piRNAs can trigger a multigenerational epigenetic memory in the germline of C. elegans.

Transgenerational effects have wide-ranging implications for human health, biological adaptation, and evolution; however, their mechanisms and biology remain poorly understood. Here, we demonstrate that a germline nuclear small RNA/chromatin pathway can maintain stable inheritance for many generations when triggered by a piRNA-dependent foreign RNA response in C. elegans. Using forward genetic screens and candidate approaches, we find that a core set of nuclear RNAi and chromatin factors is required for multigenerational inheritance of environmental RNAi and piRNA silencing. These include a germline-specific nuclear Argonaute HRDE1/WAGO-9, a HP1 ortholog HPL-2, and two putative histone methyltransferases, SET-25 and SET-32. piRNAs can trigger highly stable long-term silencing lasting at least 20 generations. Once established, this long-term memory becomes independent of the piRNA trigger but remains dependent on the nuclear RNAi/chromatin pathway. Our data present a multigenerational epigenetic inheritance mechanism induced by piRNAs.

Gene discovery and virus-induced gene silencing reveal branched pathways to major classes of bioactive diterpenoids in Euphorbia peplus.

Most macro- and polycyclic Euphorbiaceae diterpenoids derive from the common C20 precursor casbene. While the biosynthetic pathway from casbene to the lathyrane jolkinol C is characterized, pathways to other more complex classes of bioactive diterpenoids remain to be elucidated. A metabolomics-guided transcriptomic approach and a genomics approach that led to the discovery of two casbene-derived diterpenoid gene clusters yielded a total of 68 candidate genes that were transiently expressed in Nicotiana benthamiana for activity toward jolkinol C and other lathyranes. We report two short-chain dehydrogenases/reductases (SDRs), identified by RNA sequencing to be highly expressed in Euphorbia peplus latex. One of these, EpSDR-5, is a C3-ketoreductase, converting jolkinol C to the lathyrane jolkinol E. Gene function of EpSDR-5 was further confirmed by heterologous expression in Saccharomyces cerevisiae. To investigate the in vivo role of EpSDR-5, we established virus-induced gene silencing (VIGS) in E. peplus, resulting in a significant reduction in jatrophanes and a corresponding increase in ingenanes. VIGS of Casbene Synthase results in a major reduction in both jatrophanes and ingenanes, the two most abundant classes of E. peplus diterpenoids. VIGS of CYP71D365 had a similar effect, consistent with the previously determined role of this gene in the pathway to jolkinol C. These results point to jolkinol C being a branch point intermediate in the pathways to ingenanes and jatrophanes with EpSDR-5 responsible for the first step from jolkinol C to jatrophane production.

An Engineered Heat-Inducible Expression System for the Production of Casbene in Nicotiana benthamiana.

Plants respond to heat stress by producing heat-shock proteins. These are regulated by heat-shock promoters containing regulatory elements, which can be harnessed to control protein expression both temporally and spatially. In this study, we designed heat-inducible promoters to produce the diterpene casbene in Nicotiana benthamiana, through a multi-step metabolic pathway. To potentially increase gene transcription, we coupled heat-shock elements from Arabidopsis thaliana Hsp101 or Glycine max GmHsp17.3-B promoters, CAAT and TATA boxes from CaMV 35S, and the 5'UTR from the tobacco mosaic virus. The resulting four chimeric promoters fused to a green fluorescent protein (GFP) reporter showed that the variant Ara2 had the strongest fluorescent signal after heat shock. We next created a 4-gene cassette driven by the Ara2 promoter to allow for exogenous synthesis of casbene and transformed this multigene construct along with a selectable marker gene into Nicotiana benthamiana. Metabolic analysis on the transgenic lines revealed that continuous heat outperforms heat shock, with up to 1 µg/mg DW of casbene detected after 32 h of uninterrupted 40 °C heat. These results demonstrate the potential of heat-inducible promoters as synthetic biology tools for metabolite production in plants.

PRDE-1 is a nuclear factor essential for the biogenesis of Ruby motif-dependent piRNAs in C. elegans.

