Synthase Family Research Articles

Origin and early evolution of the plant terpene synthase family

As a midsized gene family conserved more by lineage than function, the typical plant terpene synthases (TPSs) could be a valuable tool to examine plant evolution. TPSs are pivotal in biosynthesis of gibberellins and related phytohormones as well as in formation of the extensive arsenal of specialized plant metabolites mediating ecological interactions whose production is often lineage specific. Yet the origin and early evolution of the TPS family is not well understood. Systematic analysis of an array of transcriptomes and sequenced genomes indicated that the TPS family originated after the divergence of land plants from charophytic algae. Phylogenetic and biochemical analyses support the hypothesis that the ancestral TPS gene encoded a bifunctional class I and II diterpene synthase producing the ent-kaurene required for phytohormone production in all extant lineages of land plants. Moreover, the ancestral TPS gene likely underwent duplication at least twice early in land plant evolution. Together these two gave rise to three TPS lineages leading to the extant TPS-c, TPS-e/f, and the remaining TPS (h/d/a/b/g) subfamilies, with the latter dedicated to secondary rather than primary metabolism while the former two contain those genes involved in ent-kaurene production. Nevertheless, parallel evolution from the ent-kaurene–producing class I and class II diterpene synthases has led to roles for TPS-e/f and -c subfamily members in secondary metabolism as well. These results clarify TPS evolutionary history and provide context for the role of these genes in producing the vast diversity of terpenoid natural products observed today in various land plant lineages.

LysX2 is a Mycobacterium tuberculosis membrane protein with an extracytoplasmic MprF-like domain

BackgroundAminoacyl-phosphatidylglycerol (aaPG) synthases are bacterial enzymes that usually catalyze transfer of aminoacyl residues to the plasma membrane phospholipid phosphatidylglycerol (PG). The result is introduction of positive charges onto the cytoplasmic membrane, yielding reduced affinity towards cationic antimicrobial peptides, and increased resistance to acidic environments. Therefore, these enzymes represent an important defense mechanism for many pathogens, including Staphylococcus aureus and Mycobacterium tuberculosis (Mtb), which are known to encode for lysyl-(Lys)-PG synthase MprF and LysX, respectively. Here, we used a combination of bioinformatic, genetic and bacteriological methods to characterize a protein encoded by the Mtb genome, Rv1619, carrying a domain with high similarity to MprF-like domains, suggesting that this protein could be a new aaPG synthase family member. However, unlike homologous domains of MprF and LysX that are positioned in the cytoplasm, we predicted that the MprF-like domain in LysX2 is in the extracytoplasmic region.ResultsUsing genetic fusions to the Escherichia coli proteins PhoA and LacZ of LysX2, we confirmed this unique membrane topology, as well as LysX and MprF as benchmarks. Expression of lysX2 in Mycobacterium smegmatis increased cell resistance to human β-defensin 2 and sodium nitrite, enhanced cell viability and delayed biofilm formation in acidic pH environment. Remarkably, MtLysX2 significantly reduced the negative charge on the bacterial surface upon exposure to an acidic environment. Additionally, we found LysX2 orthologues in major human pathogens and in rapid-growing mycobacteria frequently associated with human infections, but not in environmental and non-pathogenic mycobacteria.ConclusionsOverall, our data suggest that LysX2 is a prototype of a new class within the MprF-like protein family that likely enhances survival of the pathogenic species through its catalytic domain which is exposed to the extracytoplasmic side of the cell membrane and is required to decrease the negative charge on the bacterial surface through a yet uncharacterized mechanism.

Tissue-Specific Expression of the Terpene Synthase Family Genes in Rosa chinensis and Effect of Abiotic Stress Conditions.

