Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.

Terpene synthesis stands at the forefront of modern synthetic chemistry and represents the state-of-the-art in the chemist\'s toolbox. Notwithstanding, these endeavors are inherently tied to the current availability of natural cyclic building blocks. Addressing this limitation, the stereocontrolled cyclization of abundant unbiased linear terpenes emerges as a valuable tool, which is still difficult to achieve with chemical catalysts. In this study, we showcase the remarkable capabilities of squalene-hopene cyclases (SHCs) in the chemoenzymatic synthesis of head-to-tail-fused terpenes. By combining engineered SHCs and a practical reaction setup, we generate ten chiral scaffolds with >99% ee and de, at up to decagram scale. Our mechanistic insights suggest how cyclodextrin encapsulation of terpenes may influence the performance of the membrane-bound enzyme. Moreover, we transform the chiral templates to valuable (mero)-terpenes using interdisciplinary synthetic methods, including a catalytic ring-contraction of enol-ethers facilitated by cooperative iodine/lipase catalysis.

Wolfiporia cocos is a medicinal mushroom used in China. It biosynthesizes pachymic acid (PA), a main therapeutic triterpene associated with therapies. Nowadays, the unknown PA biosynthesis leads to difficulties in increasing its content in W. cocos. Herein, we report sequencing, assembling, and characterization of the genome and several transcriptomes of W. cocos. Sequence mining determined candidate genes that encode lanosterol synthase, sterol O-acyltransferase, and sterol C-24 methyltransferase likely involved in the steps from lanosterol to PA. Gene cluster analysis identified four CYP450 cDNAs likely involved in the biosynthesis of PA, namely WcCYP64-1, WcCYP64-2, WcCYP52, and WcCYP_FUM15, which were subjected to both overexpression and silencing in mycelia. The overexpression of each of WcCYP64-1, WcCYP52 and WcCYP_FUM15 increased the content of PA, 16α-hydroxytrametenolic acid, eburicoic acid, and tumulosic acid, while the silencing of each gene either significantly or slightly decreased the contents of these four compounds, indicating their involvement in the PA biosynthesis. In addition, different temperatures affected the expression of these genes and the formation of PA. By contrast, the overexpression and silencing of WcCYP64-2 did not alter the formation of these compounds. Taken together, these findings determine more potential steps in the biosynthetic pathway of PA for metabolic engineering.

CONCLUSIONS: The small-molecule glucosyltransferase loss-of-function mutant ugt76b1 exhibits both SID2- or NPR1-dependent and independent facets of enhanced plant immunity, whereupon FMO1 is required for the SID2 and NPR1 independence. The small-molecule glucosyltransferase UGT76B1 inactivates salicylic acid (SA), isoleucic acid (ILA), and N-hydroxypipecolic acid (NHP). ugt76b1 loss-of-function plants manifest an enhanced defense status. Thus, we were interested how UGT76B1 genetically integrates in defense pathways and whether all impacts depend on SA and NHP. We study the integration of UGT76B1 by transcriptome analyses of ugt76b1. The comparison of transcripts altered by the loss of UGT76B1 with public transcriptome data reveals both SA-responsive, ISOCHORISMATE SYNTHASE 1/SALICYLIC ACID INDUCTION DEFICIENT 2 (ICS1/SID2)- and NON EXPRESSOR OF PR GENES 1 (NPR1)-dependent, consistent with the role of UGT76B1 in glucosylating SA, and SA-non-responsive, SID2/NPR1-independent genes. We also discovered that UGT76B1 impacts on a group of genes showing non-SA-responsiveness and regulation by infections independent from SID2/NPR1. Enhanced resistance of ugt76b1 against Pseudomonas syringae is partially independent from SID2 and NPR1. In contrast, the ugt76b1-activated resistance is completely dependent on FMO1 encoding the NHP-synthesizing FLAVIN-DEPENDENT MONOOXYGENASE 1). Moreover, FMO1 ranks top among the ugt76b1-induced SID2- and NPR1-independent pathogen responsive genes, suggesting that FMO1 determines the SID2- and NPR1-independent effect of ugt76b1. Furthermore, the genetic study revealed that FMO1, ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), SID2, and NPR1 are required for the SA-JA crosstalk and senescence development of ugt76b1, indicating that EDS1 and FMO1 have a similar effect like stress-induced SA biosynthesis (SID2) or the key SA signaling regulator NPR1. Thus, UGT76B1 influences both SID2/NPR1-dependent and independent plant immunity, and the SID2/NPR1 independence is relying on FMO1 and its product NHP, another substrate of UGT76B1.

Pseudouridine is an abundant mRNA modification found in diverse organisms ranging from bacteria and viruses to multicellular plants and humans. New developments in pseudouridine profiling provide quantitative tools to map mRNA pseudouridylation sites. Sparse biochemical studies establish the potential for mRNA pseudouridylation to affect most stages of the mRNA life cycle from birth to death. This recent progress sets the stage for deeper investigations into the molecular and cellular functions of specific mRNA pseudouridines, including in disease.

