Multigenerational Regulation of the Caenorhabditis elegans Chromatin Landscape by Germline Small RNAs.

In animals, small noncoding RNAs that are expressed in the germline and transmitted to progeny control gene expression to promote fertility. Germline-expressed small RNAs, including endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs), drive the repression of deleterious transcripts such as transposons, repetitive elements, and pseudogenes. Recent studies have highlighted an important role for small RNAs in transgenerational epigenetic inheritance via regulation of heritable chromatin marks; therefore, small RNAs are thought to convey an epigenetic memory of genomic self and nonself elements. Small RNA pathways are highly conserved in metazoans and have been best described for the model organism Caenorhabditis elegans. In this review, we describe the biogenesis, regulation, and function of C. elegans endo-siRNAs and piRNAs, along with recent insights into how these distinct pathways are integrated to collectively regulate germline gene expression, transgenerational epigenetic inheritance, and ultimately, animal fertility.

The Gene-Silencing Protein MORC-1 Topologically Entraps DNA and Forms Multimeric Assemblies to Cause DNA Compaction.

Microrchidia (MORC) ATPases are critical for gene silencing and chromatin compaction in multiple eukaryotic systems, but the mechanisms by which MORC proteins act are poorly understood. Here, we apply a series of biochemical, single-molecule, and cell-based imaging approaches to better understand the function of the Caenorhabditis elegans MORC-1 protein. We find that MORC-1 binds to DNA in a length-dependent but sequence non-specific manner and compacts DNA by forming DNA loops. MORC-1 molecules diffuse along DNA but become static as they grow into foci that are topologically entrapped on DNA. Consistent with the observed MORC-1 multimeric assemblies, MORC-1 forms nuclear puncta in cells and can also form phase-separated droplets in vitro. We also demonstrate that MORC-1 compacts nucleosome templates. These results suggest that MORCs affect genome structure and gene silencing by forming multimeric assemblages to topologically entrap and progressively loop and compact chromatin.

FUS Regulates Activity of MicroRNA-Mediated Gene Silencing.

MicroRNA-mediated gene silencing is a fundamental mechanism in the regulation of gene expression. It remains unclear how the efficiency of RNA silencing could be influenced by RNA-binding proteins associated with the microRNA-induced silencing complex (miRISC). Here we report that fused in sarcoma (FUS), an RNA-binding protein linked to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), interacts with the core miRISC component AGO2 and is required for optimal microRNA-mediated gene silencing. FUS promotes gene silencing by binding to microRNA and mRNA targets, as illustrated by its action on miR-200c and its target ZEB1. A truncated mutant form of FUS that leads its carriers to an aggressive form of ALS, R495X, impairs microRNA-mediated gene silencing. The C. elegans homolog fust-1 also shares a conserved role in regulating the microRNA pathway. Collectively, our results suggest a role for FUS in regulating the activity of microRNA-mediated silencing.

daf-16/FOXO blocks adult cell fate in Caenorhabditis elegans dauer larvae via lin-41/TRIM71.

Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16/FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp. Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29. lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development, lin-41 opposes adult cell fate by inhibiting the translation of lin-29, which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41, but does not require lin-29. Taken together, this work demonstrates a novel role for daf-16/FOXO as a heterochronic gene that promotes expression of lin-41/TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.

Compartmentalization and metabolic regulation of glycolysis.

Hypoxia inhibits the tricarboxylic acid (TCA) cycle and leaves glycolysis as the primary metabolic pathway responsible for converting glucose into usable energy. However, the mechanisms that compensate for this loss in energy production due to TCA cycle inactivation remain poorly understood. Glycolysis enzymes are typically diffuse and soluble in the cytoplasm under normoxic conditions. In contrast, recent studies have revealed dynamic compartmentalization of glycolysis enzymes in response to hypoxic stress in yeast, C. elegans and mammalian cells. These messenger ribonucleoprotein (mRNP) structures, termed glycolytic (G) bodies in yeast, lack membrane enclosure and display properties of phase-separated biomolecular condensates. Disruption of condensate formation correlates with defects such as impaired synaptic function in C. elegans neurons and decreased glucose flux in yeast. Concentrating glycolysis enzymes into condensates may lead to their functioning as 'metabolons' that enhance rates of glucose utilization for increased energy production. Besides condensates, glycolysis enzymes functionally associate in other organisms and specific tissues through protein-protein interactions and membrane association. However, as discussed in this Review, the functional consequences of coalescing glycolytic machinery are only just beginning to be revealed. Through ongoing studies, we anticipate the physiological importance of metabolic regulation mediated by the compartmentalization of glycolysis enzymes will continue to emerge.

