any adhesion booster, sandblasted rebonded brackets yield a significantly lower bond strength than new brackets; (3) Enhance LC failed to improve the bond strength of rebonded brackets; (4) Ortho Solo increased the bond strength of rebonded brackets significantly; and (5) brackets rebonded with Ortho Solo yielded comparable bond strength as new brackets without any adhesion booster.