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Summary tiny-count is a highly flexible counting tool that allows for hierarchical classification and quantification of small RNA reads from high-throughput sequencing data. Selection rules can be used to filter reads by 5’ nucleotide, length, position of alignments in relation to reference features, and by the number of mismatches to reference sequences. tiny-count can quantify reads aligned to a genome or directly to small RNA or transcript sequences. With tiny-count, users can quantify a single class of small RNAs or multiple classes in parallel. tiny-count can resolve distinct classes of small RNAs, for example piRNAs and siRNAs, produced from the same locus. It can distinguish small RNAs variants, such as miRNAs and isomiRs, with single-nucleotide precision. tRNA, rRNA, and other RNA fragments can also be quantified. tiny-count can be run alone or as part of tinyRNA, a workflow that provides a basic all-in-one command line-based solution for small RNA-seq data analysis, with documentation and statistics generated at each step for accurate and reproducible results. Availability tiny-count and other tinyRNA tools are implemented in Python, C ++, Cython, and R, and the workflow is coordinated with CWL. tiny-count and tinyRNA are free and open-source software distributed under the GPLv3 license. tiny-count can be installed via Bioconda (https://anaconda.org/bioconda/tiny-count) and both tiny-count and tinyRNA documentation and software downloads are available at https://github.com/MontgomeryLab/tinyRNA. Supplementary information Reference data, including genome sequences and features tables, for certain species can be found at https://www.MontgomeryLab.org.

SummarytinyRNA performs precision analysis of small RNAs, including miRNAs, piRNAs, and siRNAs, from high-throughput sequencing experiments. At the core of tinyRNA is a highly flexible counting utility, tiny-count, that allows for hierarchical assignment of small RNA reads to features based on positional information, extent of feature overlap, 5’ nucleotide, length, and strandedness. tinyRNA provides an all-in-one solution for small RNA-seq data analysis, with documentation and statistics generated at each step for accurate, reproducible results.Availability and ImplementationtinyRNA tools are implemented in Python and R, and the pipeline workflow is coordinated with CWL. tinyRNA is free and open-source software distributed under the GPLv3 license. tinyRNA is available at https://github.com/MontgomeryLab/tinyRNA.Contacttai.montgomery@colostate.eduSupplementary informationReference data, including genome sequences and features tables, for certain species can be found at https://www.MontgomeryLab.org.

Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) are distinct classes of small RNAs required for proper germline development. To identify the roles of piRNAs and siRNAs in regulating gene expression in Caenorhabditis elegans, we subjected small RNAs and mRNAs from the gonads of piRNA and siRNA defective mutants to high-throughput sequencing. We show that piRNAs and an abundant class of siRNAs known as WAGO-class 22G-RNAs are required for proper expression of spermatogenic and oogenic genes. WAGO-class 22G-RNAs are also broadly required for transposon silencing, whereas piRNAs are largely dispensable. piRNAs, however, have a critical role in controlling histone gene expression. In the absence of piRNAs, histone mRNAs are misrouted into the nuclear RNAi pathway involving the Argonaute HRDE-1, concurrent with a reduction in the expression of many histone mRNAs. We also show that high-level gene expression in the germline is correlated with high level 22G-RNA production. However, most highly expressed genes produce 22G-RNAs through a distinct pathway that presumably involves the Argonaute CSR-1. In contrast, genes targeted by the WAGO branch of the 22G-RNA pathway are typically poorly expressed and respond unpredictably to loss of 22G-RNAs. Our results point to broad roles for piRNAs and siRNAs in controlling gene expression in the C. elegans germline.

A major challenge to our understanding of translational control has been deconvolving the individual impact specific regulatory factors have on the complex dynamics of mRNA translation. MicroRNAs (miRNAs), for example, guide Argonaute and associated proteins to target mRNAs, where they direct gene silencing in multiple ways that are not well understood. To better deconvolve these dynamics, we have developed technology to directly visualize and quantify the impact of human Argonaute2 (Ago2) on the translation and subcellular localization of individual reporter mRNAs in living cells. We show that our combined translation and Ago2 tethering system reflects endogenous miRNA-mediated gene silencing. Using the system, we find that Ago2 association leads to progressive silencing of translation at individual mRNA. The timescale of silencing was similar to that of translation, consistent with a role for Ago2 in blocking translation initiation, leading to ribosome runoff. At early time points, we observed occasional brief bursts of translational activity at Ago2-tethered mRNAs undergoing silencing, suggesting that translational repression may initially be reversible. At later time points, Ago2-tethered mRNAs cluster and coalesce with endogenous P-bodies, where a translationally silent state is maintained. These results provide a framework for exploring miRNA-mediated gene regulation in live cells at the single-molecule level. Furthermore, our tethering-based, single-molecule reporter system will likely have wide-ranging application in studying general RNA-protein interactions.

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 inCaenorhabditis 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 ofsnpc-1.3leads to depletion of male piRNAs and defects in male-dependent fertility. Furthermore, TRA-1, a master regulator of sex determination, binds to thesnpc-1.3promoter and represses its expression during oogenesis. Loss of TRA-1 targeting causes ectopic expression ofsnpc-1.3and male piRNAs during oogenesis. Thus, sexually dimorphic regulation ofsnpc-1.3expression coordinates male and female piRNA expression during germline development.

