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.

PQN-59 and GTBP-1 contribute to stress granule formation but are not essential for their assembly in C. elegans embryos.

When exposed to stressful conditions, eukaryotic cells respond by inducing the formation of cytoplasmic ribonucleoprotein complexes called stress granules. Here, we use C. elegans to study two proteins that are important for stress granule assembly in human cells - PQN-59, the human UBAP2L ortholog, and GTBP-1, the human G3BP1 and G3BP2 ortholog. Both proteins assemble into stress granules in the embryo and in the germline when C. elegans is exposed to stressful conditions. Neither of the two proteins is essential for the assembly of stress-induced granules, as shown by the single and combined depletions by RNAi, and neither pqn-59 nor gtbp-1 mutant embryos show higher sensitivity to stress than control embryos. We find that pqn-59 mutants display reduced progeny and a high percentage of embryonic lethality, phenotypes that are not dependent on stress exposure and that are not shared with gtbp-1 mutants. Our data indicate that, in contrast to human cells, PQN-59 and GTBP-1 are not required for stress granule formation but that PQN-59 is important for C. elegans development.

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.

Rapid Degradation of Caenorhabditis elegans Proteins at Single-Cell Resolution with a Synthetic Auxin.

As developmental biologists in the age of genome editing, we now have access to an ever-increasing array of tools to manipulate endogenous gene expression. The auxin-inducible degradation system allows for spatial and temporal control of protein degradation via a hormone-inducible Arabidopsis F-box protein, transport inhibitor response 1 (TIR1). In the presence of auxin, TIR1 serves as a substrate-recognition component of the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), ubiquitinating auxin-inducible degron (AID)-tagged proteins for proteasomal degradation. Here, we optimize the Caenorhabditis elegans AID system by utilizing 1-naphthaleneacetic acid (NAA), an indole-free synthetic analog of the natural auxin indole-3-acetic acid (IAA). We take advantage of the photostability of NAA to demonstrate via quantitative high-resolution microscopy that rapid degradation of target proteins can be detected in single cells within 30 min of exposure. Additionally, we show that NAA works robustly in both standard growth media and physiological buffer. We also demonstrate that K-NAA, the water-soluble, potassium salt of NAA, can be combined with microfluidics for targeted protein degradation in C. elegans larvae. We provide insight into how the AID system functions in C. elegans by determining that TIR1 depends on C. elegans SKR-1/2, CUL-1, and RBX-1 to degrade target proteins. Finally, we present highly penetrant defects from NAA-mediated degradation of the FTZ-F1 nuclear hormone receptor, NHR-25, during C. elegans uterine-vulval development. Together, this work improves our use and understanding of the AID system for dissecting gene function at the single-cell level during C. elegans development.

Inducing RNAi in Caenorhabditis elegans by Injection of dsRNA.

In Caenorhabditis elegans, long double-stranded RNAs (dsRNAs) are overwhelmingly the trigger of choice for inducing RNA interference (RNAi). Although injection of dsRNA into the somatic or germline tissues of animals requires both specific equipment and technical skills, the ability of C. elegans to amplify the initial dsRNA trigger and to transmit the RNAi activity to other somatic tissues and to the progeny of injected animals is one of the main advantages of using C. elegans as a model system. The direct injection of dsRNA into parental animals is the most reliable method for RNAi and also presents the least experiment-to-experiment and animal-to-animal variability.

Cell death machinery makes life more robust.

CED-3, a protein that is essential for programmed cell death, also has an unexpected role in the regulation of non-apoptotic genes during normal development.

LIN-42, the Caenorhabditis elegans PERIOD homolog, negatively regulates microRNA transcription.

During C. elegans development, microRNAs (miRNAs) function as molecular switches that define temporal gene expression and cell lineage patterns in a dosage-dependent manner. It is critical, therefore, that the expression of miRNAs be tightly regulated so that target mRNA expression is properly controlled. The molecular mechanisms that function to optimize or control miRNA levels during development are unknown. Here we find that mutations in lin-42, the C. elegans homolog of the circadian-related period gene, suppress multiple dosage-dependent miRNA phenotypes including those involved in developmental timing and neuronal cell fate determination. Analysis of mature miRNA levels in lin-42 mutants indicates that lin-42 functions to attenuate miRNA expression. Through the analysis of transcriptional reporters, we show that the upstream cis-acting regulatory regions of several miRNA genes are sufficient to promote highly dynamic transcription that is coupled to the molting cycles of post-embryonic development. Immunoprecipitation of LIN-42 complexes indicates that LIN-42 binds the putative cis-regulatory regions of both non-coding and protein-coding genes and likely plays a role in regulating their transcription. Consistent with this hypothesis, analysis of miRNA transcriptional reporters in lin-42 mutants indicates that lin-42 regulates miRNA transcription. Surprisingly, strong loss-of-function mutations in lin-42 do not abolish the oscillatory expression patterns of lin-4 and let-7 transcription but lead to increased expression of these genes. We propose that lin-42 functions to negatively regulate the transcriptional output of multiple miRNAs and mRNAs and therefore coordinates the expression levels of genes that dictate temporal cell fate with other regulatory programs that promote rhythmic gene expression.