Rose (Rosa chinensis) is one of the most famous ornamental plants worldwide, with a variety of colors and fragrances. Terpene synthases (TPSs) play critical roles in the biosynthesis of terpenes. In this work, we report a comprehensive study on the genome-wide identification and characterization of the TPS family in R. chinensis. We identified 49 TPS genes in the R. chinensis genome, and they were grouped into five subfamilies (TPS-a, TPS-b, TPS-c, TPS-g and TPS-e/f). Phylogenetics, gene structure and conserved motif analyses indicated that the RcTPS genes possessed relatively conserved gene structures and the RcTPS proteins contained relatively conserved motifs. Multiple putative cis-acting elements involved in the stress response were identified in the promoter region of RcTPS genes, suggesting that some could be regulated by stress. The expression profile of RcTPS genes showed that they were predominantly expressed in the petals of open flowers, pistils, leaves and roots. Under osmotic and heat stresses, the expression of most RcTPS genes was upregulated. These data provide a useful foundation for deciphering the functional roles of RcTPS genes during plant growth as well as addressing the link between terpene biosynthesis and abiotic stress responses in roses.

Genome-Wide Identification and Expression Analysis of Pseudouridine Synthase Family in Arabidopsis and Maize.

Pseudouridine (Ψ), the isomer of uridine (U), is the most abundant type of RNA modification, which is crucial for gene regulation in various cellular processes. Pseudouridine synthases (PUSs) are the key enzymes for the U-to-Ψ conversion. However, little is known about the genome-wide features and biological function of plant PUSs. In this study, we identified 20 AtPUSs and 22 ZmPUSs from Arabidopsis and maize (Zea mays), respectively. Our phylogenetic analysis indicated that both AtPUSs and ZmPUSs could be clustered into six known subfamilies: RluA, RsuA, TruA, TruB, PUS10, and TruD. RluA subfamily is the largest subfamily in both Arabidopsis and maize. It’s noteworthy that except the canonical XXHRLD-type RluAs, another three conserved RluA variants, including XXNRLD-, XXHQID-, and XXHRLG-type were also identified in those key nodes of vascular plants. Subcellular localization analysis of representative AtPUSs and ZmPUSs in each subfamily revealed that PUS proteins were localized in different organelles including nucleus, cytoplasm and chloroplasts. Transcriptional expression analysis indicated that AtPUSs and ZmPUSs were differentially expressed in various tissues and diversely responsive to abiotic stresses, especially suggesting their potential roles in response to heat and salt stresses. All these results would facilitate the functional identification of these pseudouridylation in the future.

Genome-wide analysis of sucrose synthase family in soybean and their expression in response to abiotic stress and seed development

The sucrose synthase (SS) is an important enzyme family which play a vital role in sugar metabolism to improve the fruit quality of the plants. In many plant species, the members of SS family have been investigated but the detailed information is not available in legumes particularly and Glycine max specifically. In the present study, we found thirteen SS members (GmSS1-GmSS13) in G. max genome. High conserved regions were present in the GmSS sequences that may due to the selection pressure during evolutionary events. The segmental duplication was the major factor to increase the number of GmSS family members. The identified thirteen GmSS genes were divided into Class I, Class II and Class III with variable numbers of genes in each class. The protein interaction of GmSS gave the co-expression of sucrose synthase with glucose-1-phosphate adenylyltransferase while SLAC and REL test found number of positive sites in the coding sequences of SS family members. All the GmSS family members except GmSS7 and few of class III members, were highly expressed in all the soybean tissues. The expression of the class I members decreased during seed development, whireas, the class II members expression increased during the seed developing, may involve in sugar metabolism during seed development. Solexa sequencing libraries of acidic condition (pH 4.2) stress samples showed that the expression of class I GmSS genes increased 1- to 2-folds in treated samples than control. The differential expression pattern was observed between the members of a paralogous. This study provides detailed genome-wide analysis of GmSS family in soybean that will provide new insights for future evolutionary and soybean breeding to improve the plant growth and development.

Differential Expression of 1-Aminocyclopropane-1-carboxylate Synthase and Oxidase Gene Family in Micro-Tom Tomato Leaves and Roots Under Short Daily UV Radiation

Differential Expression of 1-Aminocyclopropane-1-carboxylate Synthase and Oxidase Gene Family in Micro-Tom Tomato Leaves and Roots Under Short Daily UV Radiation

Diagnostic Value of Circulating Antigens in the Serum of Piglets with Experimental Acute Toxoplasmosis.