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 variant of a conserved subunit of the snRNA activating protein complex, as a male-specific piRNA transcription factor inC. elegans. Binding of SNPC-1.3 at male piRNA loci drives spermatogenic piRNA transcription and requires the core piRNA transcription factor SNPC-4. Loss ofsnpc-1.3leads to depletion of male piRNAs and defects in male-dependent fertility. Furthermore, TRA-1, a master regulator of sex determination, binds to thesnpc-1.3promoter and represses its expression during oogenesis. Loss of TRA-1 targeting causes ectopic expression ofsnpc-1.3and male piRNAs during oogenesis. Thus, sexual dimorphic regulation ofsnpc-1.3coordinates male and female piRNA expression during germline development.

Piwi-interacting RNAs (piRNAs) are a largely germline-specific class of small RNAs found in animals. Although piRNAs are best known for silencing transposons, they regulate many different biological processes. Here we identify a role for piRNAs in preventing runaway amplification of small interfering RNAs (siRNAs) from certain genes, including ribosomal RNAs (rRNAs) and histone mRNAs. In Caenorhabditis elegans, rRNAs and some histone mRNAs are heavily targeted by piRNAs, which facilitates their entry into an endogenous RNA interference (RNAi) pathway involving a class of siRNAs called 22G-RNAs. Under normal conditions, rRNAs and histone mRNAs produce relatively low levels of 22G-RNAs. But if piRNAs are lost, 22G-RNA production is highly elevated. We show that 22G-RNAs produced downstream of piRNAs likely function in a feed-forward amplification circuit. Thus, our results suggest that piRNAs facilitate low-level 22G-RNA production while simultaneously obstructing the 22G-RNA machinery to prevent runaway amplification from certain RNAs. Histone mRNAs and rRNAs are unique from other cellular RNAs in lacking polyA tails, which may promote feed-forward amplification of 22G-RNAs. In support of this, we show that the subset of histone mRNAs that contain polyA tails are largely resistant to silencing in piRNA mutants.

piRNAs play a critical role in the regulation of transposons and other germline genes. In Caenorhabditis elegans, regulation of piRNA target genes is mediated by the mutator complex, which synthesizes high levels of siRNAs through the activity of an RNA-dependent RNA polymerase. However, the steps between mRNA recognition by the piRNA pathway and siRNA amplification by the mutator complex are unknown. Here, we identify the Tudor domain protein, SIMR-1, as acting downstream of piRNA production and upstream of mutator complex-dependent siRNA biogenesis. Interestingly, SIMR-1 also localizes to distinct subcellular foci adjacent to P granules and Mutator foci, two phase-separated condensates that are the sites of piRNA-dependent mRNA recognition and mutator complex-dependent siRNA amplification, respectively. Thus, our data suggests a role for multiple perinuclear condensates in organizing the piRNA pathway and promoting mRNA regulation by the mutator complex.

SUMMARY Piwi-interacting RNAs (piRNAs) regulate many biological processes through mechanisms that are not fully understood. In Caenorhabditis elegans, piRNAs intersect the endogenous RNA interference (RNAi) pathway, involving a distinct class of small RNAs called 22G-RNAs, to regulate gene expression in the germline. In the absence of piRNAs, 22G-RNA production from many genes is reduced, pointing to a role for piRNAs in facilitating endogenous RNAi. Here, however, we show that many genes gain, rather than lose, 22G-RNAs in the absence of piRNAs, which is in some instances coincident with RNA silencing. Aberrant 22G-RNA production is somewhat stochastic but once established can occur within a population for at least 50 generations. Thus, piRNAs both promote and suppress 22G-RNA production and gene silencing. rRNAs and histones are hypersusceptible to aberrant silencing, but we do not find evidence that their misexpression is the primary cause of the transgenerational sterility observed in piRNA-defective mutants., Graphical Abstract, In brief Montgomery et al. show that piRNAs both promote and suppress siRNA production and RNA silencing in C. elegans. Gain or loss of siRNAs occurs somewhat stochastically in piRNA-defective mutants but once established, it occurs across numerous generations.

Caenorhabditis elegans contains 25 Argonautes, of which, ALG-1 and ALG-2 are known to primarily interact with miRNAs. ALG-5 belongs to the AGO subfamily of Argonautes that includes ALG-1 and ALG-2, but its role in small RNA pathways is unknown. We analyzed by high-throughput sequencing the small RNAs associated with ALG-5, ALG-1 and ALG-2, as well as changes in mRNA expression in alg-5, alg-1 and alg-2 mutants. We show that ALG-5 defines a distinct branch of the miRNA pathway affecting the expression of genes involved in immunity, defense, and development. In contrast to ALG-1 and ALG-2, which associate with most miRNAs and have general roles throughout development, ALG-5 interacts with only a small subset of miRNAs and is specifically expressed in the germline where it localizes alongside the piRNA and siRNA machinery at P granules. alg-5 is required for optimal fertility and mutations in alg-5 lead to a precocious transition from spermatogenesis to oogenesis. Our results provide a near-comprehensive analysis of miRNA-Argonaute interactions in C. elegans and reveal a new role for miRNAs in the germline.

