Comprehensive map of ribosomal 2'-O-methylation and C/D box snoRNAs in Drosophila melanogaster.

During their maturation, ribosomal RNAs (rRNAs) are decorated by hundreds of chemical modifications that participate in proper folding of rRNA secondary structures and therefore in ribosomal function. Along with pseudouridine, methylation of the 2'-hydroxyl ribose moiety (Nm) is the most abundant modification of rRNAs. The majority of Nm modifications in eukaryotes are placed by Fibrillarin, a conserved methyltransferase belonging to a ribonucleoprotein complex guided by C/D box small nucleolar RNAs (C/D box snoRNAs). These modifications impact interactions between rRNAs, tRNAs and mRNAs, and some are known to fine tune translation rates and efficiency. In this study, we built the first comprehensive map of Nm sites in Drosophila melanogaster rRNAs using two complementary approaches (RiboMethSeq and Nanopore direct RNA sequencing) and identified their corresponding C/D box snoRNAs by whole-transcriptome sequencing. We de novo identified 61 Nm sites, from which 55 are supported by both sequencing methods, we validated the expression of 106 C/D box snoRNAs and we predicted new or alternative rRNA Nm targets for 31 of them. Comparison of methylation level upon different stresses show only slight but specific variations, indicating that this modification is relatively stable in D. melanogaster. This study paves the way to investigate the impact of snoRNA-mediated 2'-O-methylation on translation and proteostasis in a whole organism.

Pipecolic acid synthesis is required for systemic acquired resistance and plant-to-plant-induced immunity in barley.

Defense responses in plants are based on complex biochemical processes. Systemic acquired resistance (SAR) helps to fight infections by (hemi-)biotrophic pathogens. One important signaling molecule in SAR is pipecolic acid (Pip), accumulation of which is dependent on the aminotransferase ALD1 in Arabidopsis. While exogenous Pip primes defense responses in the monocotyledonous cereal crop barley (Hordeum vulgare), it is currently unclear if endogenous Pip plays a role in disease resistance in monocots. Here, we generated barley ald1 mutants using CRISPR/Cas9, and assessed their capacity to mount SAR. Endogenous Pip levels were reduced after infection of the ald1 mutant, and this altered systemic defense against the fungus Blumeria graminis f. sp. hordei. Furthermore, Hvald1 plants did not emit nonanal, one of the key volatile compounds that are normally emitted by barley plants after the activation of SAR. This resulted in the inability of neighboring plants to perceive and/or respond to airborne cues and prepare for an upcoming infection, although HvALD1 was not required in the receiver plants to mediate the response. Our results highlight the crucial role of endogenous HvALD1 and Pip for SAR, and associate Pip, in particular together with nonanal, with plant-to-plant defense propagation in the monocot crop barley.

New molecules in plant defence against pathogens.

Plants host a multipart immune signalling network to ward off pathogens. Pathogen attack upon plant tissues can often lead to an amplified state of (induced) defence against subsequent infections in distal tissues; this is known as systemic acquired resistance (SAR). The interaction of plants with beneficial microbes of the rhizosphere microbiome can also lead to an induced resistance in above-ground plant tissues, known as induced systemic resistance. Second messengers such as calcium (Ca2+), reactive oxygen species (ROS), and nitric oxide (NO) are necessary for cell-to-cell signal propagation during SAR and show emergent roles in the mediation of other SAR metabolites. These include the lysine-derived signals pipecolic acid (Pip) and N-hydroxypipecolic acid (NHP), which are key signalling metabolites in SAR. Emerging evidence additionally pinpoints plant volatiles as modulators of defence signalling within and between plants. Plant volatile organic compounds (VOCs) such as monoterpenes can promote SAR by functioning through ROS. Furthermore, plant-derived and additionally also microbial VOCs can target both salicylic acid and jasmonic acid signalling pathways in plants and modulate defence against pathogens. In this review, an overview of recent findings in induced defence signalling, with a particular focus on newer signalling molecules and how they integrate into these networks is discussed.

Immunity-associated volatile emissions of ß-ionone and nonanal propagate defence responses in neighbouring barley plants.

Plants activate biochemical responses to combat stress. (Hemi-)biotrophic pathogens are fended off by systemic acquired resistance (SAR), a primed state allowing plants to respond faster and more strongly upon subsequent infection. Here, we show that SAR-like defences in barley (Hordeum vulgare) are propagated between neighbouring plants, which respond with enhanced resistance to the volatile cues from infected senders. The emissions of the sender plants contained 15 volatile organic compounds (VOCs) associated with infection. Two of these, ß-ionone and nonanal, elicited resistance upon plant exposure. Whole-genome transcriptomics analysis confirmed that interplant propagation of defence in barley is established as a form of priming. Although gene expression changes were more pronounced after challenge infection of the receiver plants with Blumeria graminis f. sp. hordei, differential gene expression in response to the volatile cues of the sender plants included an induction of HISTONE DEACETYLASE 2 (HvHDA2) and priming of TETRATRICOPEPTIDE REPEAT-LIKE superfamily protein (HvTPL). Because HvHDA2 and HvTPL transcript accumulation was also enhanced by exposure of barley to ß-ionone and nonanal, our data identify both genes as possible defence/priming markers in barley. Our results suggest that VOCs and plant-plant interactions are relevant for possible crop protection strategies priming defence responses in barley.

Loss of Mediator complex subunit 13 (MED13) promotes resistance to alkylation through cyclin D1 upregulation.