Toxoplasmosis, caused by Toxoplasma gondii, an apicomplexan parasite, infects all warm-blooded animals, including a third of the human population. Laboratory diagnosis of acute toxoplasmosis is based on the detection of anti-T. gondii IgM and IgG and T. gondii nucleic acid; however, these assays have certain limitations. Circulating Ags (CAgs) are reliable diagnostic indicators of acute infection. In this study, we established a model of acute T. gondii infection in Large White pigs. CAg levels peaked between 3 and 5 d after inoculation, and 28 CAgs were identified using an immunoprecipitation-shotgun approach, among which dolichol-phosphate-mannose synthase family protein (TgDPM), C3HC zinc finger-like protein (TgZFLP3), and ribosomal protein RPL7 (TgRPL7) were selected to further investigate their value in the diagnosis of acute toxoplasmosis. Immunofluorescence assays revealed that TgDPM and TgRPL7 were localized in the membrane surface, while TgZFLP3 was localized in the apical end. Western blotting revealed the presence of the three proteins in the serum during acute infection. Indirect ELISA results indicate that TgZFLP3 is likely to be a novel candidate for the diagnosis of acute toxoplasmosis. However, these three proteins may not be useful as candidate vaccines against toxoplasmosis owing to their low protective ability. In addition, deletion of the zflp3 gene partially attenuated virulence in Kunming mice. Collectively, we identified 28 CAgs in the serum of piglets with experimental acute toxoplasmosis and confirmed that TgZFLP3 is a potential biomarker for acute T. gondii infection. The results of this study provide data to improve the detection efficiency of acute toxoplasmosis.

Structural and functional characterization of β-cyanoalanine synthase from Tetranychus urticae

Tetranychus urticae is a polyphagous spider mite that can feed on more than 1100 plant species including cyanogenic plants. The herbivore genome contains a horizontally acquired gene tetur10g01570 (TuCAS) that was previously shown to participate in cyanide detoxification. To understand the structure and determine the function of TuCAS in T. urticae, crystal structures of the protein with lysine conjugated pyridoxal phosphate (PLP) were determined. These structures reveal extensive TuCAS homology with the β-substituted alanine synthase family, and they show that this enzyme utilizes a similar chemical mechanism involving a stable α-aminoacrylate intermediate in β-cyanoalanine and cysteine synthesis. We demonstrate that TuCAS is more efficient in the synthesis of β-cyanoalanine, which is a product of the detoxification reaction between cysteine and cyanide, than in the biosynthesis of cysteine. Also, the enzyme carries additional enzymatic activities that were not previously described. We show that TuCAS can detoxify cyanide using O-acetyl-L-serine as a substrate, leading to the direct formation of β-cyanoalanine. Moreover, it catalyzes the reaction between the TuCAS-bound α-aminoacrylate intermediate and aromatic compounds with a thiol group. In addition, we have tested several compounds as TuCAS inhibitors. Overall, this study identifies additional functions for TuCAS and provides new molecular insight into the xenobiotic metabolism of T. urticae.

Function and transcriptional regulation of CsKCS20 in the elongation of very-long-chain fatty acids and wax biosynthesis in Citrus sinensis flavedo.

Cuticular wax on plant aerial surfaces plays a vital role in the defense against various stresses, and the genes related to wax metabolism have been well documented in several model plants. However, there is very limited research on the key enzymes and transcription factors (TFs) associated with carbon chain distribution and wax biosynthesis in citrus fruit. In this study, an analysis of wax metabolites indicated that even carbon-chain (C24-C28) metabolites are the dominant wax components in citrus fruit, and a 3-ketoacyl-CoA synthase (KCS) family gene (CsKCS20) plays an important role in the carbon chain distribution during wax biosynthesis in a wax-deficient mutant (MT). Expression of CsKCS20 in yeast indicated that CsKCS20 can catalyze the biosynthesis of C22 and C24 very-long-chain fatty acids (VLCFAs). In addition, transcriptome and sequence analysis indicated that the differential expression of CsKCS20 between the wild-type (WT) and MT fruit can be partly attributed to the regulation of CsMYB96, which was further confirmed by yeast one-hybrid (Y1H) assays, electrophoretic mobility shift assays (EMSAs) and dual luciferase assays. The functions of CsMYB96 and CsKCS20 in wax biosynthesis were further validated by heterologous expression in Arabidopsis. In summary, this study elucidates the important roles of CsKCS20 and CsMYB96 in regulating VLCFA elongation and cuticular wax biosynthesis, which provides new directions for the improvement of citrus fruit wax quality in genetic breeding programs.