It emerges that strings of nucleotides are added to messenger RNAs that are undergoing silencing in nematode worms. The composition of these nucleotide tails promotes the formation of small RNAs that drive heritable gene regulation. PolyUG tails trigger transgenerational mRNA silencing.

piRNAs play a critical role in the regulation of transposons and other germline genes. In Caenorhabditis elegans, regulation of piRNA target genes is mediated by the mutator complex, which synthesizes high levels of siRNAs through the activity of an RNA-dependent RNA polymerase. However, the steps between mRNA recognition by the piRNA pathway and siRNA amplification by the mutator complex are unknown. Here, we identify the Tudor domain protein, SIMR-1, as acting downstream of piRNA production and upstream of mutator complex-dependent siRNA biogenesis. Interestingly, SIMR-1 also localizes to distinct subcellular foci adjacent to P granules and Mutator foci, two phase-separated condensates that are the sites of piRNA-dependent mRNA recognition and mutator complex-dependent siRNA amplification, respectively. Thus, our data suggests a role for multiple perinuclear condensates in organizing the piRNA pathway and promoting mRNA regulation by the mutator complex., eLife digest In the biological world, a process known as RNA interference helps cells to switch genes on and off and to defend themselves against harmful genetic material. This mechanism works by deactivating RNA sequences, the molecular templates cells can use to create proteins. Overall, RNA interference relies on the cell creating small RNA molecules that can target and inhibit the harmful RNA sequences that need to be silenced. More precisely, in round worms such as Caenorhabditis elegans, RNA interference happens in two steps. First, primary small RNAs identify the target sequences, which are then combatted by newly synthetised, secondary small RNAs. A number of proteins are also involved in both steps of the process. RNA interference is particularly important to preserve fertility, guarding sex cells against ‘rogue’ segments of genetic information that could be passed on to the next generation. In future sex cells, the proteins involved in RNA interference cluster together, forming a structure called a germ granule. Yet, little is known about the roles and identity of these proteins. To fill this knowledge gap, Manage et al. focused on the second stage of the RNA interference pathway in the germ granules of C. elegans, examining the molecules that physically interact with a key protein. This work revealed a new protein called SIMR-1. Looking into the role of SIMR-1 showed that the protein is required to amplify secondary small RNAs, but not to identify target sequences. However, it only promotes the creation of secondary small RNAs if a specific subtype of primary small RNAs have recognized the target RNAs for silencing. Further experiments also showed that within the germ granule, SIMR-1 is present in a separate substructure different from any compartment previously identified. This suggests that each substep of the RNA interference process takes place at a different location in the granule. In both C. elegans and humans, disruptions in the RNA interference pathway can lead to conditions such as cancer or infertility. Dissecting the roles of the proteins involved in this process in roundworms may help to better grasp how this process unfolds in mammals, and how it could be corrected in the case of disease.

Reversible changes in gene expression independent of the genetic code can be transmitted from one generation to the next via poorly understood mechanisms. In worms, a histone-modifying enzyme is necessary to keep small RNA-guided transgenerational gene silencing in check.

SUMMARY The germline contains an immortal cell lineage that ensures the faithful transmission of genetic and, in some instances, epigenetic information from one generation to the next. Here, we show that in Caenorhabditis elegans, the small RNA 3′-2′-O-methyltransferase henn-1/HEN1 is required for sustained fertility across generations. In the absence of henn-1, animals become progressively less fertile, becoming sterile after ~30 generations at 25°C. Sterility in henn-1 mutants is accompanied by severe defects in germline proliferation and maintenance. The requirement for henn-1 in transgenerational fertility is likely due to its role in methylating and, thereby, stabilizing Piwi-interacting RNAs (piRNAs). However, despite being essential for piRNA stability in embryos, henn-1 is not required for piRNA stability in adults. Thus, we propose that methylation is important for the role of piRNAs in establishing proper gene silencing during early stages of development but is dispensable for their role in the proliferated germline., In Brief Svendsen et al. identify a requirement for the small RNA methyltransferase HENN-1 in germline immortality. HENN-1 is required for piRNA stability during embryogenesis but is dispensable in the adult germline, pointing to a role for piRNAs in establishing a gene regulatory network in embryos that protects the germline throughout development., Graphical Abstract

Argonaute (AGO) proteins partner with microRNAs (miRNAs) to target specific genes for post-transcriptional regulation. During larval development in Caenorhabditis elegans, Argonaute-Like Gene 1 (ALG-1) is the primary mediator of the miRNA pathway, while the related ALG-2 protein is largely dispensable. Here we show that in adult C. elegans these AGOs are differentially expressed and, surprisingly, work in opposition to each other; alg-1 promotes longevity, whereas alg-2 restricts lifespan. Transcriptional profiling of adult animals revealed that distinct miRNAs and largely non-overlapping sets of protein-coding genes are misregulated in alg-1 and alg-2 mutants. Interestingly, many of the differentially expressed genes are downstream targets of the Insulin/ IGF-1 Signaling (IIS) pathway, which controls lifespan by regulating the activity of the DAF-16/ FOXO transcription factor. Consistent with this observation, we show that daf-16 is required for the extended lifespan of alg-2 mutants. Furthermore, the long lifespan of daf-2 insulin receptor mutants, which depends on daf-16, is strongly reduced in animals lacking alg-1 activity. This work establishes an important role for AGO-mediated gene regulation in aging C. elegans and illustrates that the activity of homologous genes can switch from complementary to antagonistic, depending on the life stage.