The full-length transcriptome of C. elegans using direct RNA sequencing.

Current transcriptome annotations have largely relied on short read lengths intrinsic to the most widely used high-throughput cDNA sequencing technologies. For example, in the annotation of the Caenorhabditis elegans transcriptome, more than half of the transcript isoforms lack full-length support and instead rely on inference from short reads that do not span the full length of the isoform. We applied nanopore-based direct RNA sequencing to characterize the developmental polyadenylated transcriptome of C. elegans Taking advantage of long reads spanning the full length of mRNA transcripts, we provide support for 23,865 splice isoforms across 14,611 genes, without the need for computational reconstruction of gene models. Of the isoforms identified, 3452 are novel splice isoforms not present in the WormBase WS265 annotation. Furthermore, we identified 16,342 isoforms in the 3' untranslated region (3' UTR), 2640 of which are novel and do not fall within 10 bp of existing 3'-UTR data sets and annotations. Combining 3' UTRs and splice isoforms, we identified 28,858 full-length transcript isoforms. We also determined that poly(A) tail lengths of transcripts vary across development, as do the strengths of previously reported correlations between poly(A) tail length and expression level, and poly(A) tail length and 3'-UTR length. Finally, we have formatted this data as a publicly accessible track hub, enabling researchers to explore this data set easily in a genome browser.

The microRNA miR-1 regulates a MEF-2-dependent retrograde signal at neuromuscular junctions.

We show that miR-1, a conserved muscle-specific microRNA, regulates aspects of both pre- and postsynaptic function at C. elegans neuromuscular junctions. miR-1 regulates the expression level of two nicotinic acetylcholine receptor (nAChR) subunits (UNC-29 and UNC-63), thereby altering muscle sensitivity to acetylcholine (ACh). miR-1 also regulates the muscle transcription factor MEF-2, which results in altered presynaptic ACh secretion, suggesting that MEF-2 activity in muscles controls a retrograde signal. The effect of the MEF-2-dependent retrograde signal on secretion is mediated by the synaptic vesicle protein RAB-3. Finally, acute activation of levamisole-sensitive nAChRs stimulates MEF-2-dependent transcriptional responses and induces the MEF-2-dependent retrograde signal. We propose that miR-1 refines synaptic function by coupling changes in muscle activity to changes in presynaptic function.

A kinesin Klp10A mediates cell cycle-dependent shuttling of Piwi between nucleus and nuage.

The piRNA pathway protects germline genomes from selfish genetic elements (e.g. transposons) through their transcript cleavage in the cytoplasm and/or their transcriptional silencing in the nucleus. Here, we describe a mechanism by which the nuclear and cytoplasmic arms of the piRNA pathway are linked. We find that during mitosis of Drosophila spermatogonia, nuclear Piwi interacts with nuage, the compartment that mediates the cytoplasmic arm of the piRNA pathway. At the end of mitosis, Piwi leaves nuage to return to the nucleus. Dissociation of Piwi from nuage occurs at the depolymerizing microtubules of the central spindle, mediated by a microtubule-depolymerizing kinesin, Klp10A. Depletion of klp10A delays the return of Piwi to the nucleus and affects piRNA production, suggesting the role of nuclear-cytoplasmic communication in piRNA biogenesis. We propose that cell cycle-dependent communication between the nuclear and cytoplasmic arms of the piRNA pathway may play a previously unappreciated role in piRNA regulation.

Context-dependent modulation of Pol II CTD phosphatase SSUP-72 regulates alternative polyadenylation in neuronal development.

Alternative polyadenylation (APA) is widespread in neuronal development and activity-mediated neural plasticity. However, the underlying molecular mechanisms are largely unknown. We used systematic genetic studies and genome-wide surveys of the transcriptional landscape to identify a context-dependent regulatory pathway controlling APA in the Caenorhabditis elegans nervous system. Loss of function in ssup-72, a Ser5 phosphatase for the RNA polymerase II (Pol II) C-terminal domain (CTD), dampens transcription termination at a strong intronic polyadenylation site (PAS) in unc-44/ankyrin yet promotes termination at the weak intronic PAS of the MAP kinase dlk-1. A nuclear protein, SYDN-1, which regulates neuronal development, antagonizes the function of SSUP-72 and several nuclear polyadenylation factors. This regulatory pathway allows the production of a neuron-specific isoform of unc-44 and an inhibitory isoform of dlk-1. Dysregulation of the unc-44 and dlk-1 mRNA isoforms in sydn-1 mutants impairs neuronal development. Deleting the intronic PAS of unc-44 results in increased pre-mRNA processing of neuronal ankyrin and suppresses sydn-1 mutants. These results reveal a mechanism by which regulation of CTD phosphorylation controls coding region APA in the nervous system.