Evolutionary genomics of structural variation in the tea plant, <i>Camellia sinensis</i>

Structural variants (SVs) are a type of genetic variation that contribute substantially to phenotypic diversity and evolution. Further study of SVs will help us understand the influence of SVs associated with tea quality and stress resistance and provide new insight into tea plant breeding improvement and genetic research. However, SVs have not been thoroughly discovered in the tea plant genomes. Herein, we constructed a large-scale SV map across a population of 107 resequenced genomes, including both ancient and cultivated tea plants. A total of 44,240 high-confident SVs were identified, including 34,124 DEL (deletions), 4,448 DUP (duplications), 2,503 INV (inversions), 544 INS (insertions) and 2,621 TRA (translocations). In total, 12,400 protein-coding genes were overlapped with SVs, of which 49.5% were expressed in all five tea tissues. SV-based analysis of phylogenetic relationships and population structure in tea plants showed a consistent evolutionary history with the SNP-based results. We also identified SVs subject to artificial selection and found that genes under domestication were enriched in metabolic pathways involving theanine and purine alkaloids, biosynthesis of monoterpenoid, phenylpropanoid, fatty acid, and isoflavonoid, contributing to traits of agronomic interest in tea plants. In addition, a total of 27 terpene synthase (<italic>TPS</italic>) family genes were selected during domestication. These results indicate that these SVs could provide extensive genomic information for tea quality improvement.

Sphingomyelin Synthase Family and Phospholipase Cs.

The sphingomyelin synthase (SMS) gene family has three members: SMS1 and SMS2 have SM synthase activity, while SMS-related protein (SMSr) has no SM synthase activity but has ceramide phosphorylethanolamine (CPE) synthase activity in vitro. Recently, we found that SMS family members are a group of phospholipase Cs (PLC). SMS1 and SMS2 are two phosphatidylcholine (PC)-PLCs and SMSr is a phosphatidylethanolamine (PE)-PLC. SMS family members not only influence SM levels but also influence the levels of diacylglycerol (DAG), PC, PE, and glycosphingolipids, thus influencing cell functions. In this chapter, we will discuss the recent progress in the research field of SMS family and will focus on its impact on metabolic diseases.

A- and B-subgenome characterisation of sucrose synthase family proteins and functional identification of MaSUS2.2 reveals its involvement in the starch accumulation in banana fruit

ABSTRACT Sucrose synthase (SUS) is one of the most important enzymes catalysing the synthesis and cleavage of sucrose, and it also plays a crucial role in multiple plant biological processes. However, SUS has been scarcely reported in banana which accumulates large amounts of starch in its fruit. In this genome-wide study on banana, nine SUS genes were identified in each of the two subgenomes (A and B) for the first time, which were clustered into three distinct groups. Those in the group II underwent further duplications. In the transcriptome analysis of Baxijiao (BX, AAA) and Fenjiao (FJ, ABB) banana, four MaSUSs (MaSUS-2.1, -2.2, -2.3, and -3.2) and five MbSUSs (MbSUS-2.1, -2.2, -2.3, -2.4, and -3.2) were expressed during fruit development, with MaSUS2.2/MbSUS2.2 being the most highly expressed. Transient overexpression of MaSUS2.2 resulted in a significant increase in its transcription, which was consistent with significantly enhanced starch content in banana fruits. Its function was further identified by its transient silencing expression in banana fruit discs, which led to a significant reduction in total starch content. Taken together, the results show that multiple SUS genes, especially SUS2.2, play an important role in starch accumulation in banana fruit.

Activation of 1-Aminocyclopropane-1-Carboxylic Acid Synthases Sets Stomatal Density and Clustered Ratio on Leaf Epidermis of Arabidopsis in Response to Drought.