Mutations in conserved residues of the C. elegans microRNA Argonaute ALG-1 identify separable functions in ALG-1 miRISC loading and target repression.

microRNAs function in diverse developmental and physiological processes by regulating target gene expression at the post-transcriptional level. ALG-1 is one of two Caenorhabditis elegans Argonautes (ALG-1 and ALG-2) that together are essential for microRNA biogenesis and function. Here, we report the identification of novel antimorphic (anti) alleles of ALG-1 as suppressors of lin-28(lf) precocious developmental phenotypes. The alg-1(anti) mutations broadly impair the function of many microRNAs and cause dosage-dependent phenotypes that are more severe than the complete loss of ALG-1. ALG-1(anti) mutant proteins are competent for promoting Dicer cleavage of microRNA precursors and for associating with and stabilizing microRNAs. However, our results suggest that ALG-1(anti) proteins may sequester microRNAs in immature and functionally deficient microRNA Induced Silencing Complexes (miRISCs), and hence compete with ALG-2 for access to functional microRNAs. Immunoprecipitation experiments show that ALG-1(anti) proteins display an increased association with Dicer and a decreased association with AIN-1/GW182. These findings suggest that alg-1(anti) mutations impair the ability of ALG-1 miRISC to execute a transition from Dicer-associated microRNA processing to AIN-1/GW182 associated effector function, and indicate an active role for ALG/Argonaute in mediating this transition.

A genome-wide RNAi screen identifies factors required for distinct stages of C. elegans piRNA biogenesis.

In animals, piRNAs and their associated Piwi proteins guard germ cell genomes against mobile genetic elements via an RNAi-like mechanism. In Caenorhabditis elegans, 21U-RNAs comprise the piRNA class, and these collaborate with 22G RNAs via unclear mechanisms to discriminate self from nonself and selectively and heritably silence the latter. Recent work indicates that 21U-RNAs are post-transcriptional processing products of individual transcription units that produce ∼ 26-nucleotide capped precursors. However, nothing is known of how the expression of precursors is controlled or how primary transcripts give rise to mature small RNAs. We conducted a genome-wide RNAi screen to identify components of the 21U biogenesis machinery. Screening by direct, quantitative PCR (qPCR)-based measurements of mature 21U-RNA levels, we identified 22 genes important for 21U-RNA production, termed TOFUs (Twenty-One-u Fouled Ups). We also identified seven genes that normally repress 21U production. By measuring mature 21U-RNA and precursor levels for the seven strongest hits from the screen, we assigned factors to discrete stages of 21U-RNA production. Our work identifies for the first time factors separately required for the transcription of 21U precursors and the processing of these precursors into mature 21U-RNAs, thereby providing a resource for studying the biogenesis of this important small RNA class.

Inducing RNAi in C. elegans by feeding with dsRNA-expressing E. coli.

Owing to the relative ease and high-throughput nature of ingestion-mediated RNAi, the feeding of engineered Escherichia coli to wild-type and mutant Caenorhabditis elegans has developed into the most productive and common method to probe the functions of C. elegans genes. This protocol includes two variations of RNAi by feeding: one in which L1 larvae are fed dsRNA-expressing E. coli in liquid culture (commonly used to score effects of RNAi on postembryonic larval development) and another involving feeding of late larval- or adult-staged animals on standard solid medium culture plates.

A feedback circuit involving let-7-family miRNAs and DAF-12 integrates environmental signals and developmental timing in Caenorhabditis elegans.

Animal development is remarkably robust; cell fates are specified with spatial and temporal precision despite physiological and environmental contingencies. Favorable conditions cause Caenorhabditis elegans to develop rapidly through four larval stages (L1-L4) to the reproductive adult. In unfavorable conditions, L2 larvae can enter the developmentally quiescent, stress-resistant dauer larva stage, enabling them to survive for prolonged periods before completing development. A specific progression of cell division and differentiation events occurs with fidelity during the larval stages, regardless of whether an animal undergoes continuous or dauer-interrupted development. The temporal patterning of developmental events is controlled by the heterochronic genes, whose products include microRNAs (miRNAs) and regulatory proteins. One of these proteins, the DAF-12 nuclear hormone receptor, modulates the transcription of certain let-7-family miRNAs, and also mediates the choice between the continuous vs. dauer-interrupted life history. Here, we report a complex feedback loop between DAF-12 and the let-7-family miRNAs involving both the repression of DAF-12 by let-7-family miRNAs and the ligand-modulated transcriptional activation and repression of the let-7-Fam miRNAs by DAF-12. We propose that this feedback loop functions to ensure robustness of cell fate decisions and to coordinate cell fate with developmental arrest.

nhl-2 Modulates microRNA activity in Caenorhabditis elegans.

