Several recent studies have found a conserved microRNA (miRNA) family, the miR-34s, to be direct transcriptional targets of p53. miR-34 activation can recapitulate elements of p53 activity, including induction of cell-cycle arrest and promotion of apoptosis, and loss of miR-34 can impair p53-mediated cell death. These data reinforce the growing awareness that non-coding RNAs are key players in tumour development by placing miRNAs in a central role in a well-known tumour-suppressor network.
Piwi proteins, together with their bound Piwi-interacting RNAs, constitute an evolutionarily conserved, germline-specific innate immune system. The piRNA pathway is one of the key mechanisms for silencing transposable elements in the germline, thereby preserving genome integrity between generations. Recent work from several groups has significantly advanced our understanding of how piRNAs arise from discrete genomic loci, termed piRNA clusters, and how these Piwi-piRNA complexes enforce transposon silencing. Here, we discuss these recent findings, as well as highlight some aspects of piRNA biology that continue to escape our understanding.
Evidence from animal studies and human famines suggests that starvation may affect the health of the progeny of famished individuals. However, it is not clear whether starvation affects only immediate offspring or has lasting effects; it is also unclear how such epigenetic information is inherited. Small RNA-induced gene silencing can persist over several generations via transgenerationally inherited small RNA molecules in C. elegans, but all known transgenerational silencing responses are directed against foreign DNA introduced into the organism. We found that starvation-induced developmental arrest, a natural and drastic environmental change, leads to the generation of small RNAs that are inherited through at least three consecutive generations. These small, endogenous, transgenerationally transmitted RNAs target genes with roles in nutrition. We defined genes that are essential for this multigenerational effect. Moreover, we show that the F3 offspring of starved animals show an increased lifespan, corroborating the notion of a transgenerational memory of past conditions.
Huang et al. (2013) recently reported that chromatin immuno-precipitation followed by sequencing (ChIP-seq) reveals the genome-wide sites of occupancy by Piwi - a piRNA-guided Argonaute protein central to transposon silencing in Drosophila. Their study also reported that loss of Piwi causes widespread rewiring of transcriptional patterns as evidenced by changes in RNA polymerase II occupancy across the genome. Here we reanalyze their underlying deep sequencing data and report that the data do not support the author’s central conclusions.
In Drosophila cells, RNA interference (RNAi) can be triggered by synthetic long double-stranded RNAs (dsRNAs). For many Drosophila cell lines and cell types, passive dsRNA uptake is inefficient. More complete silencing responses can often be obtained in Drosophila S2 cells using transfection, perhaps because higher levels of intracellular dsRNA are achieved. In this protocol, S2 cells are transfected with dsRNA using QIAGEN’s Effectene reagent, which has proven to be reliable for many investigators. A plasmid DNA can also be included in the transfection mix to provide additional functionality. The plasmid DNA can encode, for example, a reporter of the activity of a pathway or specific transcription factor, or a marker that allows visualization of some cellular behavior or structure. It is also useful to include a plasmid that encodes a fluorescent protein simply to monitor transfection efficiency.
Pregnancy is the major modulator of mammary gland activity. It induces a tremendous expansion of the mammary epithelium and the generation of alveolar structures for milk production. Anecdotal evidence from multiparous humans, indicates that the mammary gland may react less strongly to the first pregnancy than it does to subsequent pregnancies. Here we verify that the mouse mammary gland responds more robustly to a second pregnancy, indicating that the gland retains a long-term memory of pregnancy. A comparison of genome-wide profiles of DNA methylation in isolated mammary cell types reveals substantial and long lasting alterations. Since these alterations are maintained in the absence of the signal that induced them, we term them epigenetic. The majority of alterations in DNA methylation affect sites occupied by the Stat5a transcription factor and mark specific genes that are upregulated during pregnancy. We postulate that the epigenetic memory of a first pregnancy primes the activation of gene expression networks that promote mammary gland function in subsequent reproductive cycles. More broadly, our data indicate that physiological experience can broadly alter epigenetic states, functionally modifying the capacity of the affected cells to respond to later stimulatory events.