The adjustment of stomatal density and clustered ratio on the epidermis is the important strategy for plants to respond to drought, because the stoma-based water loss is directly related to plant growth and survival under drought conditions. But the relevant adjustment mechanism still needs to be explored. 1-Aminocyclopropane-1-carboxylate (ACC) is disclosed to promote stomatal development, while in vivo ACC levels depend on activation of ACC synthase (ACS) family members. Based on the findings of ACS expression involving in drought response and several ACS activity inhibitors reducing stomatal density and cluster in drought response, here we examined how ACS activation is involved in the establishment of stomatal density and cluster on the epidermis under drought conditions. Preliminary data indicated that activation of ACS2 and/or ACS6 (ACS2/6) increased stomatal density and clustered ratio on the Arabidopsis leaf epidermis by accumulating ACC under moderate drought, and raised the survival risk of seedlings under escalated drought. Further exploration indicated that, in Arabidopsis seedlings stressed by drought, the transcription factor SPEECHLESS (SPCH), the initiator of stomatal development, activates ACS2/6 expression and ACC production; and that ACC accumulation induces Ca2+ deficiency in stomatal lineage; this deficiency inactivates a subtilisin-like protease STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1) by stabilizing the inhibition of the transcription factor GT-2 Like 1 (GTL1) on SDD1 expression, resulting in an increases of stomatal density and cluster ratio on the leaf epidermis. This work provides a novel evidence that ACS2/6 activation plays a key role in the establishment of stomatal density and cluster on the leaf epidermis of Arabidopsis in response to drought.

Genome-wide analysis of the CalS gene family in cotton reveals their potential roles in fiber development and responses to stress.

Callose deposition occurs during plant growth and development, as well as when plants are under biotic and abiotic stress. Callose synthase is a key enzyme for the synthesis of callose. In this study, 27, 28, 16, and 15 callose synthase family members were identified in Gossypium hirsutum, Gossypium barbadense, Gossypium raimondii, and Gossypium arboreum using the sequence of Arabidopsis callose synthase. The CalSs were divided into five groups by phylogenetic, gene structure, and conservative motif analysis. The conserved motifs and gene structures of CalSs in each group were highly similar. Based on the analysis of cis-acting elements, it is inferred that GhCalSs were regulated by abiotic stress. WGD/Segmental duplication promoted the amplification of the CalS gene in cotton, and purification selection had an important function in the CalS family. The transcriptome data and qRT-PCR under cold, heat, salt, and PEG treatments showed that GhCalSs were involved in abiotic stress. The expression patterns of GhCalSs were different in various tissues. We predicted that GhCalS4, which was highly expressed in fibers, had an important effect on fiber elongation. Hence, these results help us understand the role of GhCalSs in fiber development and stress response.

Phylogenetic Implications and Functional Disparity in the Chalcone synthase Gene Family of Common Seagrass Zostera marina

Chalcone synthase (CHS) family are plant type III polyketide synthases that participate in the flavonoid synthesis pathway to induce plant resistance to various biotic and abiotic stresses. Zostera marina, a common seagrass, migrated to terrestrial conditions and returned to the sea, achieving the most severe habitat shift of flowering plants. Given the special evolutionary process, we conducted genome-wide, expression and enzyme activity analyses of the ZosmaCHS family to understand its phylogenetic implications. Various duplication modes led to the expansion of 11 CHS homologs in Z. marina. Based on the phylogenetic relationships, ZosmaCHSs were classified into three clades. Further quantitative real time-PCR analyses of the ZosmaCHS homologs showed different light responses and tissue-specific expression, indicating functional diversification of the ZosmaCHSs. Moreover, the ZosmaCHS proteins clustering with the validated chalcone synthases were recombined into prokaryotic expression systems. All the recombinant proteins showed CHS activity to generate naringenin chalcone with varying catalytic efficiencies. ZosmaCHS07 was regarded as the dominant CHS because of its significant light response and the higher catalytic efficiency. Taken together, the disparity of the expression and enzyme activity indicated that sub-functionalization is the primary mechanism of the expansion of the ZosmaCHSs family.