Summary More than 2,000 C. elegans genes are targeted for RNA silencing by the mutator complex, a specialized small interfering RNA (siRNA) amplification module which is nucleated by the Q/N-rich protein MUT-16. The mutator complex localizes to Mutator foci adjacent to P granules at the nuclear periphery in germ cells [1]. Here, we show that the DEAD box RNA helicase smut-1 functions redundantly in the mutator pathway with its paralog mut-14 during RNAi. Mutations in both smut-1 and mut-14 also cause widespread loss of endogenous siRNAs. The targets of mut-14 and smut-1 largely overlap with the targets of other mutator class genes; however, the mut-14 smut-1 double mutant and the mut-16 mutant display the most dramatic depletion of siRNAs, suggesting that they act at a similarly early step in siRNA formation. mut-14 and smut-1 are predominantly expressed in the germline and, unlike other mutator class genes, are specifically required for RNAi targeting germline genes. A catalytically inactive, dominant-negative missense mutant of MUT-14 is RNAi defective in vivo; however, mutator complexes containing the mutant protein retain the ability to synthesize siRNAs in vitro. The results point to a role for mut-14 and smut-1 in initiating siRNA amplification in germ cell Mutator foci, possibly through the recruitment or retention of target mRNAs.

The nematode Caenorhabditis elegans contains each of the broad classes of eukaryotic small RNAs, including microRNAs (miRNAs), endogenous small-interfering RNAs (endo-siRNAs), and piwi-interacting RNAs (piRNAs). To better understand the evolution of these regulatory RNAs, we deep-sequenced small RNAs from C. elegans and three closely related nematodes: C. briggsae, C. remanei, and C. brenneri. The results reveal a fluid landscape of small RNA pathways with essentially no conservation of individual sequences aside from a subset of miRNAs. We identified 54 miRNA families that are conserved in each of the four species, as well as numerous miRNAs that are species-specific or shared between only two or three species. Despite a lack of conservation of individual piRNAs and siRNAs, many of the features of each pathway are conserved between the different species. We show that the genomic distribution of 26G siRNAs and the tendency for piRNAs to cluster is conserved between C. briggsae and C. elegans. We also show that, in each species, 26G siRNAs trigger stage-specific secondary siRNA formation. piRNAs in each species also trigger secondary siRNA formation from targets containing up to three mismatches. Finally, we show that the production of male- and female-specific piRNAs is conserved in all four species, suggesting distinct roles for piRNAs in male and female germlines.

piRNAs are known to silence transposable elements, but not all piRNAs match transposon sequences. Recent studies from Shen et al. (2018) and Zhang et al. (2018) identify rules for piRNA target recognition in Caenorhabditis elegans. Permissive pairing rules allow targeting of essentially all germline mRNAs, while protective mechanisms prevent silencing self-genes.

[EN] In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses., We thank Goretti Nguyen for excellent technical assistance. A. C. was supported by a postdoctoral fellowship from the Ministerio de Ciencia e Innovacion (BMC-2008-0188). H.G.-R. was the recipient of a Helen Hay Whitney Postdoctoral fellowship (F-972). This work was supported by grants from the National Science Foundation (MCB-1231726), the National Institutes of Health (AI043288), and Monsanto Corporation.

Summary Background In nematodes, plants, and fungi, RNAi is remarkably potent and persistent due to the amplification of initial silencing signals by RNA-dependent RNA polymerases (RdRPs). In Caenorhabditis elegans ( C. elegans ), the interaction between the RNA-induced silencing complex (RISC) loaded with primary small interfering RNAs (siRNAs) and the target messenger RNA (mRNA) leads to the recruitment of RdRPs and synthesis of secondary siRNAs using the target mRNA as the template. The mechanism and genetic requirements for secondary siRNA accumulation are not well understood. Results From a forward genetic screen for C. elegans genes required for RNAi, we identified rde-10 , and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the known RNAi factors RSD-2 and ERGO-1, and other candidate RNAi factors. The RNAi defective genes rde-10 and rde-11 encode a novel protein and a RING-type zinc finger domain protein, respectively. Mutations in rde-10 and rde-11 genes cause dosage-sensitive RNAi deficiencies: these mutants are resistant to low dosage but sensitive to high dosage of double-stranded RNAs. We assessed the roles of rde-10 , rde-11 , and other dosage-sensitive RNAi-defective genes rsd-2 , rsd-6 , and haf-6 in both exogenous and endogenous small RNA pathways using high-throughput sequencing and qRT-PCR. These genes are required for the accumulation of secondary siRNAs in both exogenous and endogenous RNAi pathways. Conclusions The RDE-10/RDE-11 complex is essential for the amplification of RNAi in C. elegans by promoting secondary siRNA accumulation.