Piwi-interacting RNAs (piRNA) are small regulatory RNAs with essential roles in maintaining genome integrity in animals and protists. Most Caenorhabditis elegans piRNAs are transcribed from two genomic clusters that likely contain thousands of individual transcription units; however, their biogenesis is not understood. Here we identify and characterize prde-1 (piRNA silencing-defective) as the first essential C. elegans piRNA biogenesis gene. Analysis of prde-1 provides the first direct evidence that piRNA precursors are 28- to 29-nucleotide (nt) RNAs initiating 2 nt upstream of mature piRNAs. PRDE-1 is a nuclear germline-expressed protein that localizes to chromosome IV. PRDE-1 is required specifically for the production of piRNA precursors from genomic loci containing an 8-nt upstream motif, the Ruby motif. The expression of a second class of motif-independent piRNAs is unaffected in prde-1 mutants. We exploited this finding to determine the targets of the motif-independent class of piRNAs. Together, our data provide new insights into both the biogenesis and function of piRNAs in gene regulation.

Developing a Nicotiana benthamiana transgenic platform for high-value diterpene production and candidate gene evaluation.

To engineer Nicotiana benthamiana to produce novel diterpenoids, we first aimed to increase production of the diterpenoid precursor geranylgeranyl pyrophosphate (GGPP) by up-regulation of key genes of the non-mevalonate (MEP) pathway sourced from Arabidopsis thaliana. We used transient expression to evaluate combinations of the eight MEP pathway genes plus GGPP synthase and a Jatropha curcas casbene synthase (JcCAS) to identify an optimal combination for production of casbene from GGPP. AtDXS and AtHDR together with AtGGPPS and JcCAS gave a 410% increase in casbene production compared to transient expression of JcCAS alone. This combination was cloned into a single construct using the MoClo toolkit, and stably integrated into the N. benthamiana genome. We also created multigene constructs for stable transformation of two J. curcas cytochrome P450 genes, JcCYP726A20 and JcCYP71D495 that produce the more complex diterpenoid jolkinol C from casbene when expressed transiently with JcCAS in N. benthamiana. Stable transformation of JcCYP726A20, JcCYP71D495 and JcCAS did not produce any detectable jolkinol C until these genes were co-transformed with the optimal set of precursor-pathway genes. One such stable homozygous line was used to evaluate by transient expression the involvement of an 'alkenal reductase'-like family of four genes in the further conversion of jolkinol C, leading to the demonstration that one of these performs reduction of the 12,13-double bond in jolkinol C. This work highlights the need to optimize precursor supply for production of complex diterpenoids in stable transformants and the value of such lines for novel gene discovery.

Selective inhibitors of trypanosomal uridylyl transferase RET1 establish druggability of RNA post-transcriptional modifications.

Non-coding RNAs are crucial regulators for a vast array of cellular processes and have been implicated in human disease. These biological processes represent a hitherto untapped resource in our fight against disease. In this work we identify small molecule inhibitors of a non-coding RNA uridylylation pathway. The TUTase family of enzymes is important for modulating non-coding RNA pathways in both human cancer and pathogen systems. We demonstrate that this new class of drug target can be accessed with traditional drug discovery techniques. Using the Trypanosoma brucei TUTase, RET1, we identify TUTase inhibitors and lay the groundwork for the use of this new target class as a therapeutic opportunity for the under-served disease area of African Trypanosomiasis. In a broader sense this work demonstrates the therapeutic potential for targeting RNA post-transcriptional modifications with small molecules in human disease.

Targeted kinase inhibition relieves slowness and tremor in a Drosophila model of LRRK2 Parkinson's disease.

In a number of Drosophila models of genetic Parkinson's disease (PD) flies climb more slowly than wild-type controls. However, this assay does not distinguish effects of PD-related genes on gravity sensation, "arousal", central pattern generation of leg movements, or muscle. To address this problem, we have developed an assay for the fly proboscis extension response (PER). This is attractive because the PER has a simple, well-identified reflex neural circuit, in which sucrose sensing neurons activate a pair of "command interneurons", and thence motoneurons whose activity contracts the proboscis muscle. This circuit is modulated by a single dopaminergic neuron (TH-VUM). We find that expressing either the G2019S or I2020T (but not R1441C, or kinase dead) forms of human LRRK2 in dopaminergic neurons reduces the percentage of flies that initially respond to sucrose stimulation. This is rescued fully by feeding l-DOPA and partially by feeding kinase inhibitors, targeted to LRRK2 (LRRK2-IN-1 and BMPPB-32). High-speed video shows that G2019S expression in dopaminergic neurons slows the speed of proboscis extension, makes its duration more variable, and increases the tremor. Testing subsets of dopaminergic neurons suggests that the single TH-VUM neuron is likely most important in this phenotype. We conclude the Drosophila PER provides an excellent model of LRRK2 motor deficits showing bradykinesia, akinesia, hypokinesia, and increased tremor, with the possibility to localize changes in neural signaling.

