A circadian-like gene network programs the timing and dosage of heterochronic miRNA transcription during C. elegans development.

Development relies on the exquisite control of both the timing and the levels of gene expression to achieve robust developmental transitions. How cis- and trans-acting factors control both aspects simultaneously is unclear. We show that transcriptional pulses of the temporal patterning microRNA (miRNA) lin-4 are generated by two nuclear hormone receptors (NHRs) in C. elegans, NHR-85 and NHR-23, whose mammalian orthologs, Rev-Erb and ROR, function in the circadian clock. Although Rev-Erb and ROR antagonize each other to control once-daily transcription in mammals, NHR-85/NHR-23 heterodimers bind cooperatively to lin-4 regulatory elements to induce a single pulse of expression during each larval stage. Each pulse's timing, amplitude, and duration are dictated by the phased expression of these NHRs and the C. elegans Period ortholog, LIN-42, that binds to and represses NHR-85. Therefore, during nematode temporal patterning, an evolutionary rewiring of circadian clock components couples the timing of gene expression to the control of transcriptional dosage.

An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans.

The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex [SKR-1/2-CUL-1-F-box (SCF)], targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin derivative/mutant AtTIR1 pair [C. elegans AID version 2 (C.e.AIDv2)] that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand-binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID-targets, the addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expand the utility of the AID system and broaden the number of proteins that can be effectively targeted with it.

PQN-59 antagonizes microRNA-mediated repression during post-embryonic temporal patterning and modulates translation and stress granule formation in C. elegans.

microRNAs (miRNAs) are potent regulators of gene expression that function in a variety of developmental and physiological processes by dampening the expression of their target genes at a post-transcriptional level. In many gene regulatory networks (GRNs), miRNAs function in a switch-like manner whereby their expression and activity elicit a transition from one stable pattern of gene expression to a distinct, equally stable pattern required to define a nascent cell fate. While the importance of miRNAs that function in this capacity are clear, we have less of an understanding of the cellular factors and mechanisms that ensure the robustness of this form of regulatory bistability. In a screen to identify suppressors of temporal patterning phenotypes that result from ineffective miRNA-mediated target repression, we identified pqn-59, an ortholog of human UBAP2L, as a novel factor that antagonizes the activities of multiple heterochronic miRNAs. Specifically, we find that depletion of pqn-59 can restore normal development in animals with reduced lin-4 and let-7-family miRNA activity. Importantly, inactivation of pqn-59 is not sufficient to bypass the requirement of these regulatory RNAs within the heterochronic GRN. The pqn-59 gene encodes an abundant, cytoplasmically-localized, unstructured protein that harbors three essential "prion-like" domains. These domains exhibit LLPS properties in vitro and normally function to limit PQN-59 diffusion in the cytoplasm in vivo. Like human UBAP2L, PQN-59's localization becomes highly dynamic during stress conditions where it re-distributes to cytoplasmic stress granules and is important for their formation. Proteomic analysis of PQN-59 complexes from embryonic extracts indicates that PQN-59 and human UBAP2L interact with orthologous cellular components involved in RNA metabolism and promoting protein translation and that PQN-59 additionally interacts with proteins involved in transcription and intracellular transport. Finally, we demonstrate that pqn-59 depletion reduces protein translation and also results in the stabilization of several mature miRNAs (including those involved in temporal patterning). These data suggest that PQN-59 may ensure the bistability of some GRNs that require miRNA functions by promoting miRNA turnover and, like UBAP2L, enhancing protein translation.

An Epigenetic Priming Mechanism Mediated by Nutrient Sensing Regulates Transcriptional Output during C. elegans Development.