Transacting siRNA (tasiRNA) biogenesis in Arabidopsis is initiated by microRNA (miRNA) –guided cleavage of primary transcripts. In the case of TAS3 tasiRNA formation, ARGONAUTE7 (AGO7)–miR390 complexes interact with primary transcripts at two sites, resulting in recruitment of RNA-DEPENDENT RNA POLYMERASE6 for dsRNA biosynthesis. An extensive screen for Arabidopsis mutants with specific defects in TAS3 tasiRNA biogenesis or function was done. This yielded numerous ago7 mutants, one dcl4 mutant, and two mutants that accumulated low levels of miR390. A direct genome sequencing-based approach to both map and rapidly identify one of the latter mutant alleles was developed. This revealed a G-to-A point mutation ( mir390a-1 ) that was calculated to stabilize a relatively nonpaired region near the base of the MIR390a foldback, resulting in misprocessing of the miR390/miR390* duplex and subsequent reduced TAS3 tasiRNA levels. Directed substitutions, as well as analysis of variation at paralogous miR390-generating loci ( MIR390a and MIR390b ), indicated that base pair properties and nucleotide identity within a region 4–6 bases below the miR390/miR390* duplex region contributed to the efficiency and accuracy of precursor processing.

The advent of high-throughput sequencing (HTS) methods has enabled direct approaches to quantitatively profile small RNA populations. However, these methods have been limited by several factors, including representational artifacts and lack of established statistical methods of analysis. Furthermore, massive HTS data sets present new problems related to data processing and mapping to a reference genome. Here, we show that cluster-based sequencing-by-synthesis technology is highly reproducible as a quantitative profiling tool for several classes of small RNA from Arabidopsis thaliana. We introduce the use of synthetic RNA oligoribonucleotide standards to facilitate objective normalization between HTS data sets, and adapt microarray-type methods for statistical analysis of multiple samples. These methods were tested successfully using mutants with small RNA biogenesis (miRNA-defective dcl1 mutant and siRNA-defective dcl2 dcl3 dcl4 triple mutant) or effector protein (ago1 mutant) deficiencies. Computational methods were also developed to rapidly and accurately parse, quantify, and map small RNA data.

MicroRNA (miRNA)-guided cleavage initiates entry of primary transcripts into the transacting siRNA (tasiRNA) biogenesis pathway involving RNA-DEPENDENT RNA POLYMERASE6, DICER-LIKE4, and SUPPRESSOR OF GENE SILENCING3. Arabidopsis thaliana TAS1 and TAS2 families yield tasiRNA that form through miR173-guided initiation–cleavage of primary transcripts and target several transcripts encoding pentatricopeptide repeat proteins and proteins of unknown function. Here, the TAS1c locus was modified to produce synthetic (syn) tasiRNA to target an endogenous transcript encoding PHYTOENE DESATURASE and used to analyze the role of miR173 in routing of transcripts through the tasiRNA pathway. miR173 was unique from other miRNAs in its ability to initiate TAS1c -based syn-tasiRNA formation. A single miR173 target site was sufficient to route non- TAS transcripts into the pathway to yield phased siRNA. We also show that miR173 functions in association with ARGONAUTE 1 (AGO1) during TAS1 and TAS2 tasiRNA formation, and we provide data indicating that the miR173–AGO1 complex possesses unique functionality that many other miRNA–AGO1 complexes lack.

SummaryTrans-acting siRNA form through a refined RNAi mechanism in plants. miRNA-guided cleavage triggers entry of precursor transcripts into an RNA-DEPENDENT RNA POLYMERASE6 pathway, and sets the register for phased tasiRNA formation by DICER-LIKE4. Here, we show that miR390-ARGONAUTE7 complexes function in distinct cleavage or noncleavage modes at two target sites in TAS3a transcripts. The AGO7 cleavage, but not the noncleavage, function could be provided by AGO1, the dominant miRNA-associated AGO, but only when AGO1 was guided to a modified target site through an alternate miRNA. AGO7 was highly selective for interaction with miR390, and miR390 in turn was excluded from association with AGO1 due entirely to an incompatible 5′ adenosine. Analysis of AGO1, AGO2, and AGO7 revealed a potent 5′ nucleotide discrimination function for some, although not all, ARGONAUTEs. miR390 and AGO7, therefore, evolved as a highly specific miRNA guide/effector protein pair to function at two distinct tasiRNA biogenesis steps.

AUXIN RESPONSE FACTORS (ARFs) are transcription factors involved in auxin signal transduction during many stages of plant growth development. ARF10, ARF16 and ARF17 are targeted by microRNA160 (miR160) in Arabidopsis thaliana. Here, we show that negative regulation of ARF10 by miR160 plays important roles in seed germination and post-germination. Transgenic plants expressing an miR160-resistant form of ARF10, which has silent mutations in the miRNA target site (termed mARF10), exhibited developmental defects such as serrated leaves, curled stems, contorted flowers and twisted siliques. These phenotypes were not observed in wild-type plants or plants transformed with the targeted ARF10 gene. During sensu stricto germination and post-germination, mARF10 mutant seeds and plants were hypersensitive to ABA in a dose-dependent manner. ABA hypersensitivity was mimicked in wild-type plants by exogenous auxin. In contrast, overexpression of MIR160 (35S:MIR160) resulted in reduced sensitivity to ABA during germination. Transcriptome analysis of germinating ARF10 and mARF10 seeds indicated that typical ABA-responsive genes expressed during seed maturation were overexpressed in germinating mARF10 seeds. These results indicate that negative regulation of ARF10 by miR160 plays a critical role in seed germination and post-embryonic developmental programs, at least in part by mechanisms involving interactions between ARF10-dependent auxin and ABA pathways.