Radiation‑induced dysfunction of energy metabolism in the heart results in the fibrosis of cardiac tissues.

Thoracic radiotherapy increases the risk of radiation-induced heart damage (RIHD); however, the molecular mechanisms underlying these changes are not fully understood. The aim of the present study was to investigate the effects of radiation on the mouse heart using high-throughput proteomics. Male C57BL/6J mice were used to establish a model of RIHD by exposing the entire heart to 16 Gy high-energy X-rays, and cardiac injuries were verified using a cardiac echocardiogram, as well as by measuring serum brain natriuretic peptide levels and conducting H&E and Masson staining 5 months after irradiation. Proteomics experiments were performed using the heart apex of 5-month irradiated mice and control mice that underwent sham-irradiation. The most significantly differentially expressed proteins were enriched in ‘cardiac fibrosis’ and ‘energy metabolism’. Next, the cardiac fibrosis and changes to energy metabolism were confirmed using immunohistochemistry staining and western blotting. Extracellular matrix proteins, such as collagen type 1 α 1 chain, collagen type III α 1 chain, vimentin and CCCTC-binding factor, along with metabolism-related proteins, such as fatty acid synthase and solute carrier family 25 member 1, exhibited upregulated expression following exposure to ionizing radiation. Additionally, the myocardial mitochondria inner membranes were injured, along with a decrease in ATP levels and the accumulation of lactic acid in the irradiated heart tissues. These results suggest that the high doses of ionizing radiation used lead to structural remodeling, functional injury and fibrotic alterations in the mouse heart. Radiation-induced mitochondrial damage and metabolic alterations of the cardiac tissue may thus be a pathogenic mechanism of RIHD.

Physio-biochemical and proteomic mechanisms of coronatine induced potassium stress tolerance in xylem sap of cotton

Potassium (K) is a major plant nutrient, and its deficiency can limit plant growth and development. Coronatine (COR) could increase cotton seedling tolerance to K deficiency, which was hypothesized to be in relation to improving the physiological and proteomic profile of xylem sap. To test this hypothesis, cotton seedlings growth, physio-biochemical and proteomic profile of xylem sap treated with (0 and 10 nM COR) under deficient K solution (0.05 mM KCl) were explored. Compared with control, COR treatment significantly increased lateral root number and root diameter and decreased contents of malondialdehyde (MDA), some cations like potassium and calcium in xylem sap and its volume. These morphological and physiological presentations were well evidenced by differentially expressed proteins (DEPs) in the xylem sap. For instance, increasing the average root diameter and lateral roots were related to up-regulation of cobalamin-independent methionine synthase family proteins, auxin-responsive protein, and cell wall remodeling proteins such as dirigent-like protein, laccase, and the pectin lyase; lessening xylem sap volume and some cations contents was in connections with down-regulation of uclacyanin 1 and up-regulation of calmodulin-domain protein kinase 7; the MDA content reducing was associated with many PCD-related proteins’ down-regulation or loss. Furthermore, COR potentially weakened plant defense, owing to lessening or disappearing lipid-transfer proteins, signaling proteins, and other proteins positively involved in plant defense.

Study of Terpenoid Synthesis and Prenyltransferase in Roots of Rehmannia glutinosa Based on iTRAQ Quantitative Proteomics.