Pseudouridine, one of the most abundant RNA modifications, is synthesized by stand-alone or RNA-guided pseudouridine synthases. Here, we comprehensively mapped pseudouridines in rRNAs, tRNAs and small RNAs in the archaeon Sulfolobus islandicus and identified Cbf5-associated H/ACA RNAs. Through genetic deletion and in vitro modification assays, we determined the responsible enzymes for these modifications. The pseudouridylation machinery in S. islandicus consists of the stand-alone enzymes aPus7 and aPus10, and six H/ACA RNA-guided enzymes that account for all identified pseudouridines. These H/ACA RNAs guide the modification of all eleven sites in rRNAs, two sites in tRNAs, and two sites in CRISPR RNAs. One H/ACA RNA shows exceptional versatility by targeting eight different sites. aPus7 and aPus10 are responsible for modifying positions 13, 54 and 55 in tRNAs. We identified four atypical H/ACA RNAs that lack the lower stem and the ACA motif and confirmed their function both in vivo and in vitro. Intriguingly, atypical H/ACA RNAs can be modified by Cbf5 in a guide-independent manner. Our data provide the first global view of pseudouridylation in archaea and reveal unexpected structures, substrates, and activities of archaeal H/ACA RNPs.

Triterpenes are a class of bioactive compounds with diverse biological functions, playing pivotal roles in plant defense against biotic stressors. Oxidosqualene cyclases (OSCs) serve as gatekeepers in the biosynthesis of triterpenes. In this study, we utilized a Nicotiana benthamiana heterologous expression system to characterize NaOSC1 from Nicotiana attenuata as a multifunctional enzyme capable of synthesizing lupeol, dammarenediol II, 3-alpha,20-lupanediol, and 7 other triterpene scaffolds. We also demonstrated that NaOSC2 is, in contrast, a selective enzyme, producing only the β-amyrin scaffold. Through virus-induced gene silencing and in vitro toxicity assays, we elucidated the roles of NaOSC1 and NaOSC2 in the defense of N. attenuata against Manduca sexta larvae. Metabolomic and feature-based molecular network analyses of leaves with silenced NaOSC1 and NaOSC2 unveiled 3 potential triterpene glycoside metabolite clusters. Interestingly, features identified as triterpenes within these clusters displayed a significant negative correlation with larval mass. Our study highlights the pivotal roles of NaOSC1 and NaOSC2 from N. attenuata in the initial steps of triterpene biosynthesis, subsequently influencing defense against M. sexta through the modulation of downstream triterpene glycoside compounds.

BACKGROUND: Patients with biallelic variants in the lanosterol synthase (LSS) gene has been reported to exhibit phenotypes as follows: non-syndromic form of hypotrichosis, congenital cataracts, and alopecia with intellectual disability or growth retardation. However, genotype-phenotype correlations in the LSS gene are still not completely clear.
METHODS: In this study, we reported a Chinese girl who had congenital cataracts with hypotrichosis. The trio exome sequencing was performed to elucidate the genetic cause of the patient.
RESULTS: We identified compound heterozygous variants (c.296G>A, p.G99D and c.1025T>G, p.I342S) in the LSS gene. Both variants altered the amino acid coding at highly conserved amino acid residues and were predicted to be deleterious using prediction software.
CONCLUSIONS: Our report expands the spectrum of variants in the LSS gene and will be helpful for genotype-phenotype correlations study.

The chemical modification of RNA bases represents a ubiquitous activity that spans all domains of life. Pseudouridylation is the most common RNA modification and is observed within tRNA, rRNA, ncRNA and mRNAs. Pseudouridine synthase or \'PUS\' enzymes include those that rely on guide RNA molecules and others that function as \'stand-alone\' enzymes. Among the latter, several have been shown to modify mRNA transcripts. Although recent studies have defined the structural requirements for RNA to act as a PUS target, the mechanisms by which PUS1 recognizes these target sequences in mRNA are not well understood. Here we describe the crystal structure of yeast PUS1 bound to an RNA target that we identified as being a hot spot for PUS1-interaction within a model mRNA at 2.4 Å resolution. The enzyme recognizes and binds both strands in a helical RNA duplex, and thus guides the RNA containing the target uridine to the active site for subsequent modification of the transcript. The study also allows us to show the divergence of related PUS1 enzymes and their corresponding RNA target specificities, and to speculate on the basis by which PUS1 binds and modifies mRNA or tRNA substrates.

Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N5-CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N5-CAIR mutase of the human pathogens Legionella pneumophila (LpPurE) and Burkholderia cenocepacia (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds in silico and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC50 values. Several of these compounds, including the α1-blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.