Summary MicroRNAs (miRNAs) and trans -acting siRNAs (ta-siRNAs) in plants form through distinct pathways, although they function as negative regulators of mRNA targets by similar mechanisms [1–7]. Three ta-siRNA gene families ( TAS1 , TAS2 , and TAS3 ) are known in Arabidopsis thaliana . Biogenesis of TAS3 ta-siRNAs, which target mRNAs encoding several AUXIN RESPONSE FACTORs (including ARF3/ETTIN and ARF4 [1, 8]) involves miR390-guided processing of primary transcripts, conversion of a precursor to dsRNA through RNA-DEPENDENT RNA POLYMERASE6 (RDR6) activity, and sequential DICER-LIKE4 (DCL4)-mediated cleavage events. We show that the juvenile-to-adult phase transition is normally suppressed by TAS3 ta-siRNAs, in an ARGONAUTE7-dependent manner, through negative regulation of ARF3 mRNA. Expression of a nontargeted ARF3 mutant ( ARF3mut ) in a wild-type background reproduced the phase-change phenotypes detected in rdr6-15 and dcl4-2 mutants, which lose all ta-siRNAs. Expression of either ARF3 or ARF3mut in rdr6-15 plants, in which both endogenous and transgenic copies of ARF3 were derepressed, resulted in further acceleration of phase change and severe morphological and patterning defects of leaves and floral organs. In light of the functions of ARF3 and ARF4 in organ asymmetry, these data reveal multiple roles for TAS3 ta-siRNA-mediated regulation of ARF genes in developmental timing and patterning.

Summary Germline-expressed endogenous small interfering RNAs (endo-siRNAs) transmit multigenerational epigenetic information to ensure fertility in subsequent generations. In Caenorhabditis elegans , nuclear RNAi ensures robust inheritance of endo-siRNAs and deposition of repressive H3K9me3 marks at target loci. How target silencing is maintained in subsequent generations is poorly understood. We discovered that morc-1 is essential for transgenerational fertility and acts as an effector of endo-siRNAs. Unexpectedly, morc-1 is dispensable for siRNA inheritance but is required for target silencing and maintenance of siRNA-dependent chromatin organization. A forward genetic screen identified mutations in met-1 , which encodes an H3K36 methyltransferase, as potent suppressors of morc-1(−) and nuclear RNAi mutant phenotypes. Further analysis of nuclear RNAi and morc-1(−) mutants revealed a progressive, met-1- dependent enrichment of H3K36me3, suggesting that robust fertility requires repression of MET-1 activity at nuclear RNAi targets. Without MORC-1 and nuclear RNAi, MET-1-mediated encroachment of euchromatin leads to detrimental decondensation of germline chromatin and germline mortality.

RNA silencing can be initiated by endogenous or exogenously delivered siRNAs. In Caenorhabditis elegans, RNA silencing guided by primary siRNAs is inefficient and therefore requires an siRNA amplification step involving RNA-dependent RNA polymerases (RdRPs). Many factors involved in RNA silencing localize to protein- and RNA-rich nuclear pore-associated P granules in the germline, where they are thought to surveil mRNAs as they exit the nucleus. Mutator class genes are required for siRNA-mediated RNA silencing in both germline and somatic cells, but their specific roles and relationship to other siRNA factors are unclear. Here we show that each of the six mutator proteins localizes to punctate foci at the periphery of germline nuclei. The Mutator foci are adjacent to P granules but are not dependent on core P-granule components or other RNAi pathway factors for their formation or stability. The glutamine/asparagine (Q/N)-rich protein MUT-16 is specifically required for the formation of a protein complex containing the mutator proteins, and in its absence, Mutator foci fail to form at the nuclear periphery. The RdRP RRF-1 colocalizes with MUT-16 at Mutator foci, suggesting a role for Mutator foci in siRNA amplification. Furthermore, we demonstrate that genes that yield high levels of siRNAs, indicative of multiple rounds of siRNA amplification, are disproportionally affected in mut-16 mutants compared with genes that yield low levels of siRNAs. We propose that the mutator proteins and RRF-1 constitute an RNA processing compartment required for siRNA amplification and RNA silencing.