Identification of small RNA pathway genes using patterns of phylogenetic conservation and divergence.

Genetic and biochemical analyses of RNA interference (RNAi) and microRNA (miRNA) pathways have revealed proteins such as Argonaute and Dicer as essential cofactors that process and present small RNAs to their targets. Well-validated small RNA pathway cofactors such as these show distinctive patterns of conservation or divergence in particular animal, plant, fungal and protist species. We compared 86 divergent eukaryotic genome sequences to discern sets of proteins that show similar phylogenetic profiles with known small RNA cofactors. A large set of additional candidate small RNA cofactors have emerged from functional genomic screens for defects in miRNA- or short interfering RNA (siRNA)-mediated repression in Caenorhabditis elegans and Drosophila melanogaster, and from proteomic analyses of proteins co-purifying with validated small RNA pathway proteins. The phylogenetic profiles of many of these candidate small RNA pathway proteins are similar to those of known small RNA cofactor proteins. We used a Bayesian approach to integrate the phylogenetic profile analysis with predictions from diverse transcriptional coregulation and proteome interaction data sets to assign a probability for each protein for a role in a small RNA pathway. Testing high-confidence candidates from this analysis for defects in RNAi silencing, we found that about one-half of the predicted small RNA cofactors are required for RNAi silencing. Many of the newly identified small RNA pathway proteins are orthologues of proteins implicated in RNA splicing. In support of a deep connection between the mechanism of RNA splicing and small-RNA-mediated gene silencing, the presence of the Argonaute proteins and other small RNA components in the many species analysed strongly correlates with the number of introns in those species.

Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1.

MicroRNAs (miRNAs) play essential, conserved roles in diverse developmental processes through association with the miRNA-induced silencing complex (miRISC). Whereas fundamental insights into the mechanistic framework of miRNA biogenesis and target gene silencing have been established, posttranslational modifications that affect miRISC function are less well understood. Here we report that the conserved serine/threonine kinase, casein kinase II (CK2), promotes miRISC function in Caenorhabditis elegans. CK2 inactivation results in developmental defects that phenocopy loss of miRISC cofactors and enhances the loss of miRNA function in diverse cellular contexts. Whereas CK2 is dispensable for miRNA biogenesis and the stability of miRISC cofactors, it is required for efficient miRISC target mRNA binding and silencing. Importantly, we identify the conserved DEAD-box RNA helicase, CGH-1/DDX6, as a key CK2 substrate within miRISC and demonstrate phosphorylation of a conserved N-terminal serine is required for CGH-1 function in the miRNA pathway.

A conserved upstream motif orchestrates autonomous, germline-enriched expression of Caenorhabditis elegans piRNAs.

Piwi-interacting RNAs (piRNAs) fulfill a critical, conserved role in defending the genome against foreign genetic elements. In many organisms, piRNAs appear to be derived from processing of a long, polycistronic RNA precursor. Here, we establish that each Caenorhabditis elegans piRNA represents a tiny, autonomous transcriptional unit. Remarkably, the minimal C. elegans piRNA cassette requires only a 21 nucleotide (nt) piRNA sequence and an ∼50 nt upstream motif with limited genomic context for expression. Combining computational analyses with a novel, in vivo transgenic system, we demonstrate that this upstream motif is necessary for independent expression of a germline-enriched, Piwi-dependent piRNA. We further show that a single nucleotide position within this motif directs differential germline enrichment. Accordingly, over 70% of C. elegans piRNAs are selectively expressed in male or female germline, and comparison of the genes they target suggests that these two populations have evolved independently. Together, our results indicate that C. elegans piRNA upstream motifs act as independent promoters to specify which sequences are expressed as piRNAs, how abundantly they are expressed, and in what germline. As the genome encodes well over 15,000 unique piRNA sequences, our study reveals that the number of transcriptional units encoding piRNAs rivals the number of mRNA coding genes in the C. elegans genome.

The Caenorhabditis elegans HEN1 ortholog, HENN-1, methylates and stabilizes select subclasses of germline small RNAs.