Alkylating drugs are among the most often used chemotherapeutics. While cancer cells frequently develop resistance to alkylation treatments, detailed understanding of mechanisms that lead to the resistance is limited. Here, by using genome-wide CRISPR-Cas9 based screen, we identify transcriptional Mediator complex subunit 13 (MED13) as a novel modulator of alkylation response. The alkylation exposure causes significant MED13 downregulation, while complete loss of MED13 results in reduced apoptosis and resistance to alkylating agents. Transcriptome analysis identified cyclin D1 (CCND1) as one of the highly overexpressed genes in MED13 knock-out (KO) cells, characterized by shorter G1 phase. MED13 is able to bind to CCND1 regulatory elements thus influencing the expression. The resistance of MED13 KO cells is directly dependent on the cyclin D1 overexpression, and its down-regulation is sufficient to re-sensitize the cells to alkylating agents. We further demonstrate the therapeutic potential of MED13-mediated response, by applying combinatory treatment with CDK8/19 inhibitor Senexin A. Importantly, the treatment with Senexin A stabilizes MED13, and in combination with alkylating agents significantly reduces viability of cancer cells. In summary, our findings identify novel alkylation stress response mechanism dependent on MED13 and cyclin D1 that can serve as basis for development of innovative therapeutic strategies.

Systemic propagation of immunity in plants.

Systemic immunity triggered by local plant-microbe interactions is studied as systemic acquired resistance (SAR) or induced systemic resistance (ISR) depending on the site of induction and the lifestyle of the inducing microorganism. SAR is induced by pathogens interacting with leaves, whereas ISR is induced by beneficial microbes interacting with roots. Although salicylic acid (SA) is a central component of SAR, additional signals exclusively promote systemic and not local immunity. These signals cooperate in SAR- and possibly also ISR-associated signaling networks that regulate systemic immunity. The non-SA SAR pathway is driven by pipecolic acid or its presumed bioactive derivative N-hydroxy-pipecolic acid. This pathway further regulates inter-plant defense propagation through volatile organic compounds that are emitted by SAR-induced plants and recognized as defense cues by neighboring plants. Both SAR and ISR influence phytohormone crosstalk towards enhanced defense against pathogens, which at the same time affects the composition of the plant microbiome. This potentially leads to further changes in plant defense, plant-microbe, and plant-plant interactions. Therefore, we propose that such inter-organismic interactions could be combined in potentially highly effective plant protection strategies.

New indolylarylsulfone non-nucleoside reverse transcriptase inhibitors show low nanomolar inhibition of single and double HIV-1 mutant strains.

We designed and synthesized 21 new indolylarylsulfones (IASs) as new HIV-1 NNRTIs. Among these, IAS 12 exhibited a remarkable antiviral activity against single and double mutants (K103N EC50 = <0.7 nM; Y181C EC50 = <0.7 nM; Y188L EC50 = 21.3 nM; K103N-Y181C EC50 = 6.2 nM), resulting equally or more active than previuosly reported IAS 6 and some approved anti-HIV-1 drugs. Docking and molecular dynamics simulations of compound 12 in complex with WT, Y181C, Y188L, K103N and K103N-Y181C RTs clarified a general binding mode that was consistent with biological results. Kinetic experiments disclosed that derivative 12 preferentially binds WT and K103N-Y181C RTs to binary and ternary complexes, respectively.

Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression.

Base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG) is essential for removal of aberrantly methylated DNA bases. Genome instability and accumulation of aberrant bases accompany multiple diseases, including cancer and neurological disorders. While BER is well studied on naked DNA, it remains unclear how BER efficiently operates on chromatin. Here, we show that AAG binds to chromatin and forms complex with RNA polymerase (pol) II. This occurs through direct interaction with Elongator and results in transcriptional co-regulation. Importantly, at co-regulated genes, aberrantly methylated bases accumulate towards the 3'end in regions enriched for BER enzymes AAG and APE1, Elongator and active RNA pol II. Active transcription and functional Elongator are further crucial to ensure efficient BER, by promoting AAG and APE1 chromatin recruitment. Our findings provide insights into genome stability maintenance in actively transcribing chromatin and reveal roles of aberrantly methylated bases in regulation of gene expression.

Chiral Indolylarylsulfone Non-Nucleoside Reverse Transcriptase Inhibitors as New Potent and Broad Spectrum Anti-HIV-1 Agents.

We designed and synthesized a series of chiral indolyarylsulfones (IASs) as new HIV-1 NNRTIs. The new IASs 8-37 showed potent inhibition of the HIV-1 WT NL4-3 strain and of the mutant K103N, Y181C, Y188L, and K103N-Y181C HIV-1 strains. Six racemic mixtures, 8, 23-25, 31, and 33, were separated at semipreparative level into their pure enantiomers. The (R)-8 enantiomer bearing the chiral (α-methylbenzyl) was superior to the (S)-counterpart. IAS derivatives bearing the (S) alanine unit, (S)-23, (S,R)-25, (S)-31, and (S)-33, were remarkably more potent than the corresponding (R)-enantiomers. Compound 23 protected hippocampal neuronal cells from the excitotoxic insult, while efavirenz (EFV) did not contrast the neurotoxic effect of glutamate. The present results highlight the chiral IASs as new NNRTIs with improved resistance profile against the mutant HIV-1 strains and reduced neurotoxic effects.