Small RNAs—including piRNAs, miRNAs, and endogenous siRNAs—bind Argonaute proteins to form RNA silencing complexes that target coding genes, transposons, and aberrant RNAs. To assess the requirements for endogenous siRNA formation and activity in Caenorhabditis elegans, we developed a GFP-based sensor for the endogenous siRNA 22G siR-1, one of a set of abundant siRNAs processed from a precursor RNA mapping to the X chromosome, the X-cluster. Silencing of the sensor is also dependent on the partially complementary, unlinked 26G siR-O7 siRNA. We show that 26G siR-O7 acts in trans to initiate 22G siRNA formation from the X-cluster. The presence of several mispairs between 26G siR-O7 and the X-cluster mRNA, as well as mutagenesis of the siRNA sensor, indicates that siRNA target recognition is permissive to a degree of mispairing. From a candidate reverse genetic screen, we identified several factors required for 22G siR-1 activity, including the chromatin factors mes-4 and gfl-1, the Argonaute ergo-1, and the 3′ methyltransferase henn-1. Quantitative RT–PCR of small RNAs in a henn-1 mutant and deep sequencing of methylated small RNAs indicate that siRNAs and piRNAs that associate with PIWI clade Argonautes are methylated by HENN-1, while siRNAs and miRNAs that associate with non-PIWI clade Argonautes are not. Thus, PIWI-class Argonaute proteins are specifically adapted to associate with methylated small RNAs in C. elegans., Author Summary RNA interference (RNAi) is the process in which endogenous small RNA pathways are exploited by researchers to direct RNA silencing of particular genes. Plants and animals use endogenous RNA silencing pathways for protection against viruses and transposable elements and to regulate genes during development. The features that route genes into specific RNA silencing pathways are poorly understood. Furthermore, it is not clear how small RNAs identify target mRNAs and how they repress their activity. Here, we show that a single siRNA target site is sufficient to trigger gene silencing in C. elegans without requiring perfect complementarity for target recognition. We also discovered an endogenous siRNA that acts in trans to initiate siRNA amplification. Finally, we show that siRNAs and PIWI-interacting RNAs (piRNAs) that bind specifically to PIWI clade Argonautes are methylated by the C. elegans HEN1 ortholog HENN-1.

Argonaute-associated siRNAs and Piwi-associated piRNAs have overlapping roles in silencing mobile genetic elements in animals. In Caenorhabditis elegans , mutator ( mut ) class genes mediate siRNA-guided repression of transposons as well as exogenous RNAi, but their roles in endogenous RNA silencing pathways are not well-understood. To characterize the endogenous small RNAs dependent on mut class genes, small RNA populations from a null allele of mut-16 as well as a regulatory mut-16 ( mg461 ) allele that disables only somatic RNAi were subjected to deep sequencing. Additionally, each of the mut class genes was tested for a requirement in 26G siRNA pathways. The results indicate that mut-16 is an essential factor in multiple endogenous germline and somatic siRNA pathways involving several distinct Argonautes and RNA-dependent RNA polymerases. The results also reveal essential roles for mut-2 and mut-7 in the ERGO-1 class 26G siRNA pathway and less critical roles for mut-8 , mut-14 , and mut-15 . We show that transposons are hypersusceptible to mut-16 –dependent silencing and identify a requirement for the siRNA machinery in piRNA biogenesis from Tc1 transposons. We also show that the soma-specific mut-16 ( mg461 ) mutant allele is present in multiple C. elegans laboratory strains.

[EN] NA interference pathways can involve amplification of secondary siRNAs by RNA-dependent RNA polymerases. In plants, RDR6-dependent secondary siRNAs arise from transcripts targeted by some microRNAs (miRNAs). Here, Arabidopsis thaliana secondary siRNAs from mRNA as well as trans-acting siRNAs are shown to be triggered through initial targeting by a 22-nucleotide (nt) miRNA that associates with AGO1. In contrast to canonical 21-nt miRNAs, 22-nt miRNAs primarily arise from foldback precursors containing asymmetric bulges. Using artificial miRNA constructs, conversion of asymmetric foldbacks to symmetric foldbacks resulted in the production of 21-nt forms of miR173, miR472 and miR828. Both 21- and 22-nt forms associated with AGO 1 and guided accurate slicer activity, but only 22-nt forms were competent to trigger RDR6-dependent siRNA production from target RNA. These data suggest that AGO 1 functions differentially with 21- and 22-nt miRNAs to engage the RDR6-associated amplification apparatus., A. C. was supported by a postdoctoral fellowship from the Spanish Ministerio de Ciencia e Innovacion (BMC-2008-0188). This work was supported by grants from the US National Science Foundation (MCB-0618433 and MCB-0956526), the US National Institutes of Health (AI43288) and Monsanto Corporation.

MicroRNAs and trans-acting siRNAs (ta-siRNAs) have important regulatory roles in development. Unlike other developmentally important regulatory molecules, small RNAs are not known to act as mobile signals during development. Here, we show that low-abundant, conserved ta-siRNAs, termed tasiR-ARFs, move intercellularly from their defined source of biogenesis on the upper (adaxial) side of leaves to the lower (abaxial) side to create a gradient of small RNAs that patterns the abaxial determinant AUXIN RESPONSE FACTOR3. Our observations have important ramifications for the function of small RNAs and suggest they can serve as mobile, instructive signals during development.

AUXIN RESPONSE FACTORS (ARFs) are transcription factors involved in auxin signal transduction during many stages of plant growth development. ARF10, ARF16 and ARF17 are targeted by microRNA160 (miR160) in Arabidopsis thaliana. Here, we show that negative regulation of ARF10 by miR160 plays important roles in seed germination and post-germination. Transgenic plants expressing an miR160-resistant form of ARF10, which has silent mutations in the miRNA target site (termed mARF10), exhibited developmental defects such as serrated leaves, curled stems, contorted flowers and twisted siliques. These phenotypes were not observed in wild-type plants or plants transformed with the targeted ARF10 gene. During sensu stricto germination and post-germination, mARF10 mutant seeds and plants were hypersensitive to ABA in a dose-dependent manner. ABA hypersensitivity was mimicked in wild-type plants by exogenous auxin. In contrast, overexpression of MIR160 (35S:MIR160) resulted in reduced sensitivity to ABA during germination. Transcriptome analysis of germinating ARF10 and mARF10 seeds indicated that typical ABA-responsive genes expressed during seed maturation were overexpressed in germinating mARF10 seeds. These results indicate that negative regulation of ARF10 by miR160 plays a critical role in seed germination and post-embryonic developmental programs, at least in part by mechanisms involving interactions between ARF10-dependent auxin and ABA pathways.