Small RNAs regulate diverse biological processes by directing effector proteins called Argonautes to silence complementary mRNAs. Maturation of some classes of small RNAs involves terminal 2'-O-methylation to prevent degradation. This modification is catalyzed by members of the conserved HEN1 RNA methyltransferase family. In animals, Piwi-interacting RNAs (piRNAs) and some endogenous and exogenous small interfering RNAs (siRNAs) are methylated, whereas microRNAs are not. However, the mechanisms that determine animal HEN1 substrate specificity have yet to be fully resolved. In Caenorhabditis elegans, a HEN1 ortholog has not been studied, but there is evidence for methylation of piRNAs and some endogenous siRNAs. Here, we report that the worm HEN1 ortholog, HENN-1 (HEN of Nematode), is required for methylation of C. elegans small RNAs. Our results indicate that piRNAs are universally methylated by HENN-1. In contrast, 26G RNAs, a class of primary endogenous siRNAs, are methylated in female germline and embryo, but not in male germline. Intriguingly, the methylation pattern of 26G RNAs correlates with the expression of distinct male and female germline Argonautes. Moreover, loss of the female germline Argonaute results in loss of 26G RNA methylation altogether. These findings support a model wherein methylation status of a metazoan small RNA is dictated by the Argonaute to which it binds. Loss of henn-1 results in phenotypes that reflect destabilization of substrate small RNAs: dysregulation of target mRNAs, impaired fertility, and enhanced somatic RNAi. Additionally, the henn-1 mutant shows a weakened response to RNAi knockdown of germline genes, suggesting that HENN-1 may also function in canonical RNAi. Together, our results indicate a broad role for HENN-1 in both endogenous and exogenous gene silencing pathways and provide further insight into the mechanisms of HEN1 substrate discrimination and the diversity within the Argonaute family.

A maternally programmed intergenerational mechanism enables male offspring to make piRNAs from Y-linked precursor RNAs in Drosophila.

In animals, PIWI-interacting RNAs (piRNAs) direct PIWI proteins to silence complementary targets such as transposons. In Drosophila and other species with a maternally specified germline, piRNAs deposited in the egg initiate piRNA biogenesis in the progeny. However, Y chromosome loci cannot participate in such a chain of intergenerational inheritance. How then can the biogenesis of Y-linked piRNAs be initiated? Here, using Suppressor of Stellate (Su(Ste)), a Y-linked Drosophila melanogaster piRNA locus as a model, we show that Su(Ste) piRNAs are made in the early male germline via 5'-to-3' phased piRNA biogenesis initiated by maternally deposited 1360/Hoppel transposon piRNAs. Notably, deposition of Su(Ste) piRNAs from XXY mothers obviates the need for phased piRNA biogenesis in sons. Together, our study uncovers a developmentally programmed, intergenerational mechanism that allows fly mothers to protect their sons using a Y-linked piRNA locus.

High-resolution profiling and discovery of planarian small RNAs.

Freshwater planarian flatworms possess uncanny regenerative capacities mediated by abundant and collectively totipotent adult stem cells. Key functions of these cells during regeneration and tissue homeostasis have been shown to depend on PIWI, a molecule required for Piwi-interacting RNA (piRNA) expression in planarians. Nevertheless, the full complement of piRNAs and microRNAs (miRNAs) in this organism has yet to be defined. Here we report on the large-scale cloning and sequencing of small RNAs from the planarian Schmidtea mediterranea, yielding altogether millions of sequenced, unique small RNAs. We show that piRNAs are in part organized in genomic clusters and that they share characteristic features with mammalian and fly piRNAs. We further identify 61 novel miRNA genes and thus double the number of known planarian miRNAs. Sequencing, as well as quantitative PCR of small RNAs, uncovered 10 miRNAs enriched in planarian stem cells. These miRNAs are down-regulated in animals in which stem cells have been abrogated by irradiation, and thus constitute miRNAs likely associated with specific stem-cell functions. Altogether, we present the first comprehensive small RNA analysis in animals belonging to the third animal superphylum, the Lophotrochozoa, and single out a number of miRNAs that may function in regeneration. Several of these miRNAs are deeply conserved in animals.

26G endo-siRNAs regulate spermatogenic and zygotic gene expression in Caenorhabditis elegans.