Rehmannia glutinosa has important medicinal value; terpenoid is one of the main active components in R. glutinosa. In this study, iTRAQ technique was used to analyze the relative abundance of proteins in roots of R. glutinosa, and 6,752 reliable proteins were quantified. GO enrichment results indicated that most proteins were involved in metabolic process or cellular process, 57.63% proteins had catalytic activity, and 65.80% proteins were enriched in membrane-bounded organelle. In roots of R. glutinosa, there were 38 KEGG enrichments with significance, more DEPs were found in some pathways, especially the proteasome pathway and TCA cycle with 15.0% DEPs between elongation stage and expansion stage of roots. Furthermore, five KEGG pathways of terpenoid synthesis were found. Most prenyltransferases belong to FPP/GGPP synthase family, involved in terpenoid backbone biosynthesis, and all interacted with biotin carboxylase CAC2. Compared with that at the elongation stage, many prenyltransferases exhibited higher expression at the expansion stage or maturation stage of roots. In addition, eight FPP/GGPP synthase encoding genes were cloned from R. glutinosa, namely FPPS, FPPS1, GGPS, GGPS3, GGPS4, GGPS5, GPPS and GPPS2, introns were also found in FPPS, FPPS1, GGPS5 and GGPS2, and FPP/GPP synthases were more conservative in organisms, especially in viridiplantae, in which the co-occurrence of GPPS or GPPS2 was significantly higher in plants. Further analysis found that FPP/GGPP synthases of R. glutinosa were divided into three kinds, GGPS, GPPS and FPPS, and their gene expression was significantly diverse in different varieties, growth periods, or tissues of R. glutinosa. Compared with that of GGPS, the expression of GPPS and FPPS was much higher in R. glutinosa, especially at the expansion stage and maturation stage. Thus, the synthesis of terpenoids in roots of R. glutinosa is intricately regulated and needs to be further studied.

MO18-6 The tumor suppression potential of RNA pseudouridine synthase family via microRNA regulation

MicroRNAs are non-coding RNAs consisting of about 22 nucleotides that regulate a large number of mRNAs. Among them, let-7 is one of the first microRNAs to be identified and has tumor suppressive potential through regulation of KRAS and MYC mRNAs. We have shown that TruB1, one of the tRNA pseudouridine synthetases, regulates let-7 relatively specifically by cell-based screening (Kurimoto et al. EMBO J. 2020). In the present study, to evaluate the specificity of the let-7-mediated tumor suppressor ability of TruB1, we examined the effects of whole RNA pseudouridine synthetase on the regulation of its let-7 in vitro experiments and in the reported literatures. The results showed that DKC1 and PUS10 also regulate microRNAs of different sub-subset from let-7 in the reported literatures, whereas TruB2 may regulate microRNA subset including let-7. We will discuss about clinical impact of TruB2 on several cancers by TCGA database. These new functions of RNA-pseudouridine enzymes might have the impact on the tumor-suppressive potential. These results are significant from a molecular biological point of view, leading to a clarification of the regulatory mechanism.

Discovery and Functional Characterization of a Diverse Diterpene Synthase Family in the Medicinal Herb Isodon lophanthoides Var. gerardiana.

Isodon lophanthoides var. gerardiana (Lamiaceae), also named xihuangcao, is a traditional Chinese medicinal herb that exhibits a broad range of pharmacological activities. Abietane-type diterpenoids are the characteristic constituents of I. lophanthoides, yet their biosynthesis has not been elucidated. Although the aerial parts are the most commonly used organs of I. lophanthoides, metabolite profiling by gas chromatography-mass spectrometry showed the underground parts also contain large amounts of labdane diterpenoids including abietatriene, miltiradiene and ferruginol, which are distinct from the 13-hydroxy-8(14)-abietene detected in the aerial parts. Comparative transcriptome analysis of root and leaf samples identified a diverse diterpene synthase family including 6 copalyl diphosphate synthase (IlCPS1-6) and 5 kaurene synthase-like (IlKSL1-5). Here we report the functional characterization of six of these enzymes using yeast heterologous expression system. Both IlCPS1 and IlCPS3 synthesized (+)-copalyl diphosphate (CPP), in combination with IlKSL1 resulted in miltiradiene, precursor of abietane-type diterpenoids, while coupling with IlKSL5 led to the formation of hydroxylated diterpene scaffold nezukol. Expression profiling and phylogenetic analysis further support the distinct evolutionary relationship and spatial distribution of IlCPS1 and IlCPS3. IlCPS2 converted GGPP into labda-7,13E-dien-15-ol diphosphate. IlCPS6 was identified as ent-CPS, indicating a role in gibberellin metabolism. We further identified a single residue that determined the water addition of nezukol synthase IlKSL5. Substitution of alanine 513 with isoleucine completely altered the product outcome from hydroxylated nezukol to isopimara-7,15-diene. Together, these findings elucidated the early steps of bioactive abietane-type diterpenoid biosynthesis in I. lophanthoides and the catalytic mechanism of nezukol synthase.