The maturation and quality control of mRNA in eukaryotes is a tightly regulated, multistep process that begins on nascent transcripts. A 7-methyl guanosine (7MeG) cap structure is added to the 5′ end of pre-mRNA as it emerges from the exit channel of RNA Polymerase II. The multifunctional nuclear cap-binding complex (CBC), consisting of two protein subunits (CBP80 and CBP20), assembles at the pre-mRNA cap early during transcript formation and helps recruit the splicesome machinery to the cap-proximal intron (1, 2). Termination of transcription involves cleavage and polyadenylation at the 3′ end, and the mature mRNA is retained in the nucleus or exported to the cytoplasm. In either case, the mRNA undergoes a pioneer round of translation and surveillance by the nonsense-mediated mRNA decay (NMD) pathway to eliminate defective or misspliced transcripts. Although the CBC localizes primarily to the nucleus, it remains associated with mRNAs during export to the cytoplasm and during the pioneer round of translation and mRNA surveillance. After the first round of translation, the CBC is replaced by the eukaryotic initiation complex eIF4F, and the mRNA steady-state translation initiation complex is formed (3). But not all RNA Pol II transcripts are predestined for translation. Primary transcripts for microRNA (pri-miRNA) are retained in the nucleus, where they are processed into ≈21- to 22-nt miRNA that generally function as posttranscriptional regulators of mRNA expression. Although it is known that pri-miRNA transcripts form by RNA Pol II transcription, the extent to which pri-miRNA and pre-mRNA transcripts share common processing components is far from settled. In this issue of PNAS, Laubinger et al. (4) identify an important relationship between mRNA maturation and miRNA primary transcript processing in plants.

Endogenous small interfering RNAs (siRNAs) are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs) of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt) endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7–dependent siRNAs require Dicer. We identify that the eri-6/7–dependent siRNAs have a passenger strand that is ∼19 nt and is inset by ∼3–4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7–dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA–associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation., Author Summary Endogenous small interfering RNAs (siRNAs) are a class of small RNAs present in fungi, plants, and animals. Small RNAs, including microRNAs, are known to regulate the expression levels of genes, silence invading elements such as transposons, and act in cell division. However, the function of many endogenous siRNAs is unknown. We have found that the ERI-6/7 helicase is required for a subset of endogenous siRNAs present in the nematode Caenorhabditis elegans. The ERI-6/7 helicase acts in a pathway together with the Argonaute protein ERGO-1 to produce two types of siRNAs: a primary class of 26 nucleotides in length present in oocytes and embryos, and a class of 22 nucleotide siRNAs present in later stages of development. These siRNAs correspond to only about one hundred genes. Interestingly, we found that these genes fall into groups of genes that contain nearly identical DNA sequences. The sequences of these genes are not conserved in other organisms, not even in related nematodes. These results point to a potential function of these endogenous siRNAs: silencing of recently acquired, duplicated genes. Our work demonstrates a new role of small RNAs, different from known functions in transposon silencing and regulation of gene expression.

SummarypiRNAs silence foreign genes, such as transposons, to preserve genome integrity, but they also target endogenous mRNAs by mechanisms that are poorly understood. Caenorhabditis elegans piRNAs interact with both transposon and nontransposon mRNAs to initiate sustained silencing via the RNAi pathway. To assess the dysregulation of gene silencing caused by lack of piRNAs, we restored RNA silencing in RNAi-defective animals in the presence or absence of piRNAs. In the absence of piRNAs and a cellular memory of piRNA activity, essential and conserved genes are misrouted into the RNAi pathway to produce siRNAs that bind the nuclear Argonaute HRDE-1, resulting in dramatic defects in germ cell proliferation and function such that the animals are sterile. Inactivation of RNAi suppresses sterility, indicating that aberrant siRNAs produced in the absence of piRNAs target essential genes for silencing. Thus, by reanimating RNAi, we uncovered a role for piRNAs in protecting essential genes from RNA silencing.

Translation inhibition is a major but poorly understood mode of action of micro(mi)RNAs in plants and animals. In particular, the subcellular location where this process takes place is unknown. Here we show that the translation inhibition but not the mRNA cleavage activity of Arabidopsis miRNAs requires ALTERED MERISTEM PROGRAM1 (AMP1). AMP1 encodes an integral membrane protein associated with endoplasmic reticulum (ER) and ARGONAUTE1, the miRNA effector and a peripheral ER membrane protein. Large differences in polysome association of miRNA target RNAs are found between wild type and the amp1 mutant for membrane-bound but not total polysomes. This, together with AMP1-independent recruitment of miRNA target transcripts to membrane fractions, shows that miRNAs inhibit the translation of target RNAs on the ER. This study demonstrates that translation inhibition is an important activity of plant miRNAs, reveals the subcellular location of this activity, and uncovers a previously unknown function of the ER.