Although precise tuning of gene expression levels is critical for most developmental pathways, the mechanisms by which the transcriptional output of dosage-sensitive molecules is established or modulated by the environment remain poorly understood. Here, we provide a mechanistic framework for how the conserved transcription factor BLMP-1/Blimp1 operates as a pioneer factor to decompact chromatin near its target loci during embryogenesis (hours prior to major transcriptional activation) and, by doing so, regulates both the duration and amplitude of subsequent target gene transcription during post-embryonic development. This priming mechanism is genetically separable from the mechanisms that establish the timing of transcriptional induction and functions to canalize aspects of cell-fate specification, animal size regulation, and molting. A key feature of the BLMP-1-dependent transcriptional priming mechanism is that chromatin decompaction is initially established during embryogenesis and maintained throughout larval development by nutrient sensing. This anticipatory mechanism integrates transcriptional output with environmental conditions and is essential for resuming normal temporal patterning after animals exit nutrient-mediated developmental arrests.

A Caenorhabditis elegans Model for Integrating the Functions of Neuropsychiatric Risk Genes Identifies Components Required for Normal Dendritic Morphology.

Analysis of patient-derived DNA samples has identified hundreds of variants that are likely involved in neuropsychiatric diseases such as autism spectrum disorder (ASD) and schizophrenia (SCZ). While these studies couple behavioral phenotypes to individual genotypes, the number and diversity of candidate genes implicated in these disorders highlights the fact that the mechanistic underpinnings of these disorders are largely unknown. Here, we describe a RNAi-based screening platform that uses C. elegans to screen candidate neuropsychiatric risk genes (NRGs) for roles in controlling dendritic arborization. To benchmark this approach, we queried published lists of NRGs whose variants in ASD and SCZ are predicted to result in complete or partial loss of gene function. We found that a significant fraction (>16%) of these candidate NRGs are essential for dendritic development. Furthermore, these gene sets are enriched for dendritic arbor phenotypes (>14 fold) when compared to control RNAi datasets of over 500 human orthologs. The diversity of PVD structural abnormalities observed in these assays suggests that the functions of diverse NRGs (encoding transcription factors, chromatin remodelers, molecular chaperones and cytoskeleton-related proteins) converge to regulate neuronal morphology and that individual NRGs may play distinct roles in dendritic branching. We also demonstrate that the experimental value of this platform by providing additional insights into the molecular frameworks of candidate NRGs. Specifically, we show that ANK2/UNC-44 function is directly integrated with known regulators of dendritic arborization and suggest that altering the dosage of ARID1B/LET-526 expression during development affects neuronal morphology without diminishing aspects of cell fate specification.

The Full-Size ABCG Transporter of Medicago truncatula Is Involved in Strigolactone Secretion, Affecting Arbuscular Mycorrhiza.

Strigolactones (SLs) are plant-derived signaling molecules that stimulate the hyphal branching of arbuscular mycorrhizal fungi (AMF), and consequently promote symbiotic interaction between the fungus and the plant. Currently, our knowledge on the molecular mechanism of SL transport is restricted to the Solanaceae family. In the Solanaceae family, SL translocation toward the rhizosphere occurs through the exodermis via hypodermal passage cells and involves a member of the G subfamily, of the ATP-binding cassette (ABC) membrane transporters. Most Fabaceae species, including those that are agriculturally important, have a different root anatomy compared to most angiosperm plants (i.e., lacking an exodermis). Thus, we have investigated how SL transport occurs in the model legume Medicago truncatula. Here, we show that overexpression of a SL transporter from petunia (PaPDR1) enhances AMF colonization rates in M. truncatula. This result demonstrates the importance of ABCG proteins for the translocation of orobanchol-type molecules to facilitate arbuscular mycorrhiza, regardless of root anatomy and phylogenetic relationships. Moreover, our research has led to the identification of Medicago ABCG59, a close homologue of Petunia PDR1, that exhibits root specific expression and is up-regulated by phosphate starvation as well as in the presence of rac-GR24, a synthetic SL. Its promoter is active in cortical cells, root tips, and the meristematic zone of nodules. The mtabcg59 loss-of-function mutant displayed a reduced level of mycorrhization compared to the WT plants but had no impact on the number of nodules after Sinorhizobium meliloti inoculation. The reduced mycorrhization indicates that less SLs are secreted by the mutant plants, which is in line with the observation that mtabcg59 exudates exhibit a reduced stimulatory effect on the germination of the parasitic plant Phelipanche ramosa compared to the corresponding wild type.