Endogenous small interfering RNAs (endo-siRNAs) regulate diverse gene expression programs in eukaryotes by either binding and cleaving mRNA targets or mediating heterochromatin formation; however, the mechanisms of endo-siRNA biogenesis, sorting, and target regulation remain poorly understood. Here we report the identification and function of a specific class of germline-generated endo-siRNAs in Caenorhabditis elegans that are 26 nt in length and contain a guanine at the first nucleotide position (i.e., 26G RNAs). 26G RNAs regulate gene expression during spermatogenesis and zygotic development, and their biogenesis requires the ERI-1 exonuclease and the RRF-3 RNA-dependent RNA polymerase (RdRP). Remarkably, we identified two nonoverlapping subclasses of 26G RNAs that sort into specific RNA-induced silencing complexes (RISCs) and differentially regulate distinct mRNA targets. Class I 26G RNAs target genes are expressed during spermatogenesis, whereas class II 26G RNAs are maternally inherited and silence gene expression during zygotic development. These findings implicate a class of endo-siRNAs in the global regulation of transcriptional programs required for fertility and development.

Feedback between a retinoid-related nuclear receptor and the let-7 microRNAs controls the pace and number of molting cycles in C. elegans.

Animal development requires coordination among cyclic processes, sequential cell fate specifications, and once-a-lifetime morphogenic events, but the underlying timing mechanisms are not well understood. Caenorhabditis elegans undergoes four molts at regular 8 to 10 hour intervals. The pace of the cycle is governed by PERIOD/lin-42 and other as-yet unknown factors. Cessation of the cycle in young adults is controlled by the let-7 family of microRNAs and downstream transcription factors in the heterochronic pathway. Here, we characterize a negative feedback loop between NHR-23, the worm homolog of mammalian retinoid-related orphan receptors (RORs), and the let-7 family of microRNAs that regulates both the frequency and finite number of molts. The molting cycle is decelerated in nhr-23 knockdowns and accelerated in let-7(-) mutants, but timed similarly in let-7(-) nhr-23(-) double mutants and wild-type animals. NHR-23 binds response elements (ROREs) in the let-7 promoter and activates transcription. In turn, let-7 dampens nhr-23 expression across development via a complementary let-7-binding site (LCS) in the nhr-23 3' UTR. The molecular interactions between NHR-23 and let-7 hold true for other let-7 family microRNAs. Either derepression of nhr-23 transcripts by LCS deletion or high gene dosage of nhr-23 leads to protracted behavioral quiescence and extra molts in adults. NHR-23 and let-7 also coregulate scores of genes required for execution of the molts, including lin-42. In addition, ROREs and LCSs isolated from mammalian ROR and let-7 genes function in C. elegans, suggesting conservation of this feedback mechanism. We propose that this feedback loop unites the molting timer and the heterochronic gene regulatory network, possibly by functioning as a cycle counter.

Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants.

Caenorhabditis elegans homologues of the retinoblastoma (Rb) tumour suppressor complex specify cell lineage during development. Here we show that mutations in Rb pathway components enhance RNA interference (RNAi) and cause somatic cells to express genes and elaborate perinuclear structures normally limited to germline-specific P granules. Furthermore, particular gene inactivations that disrupt RNAi reverse the cell lineage transformations of Rb pathway mutants. These findings suggest that mutations in Rb pathway components cause cells to revert to patterns of gene expression normally restricted to germ cells. Rb may act by a similar mechanism to transform mammalian cells.

SNPC-1.3 is a sex-specific transcription factor that drives male piRNA expression in C. elegans.

Piwi-interacting RNAs (piRNAs) play essential roles in silencing repetitive elements to promote fertility in metazoans. Studies in worms, flies, and mammals reveal that piRNAs are expressed in a sex-specific manner. However, the mechanisms underlying this sex-specific regulation are unknown. Here we identify SNPC-1.3, a male germline-enriched variant of a conserved subunit of the small nuclear RNA-activating protein complex, as a male-specific piRNA transcription factor in Caenorhabditis elegans. SNPC-1.3 colocalizes with the core piRNA transcription factor, SNPC-4, in nuclear foci of the male germline. Binding of SNPC-1.3 at male piRNA loci drives spermatogenic piRNA transcription and requires SNPC-4. Loss of snpc-1.3 leads to depletion of male piRNAs and defects in male-dependent fertility. Furthermore, TRA-1, a master regulator of sex determination, binds to the snpc-1.3 promoter and represses its expression during oogenesis. Loss of TRA-1 targeting causes ectopic expression of snpc-1.3 and male piRNAs during oogenesis. Thus, sexually dimorphic regulation of snpc-1.3 expression coordinates male and female piRNA expression during germline development.