Computational approaches for understanding the diagnosis and treatment of Parkinson's disease.

This study describes how the application of evolutionary algorithms (EAs) can be used to study motor function in humans with Parkinson's disease (PD) and in animal models of PD. Human data is obtained using commercially available sensors via a range of non-invasive procedures that follow conventional clinical practice. EAs can then be used to classify human data for a range of uses, including diagnosis and disease monitoring. New results are presented that demonstrate how EAs can also be used to classify fruit flies with and without genetic mutations that cause Parkinson's by using measurements of the proboscis extension reflex. The case is made for a computational approach that can be applied across human and animal studies of PD and lays the way for evaluation of existing and new drug therapies in a truly objective way.

Interactions between a receptor tyrosine phosphatase and a cell surface ligand regulate axon guidance and glial-neuronal communication.

We developed a screening method for orphan receptor ligands, in which cell-surface proteins are expressed in Drosophila embryos from GAL4-dependent insertion lines and ligand candidates identified by the presence of ectopic staining with receptor fusion proteins. Stranded at second (Sas) binds to the receptor tyrosine phosphatase Ptp10D in embryos and in vitro. Sas and Ptp10D can interact in trans when expressed in cultured cells. Interactions between Sas and Ptp10D on longitudinal axons are required to prevent them from abnormally crossing the midline. Sas is expressed on both neurons and glia, whereas Ptp10D is restricted to CNS axons. We conducted epistasis experiments by overexpressing Sas in glia and examining how the resulting phenotypes are changed by removal of Ptp10D from neurons. We find that neuronal Ptp10D restrains signaling by overexpressed glial Sas, which would otherwise produce strong glial and axonal phenotypes.

Integration of telencephalic Wnt and hedgehog signaling center activities by Foxg1.

The forebrain is patterned along the dorsoventral (DV) axis by Sonic Hedgehog (Shh). However, previous studies have suggested the presence of an Shh-independent mechanism. Our study identifies Wnt/beta-catenin-activated from the telencephalic roof-as an Shh-independent pathway that is essential for telencephalic pallial (dorsal) specification during neurulation. We demonstrate that the transcription factor Foxg1 coordinates the activity of two signaling centers: Foxg1 is a key downstream effector of the Shh pathway during induction of subpallial (ventral) identity, and it inhibits Wnt/beta-catenin signaling through direct transcriptional repression of Wnt ligands. This inhibition restricts the dorsal Wnt signaling center to the roof plate and consequently limits pallial identities. Concomitantly to these roles, Foxg1 controls the formation of the compartment boundary between telencephalon and basal diencephalon. Altogether, these findings identify a key direct target of Foxg1, and uncover a simple molecular mechanism by which Foxg1 integrates two opposing signaling centers.

A screen of cell-surface molecules identifies leucine-rich repeat proteins as key mediators of synaptic target selection.

In Drosophila embryos and larvae, a small number of identified motor neurons innervate body wall muscles in a highly stereotyped pattern. Although genetic screens have identified many proteins that are required for axon guidance and synaptogenesis in this system, little is known about the mechanisms by which muscle fibers are defined as targets for specific motor axons. To identify potential target labels, we screened 410 genes encoding cell-surface and secreted proteins, searching for those whose overexpression on all muscle fibers causes motor axons to make targeting errors. Thirty such genes were identified, and a number of these were members of a large gene family encoding proteins whose extracellular domains contain leucine-rich repeat (LRR) sequences, which are protein interaction modules. By manipulating gene expression in muscle 12, we showed that four LRR proteins participate in the selection of this muscle as the appropriate synaptic target for the RP5 motor neuron.