Sentryn and SAD Kinase Link the Guided Transport and Capture of Dense Core Vesicles in Caenorhabditis elegans.

Dense core vesicles (DCVs) can transmit signals by releasing neuropeptides from specialized synaptic regions called active zones. DCVs reach the active zone by motorized transport through a long axon. A reverse motor frequently interrupts progress by taking DCVs in the opposite direction. "Guided transport" refers to the mechanism by which outward movements ultimately dominate to bring DCVs to the synaptic region. After guided transport, DCVs alter their interactions with motors and enter a "captured" state. The mechanisms of guided transport and capture of DCVs are unknown. Here, we discovered two proteins that contribute to both processes in Caenorhabditis elegans SAD kinase and a novel conserved protein we named Sentryn are the first proteins found to promote DCV capture. By imaging DCVs moving in various regions of single identified neurons in living animals, we found that DCV guided transport and capture are linked through SAD kinase, Sentryn, and Liprin-α. These proteins act together to regulate DCV motorized transport in a region-specific manner. Between the cell body and the synaptic region, they promote forward transport. In the synaptic region, where all three proteins are highly enriched at active zones, they promote DCV pausing by inhibiting transport in both directions. These three proteins appear to be part of a special subset of active zone-enriched proteins because other active zone proteins do not share their unique functions.

Sentryn Acts with a Subset of Active Zone Proteins To Optimize the Localization of Synaptic Vesicles in Caenorhabditis elegans.

Synaptic vesicles (SVs) transmit signals by releasing neurotransmitters from specialized synaptic regions of neurons. In the synaptic region, SVs are tightly clustered around small structures called active zones. The motor KIF1A transports SVs outward through axons until they are captured in the synaptic region. This transport must be guided in the forward direction because it is opposed by the dynein motor, which causes SVs to reverse direction multiple times en route. The core synapse stability (CSS) system contributes to both guided transport and capture of SVs. We identified Sentryn as a CSS protein that contributes to the synaptic localization of SVs in Caenorhabditis elegans Like the CSS proteins SAD Kinase and SYD-2 (Liprin-α), Sentryn also prevents dynein-dependent accumulation of lysosomes in dendrites in strains lacking JIP3. Genetic analysis showed that Sentryn and SAD Kinase each have at least one nonoverlapping function for the stable accumulation of SVs at synapses that, when combined with their shared functions, enables most of the functions of SYD-2 (Liprin-α) for capturing SVs. Also like other CSS proteins, Sentryn appears enriched at active zones and contributes to active zone structure, suggesting that it is a novel, conserved active zone protein. Sentryn is recruited to active zones by a process dependent on the active zone-enriched CSS protein SYD-2 (Liprin-α). Our results define a specialized group of active zone enriched proteins that can affect motorized transport throughout the neuron and that have roles in both guided transport and capture of SVs.

Abscisic acid - an overlooked player in plant-microbe symbioses formation?

Abscisic acid (ABA) is an ubiquitous plant hormone and one of the foremost signalling molecules, controlling plants' growth and development, as well as their response to environmental stresses. To date, the function of ABA has been extensively investigated as an abiotic stress molecule which regulates the plants' water status. However, in the context of symbiotic associations, ABA is less recognized. In contrast to well-described auxin/cytokinin and gibberellin/strigolactone involvement in symbioses, ABA has long been underestimated. Interestingly, ABA emerges as an important player in arbuscular mycorrhiza and legume-rhizobium symbiosis. The plant's use of stress hormones like ABA in regulation of those interactions directly links the efficiency of these processes to the environmental status of the plant, notably during drought stress. Here we provide an overview of ABA interplay in beneficial associations of plants with microorganisms and propose ABA as a potential factor determining whether the investment in establishing the interaction is higher than the profit coming from it.