TRIM-NHL proteins represent a large class of metazoan proteins implicated in development and disease. We demonstrate that a C. elegans TRIM-NHL protein, NHL-2, functions as a cofactor for the microRNA-induced silencing complex (miRISC) and thereby enhances the posttranscriptional repression of several genetically verified microRNA targets, including hbl-1 and let-60/Ras (by the let-7 family of microRNAs) and cog-1 (by the lsy-6 microRNA). NHL-2 is localized to cytoplasmic P-bodies and physically associates with the P-body protein CGH-1 and the core miRISC components ALG-1/2 and AIN-1. nhl-2 and cgh-1 mutations compromise the repression of microRNA targets in vivo but do not affect microRNA biogenesis, indicating a role for an NHL-2:CGH-1 complex in the effector phase of miRISC activity. We propose that the NHL-2:CGH-1 complex functions in association with mature miRISC to modulate the efficacy of microRNA:target interactions in response to physiological and developmental signals, thereby ensuring the robustness of genetic regulatory pathways regulated by microRNAs.

The microRNA-argonaute complex: a platform for mRNA modulation.

With the cloning the lin-4 gene in 1993, the possibility of an approximately 21-nucleotide RNA functioning as a regulatory molecule intrigued a relatively small number of scientists. This idea appeared to be a peculiarity of C. elegans as it was not until seven years later that the second, more conserved small RNA, let-7 was cloned. A spate of papers in 2000 and 2001 revealed that the underlying properties of the lin-4 and let-7 genes were a common facet of animal genomes and the absolute number and potential of this new class of gene products requires us to integrate them with other aspects of gene expression and evolution.(1-3) A wealth of information has accumulated in the intervening years that outline, in general, how these small RNAs are expressed and processed into a functional form. Contemporaneous to these studies, experiments also identified a cadre of evolutionarily conserved proteins, the Argonautes (Agos) that directly associate with and are required for microRNA function. Computational and experimental methods have led the identification of many functional mRNA targets. In the last few years, a significant body of work has focused on resolving two key issues: How do microRNAs function in particular genetic contexts (i.e., as "molecular switches" or "fine-tuners" of gene expression) and secondly, what facet/s of mRNA metabolism do microRNAs modulate in their role(s) as a regulatory molecule? The primary objective here is not to comprehensively compare the competing models of microRNA function (reviewed in refs. 4-6) but to frame a potential solution to these two fundamental questions by suggesting that the core microRNA-Ribonucleic-Protein Complex (microRNP), composed of the microRNA and an Ago protein, functions as a highly modifiable scaffold that associates with specific mRNAs via the bound microRNA and facilitates the localized activity of a variety of accessory proteins. The resulting composite mechanism could account for the apparent complexities of measuring microRNA activity and furthermore, accommodate the broad levels of regulation observed in vivo.

Interacting endogenous and exogenous RNAi pathways in Caenorhabditis elegans.

C. elegans contains numerous small RNAs of ~21-24 nt in length. The microRNAs (miRNAs) are small noncoding RNAs produced by DCR-1- and ALG-dependent processing of self-complementary hairpin transcripts. Endogenous small interfering RNAs (endo-siRNAs), associated with ongoing silencing of protein-coding genes in normal worms, are produced by mechanisms that involve DCR-1 but, unlike miRNAs, also involve RDE-2, RDE-3, RDE-4, RRF-1, and RRF-3. The tiny noncoding (tncRNAs) are similar to endo-siRNAs in their biogenesis except that they are derived from noncoding sequences. These endo-siRNA- and tncRNA-based endogenous RNAi pathways involve some components, including DCR-1 and RDE-4, that are shared with exogenous RNAi, and some components, including RRF-3 and ERI-1, that are specific to endogenous RNAi. rrf-3 and eri-1 mutants are enhanced for some silencing processes and defective for others, suggesting cross-regulatory interactions between RNAi pathways in C. elegans. Microarray expression profiling of RNAi-defective mutant worms further suggests diverse endogenous RNAi pathways for silencing different sets of genes.

Yeast poly(A)-binding protein, Pab1, and PAN, a poly(A) nuclease complex recruited by Pab1, connect mRNA biogenesis to export.

In eukaryotic cells, pre-mRNAs undergo extensive processing in the nucleus prior to export. Processing is subject to a quality-control mechanism that retains improperly processed transcripts at or near sites of transcription. A poly(A) tail added by the normal 3'-processing machinery is necessary but not sufficient for export. Retention depends on the exosome. In this study, we identify the poly(A)-binding protein, Pab1, and the poly(A) nuclease, PAN, as important factors that couple 3' processing to export. Pab1 contains a nonessential leucine-rich nuclear export signal and shuttles between the nucleus and the cytoplasm. It can exit the nucleus either as cargo of exportin 1 or bound to mRNA. Pab1 is essential but several bypass suppressors have been identified. Deletion of PAB1 from these bypass suppressor strains results in exosome-dependent retention at sites of transcription. Retention is also seen in cells lacking PAN, which Pab1 is thought to recruit and which may be responsible for the final step of mRNA biogenesis, trimming of the poly(A) tail to the length found on newly exported mRNAs. The studies presented here suggest that proper loading of Pab1 onto mRNAs and final trimming of the tail allows release from transcription sites and couples pre-mRNA processing to export.