In Drosophila, long-term effects of RNA interference (RNAi) must be achieved by integrating into the genome a template from which an RNAi trigger is transcribed by cellular RNA polymerases, generally RNA polymerase II or III. With encoded triggers, not only can essentially permanent silencing be achieved, but control can also be exerted over the level of trigger expression, with a resulting variation in the degree to which the target is silenced. Knockdown can also be controlled in a temporal and cell-type-dependent fashion through the use of well-established transgenic methodologies and well-tested promoters. The forms of encoded triggers vary. Long double-stranded RNAs can be expressed as extended inverted repeats. The nearest equivalent of a small interfering RNA is an artificial microRNA (miRNA) or short hairpin RNA (shRNA), where a natural miRNA backbone (also called a scaffold) is remodeled to produce a different small RNA or a small inverted repeat (<30 nucleotides) is simply expressed. This protocol describes creation of transgenic Drosophila carrying shRNA inserts in a remodeled endogenous miRNA backbone. The protocol applies to the use of miRNA-based shRNAs, but most of the vectors, principles of experimental design, and methods are also applicable to long inverted repeat transgenes.
miRNAs are essential for self-renewal and differentiation of normal and malignant stem cells by regulating the expression of key stem cell regulatory genes. Here, we report evidence implicating the miR100 in self-renewal of cancer stem-like cells (CSC). We found that miR100 expression levels relate to the cellular differentiation state, with lowest expression in cells displaying stem cell markers. Utilizing a tetracycline-inducible lentivirus to elevate expression of miR100 in human cells, we found that increasing miR100 levels decreased the production of breast CSCs. This effect was correlated with an inhibition of cancer cell proliferation in vitro and in mouse tumor xenografts due to attenuated expression of the CSC regulatory genes SMARCA5, SMARCD1, and BMPR2. Furthermore, miR100 induction in breast CSCs immediately upon their orthotopic implantation or intracardiac injection completely blocked tumor growth and metastasis formation. Clinically, we observed a significant association between miR100 expression in breast cancer specimens and patient survival. Our results suggest that miR100 is required to direct CSC self-renewal and differentiation.
The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Both small RNA species silence gene expression post-transcriptionally in association with the ARGONAUTE (AGO) family of proteins. In mammals, there are four AGO proteins (AGO1-4), of which only AGO2 possesses endonucleolytic activity. siRNAs trigger endonucleolytic cleavage of target mRNAs, mediated by AGO2, whereas miRNAs cause translational repression and mRNA decay through association with any of the four AGO proteins. Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because mouse oocytes express both miRNAs and endo-siRNAs, this phenotype could be due to the absence of either class of small RNA, or both. However, we and others demonstrated that miRNA function is suppressed in mouse oocytes, which suggested that endo-siRNAs, not miRNAs, are essential for female meiosis. To determine if this was the case we generated mice that express a catalytically inactive knock-in allele of Ago2 (Ago2ADH) exclusively in oocytes and thereby disrupted the function of siRNAs. Oogenesis and hormonal response are normal in Ago2ADH oocytes, but meiotic maturation is impaired, with severe defects in spindle formation and chromosome alignment that lead to meiotic catastrophe. The transcriptome of these oocytes is widely perturbed and shows a highly significant correlation with the transcriptome of Dicer null and Ago2 null oocytes. Expression of the mouse transcript (MT), the most abundant transposable element in mouse oocytes, is increased. This study reveals that endo-siRNAs are essential during meiosis I in mouse females, demonstrating a role for endo-siRNAs in mammals.
In animals, the three main classes of small RNAs are microRNAs, short interfering RNAs, and PIWI-interacting RNAs. All three RNA species silence gene expression post-transcriptionally through interaction with the ARGONAUTE family of proteins. In mammals in particular, microRNAs are ubiquitously expressed, are essential for development, and perform numerous functions in a variety of cells and tissues. piRNAs are expressed almost exclusively in the germline, and are essential for male fertility and defense against transposons. Endogenous siRNAs are only expressed in germ cells and embryonic stem cells and have not been ascribed a functional role. By engineering a mouse that expresses a modified ARGONAUTE protein, we disrupt the function of endo-siRNAs exclusively in oocytes and find that females are infertile. Oocytes with an impaired siRNA pathway fail to complete meiosis I, and display severe spindle formation and chromosome alignment defects. Their transcriptome is widely perturbed and expression of the most abundant transposon is increased. These findings indicate that endo-siRNAs are essential for female fertility in mouse, are required for spindle formation, chromosome congression, and defense against transposons. This study unequivocally demonstrates an essential function for siRNAs in mammals, mediated through endonucleolytic cleavage of targets, and provides an explanation for the selective pressure that one AGO protein retains catalytic activity.
Short hairpin RNAs (shRNAs) provide powerful experimental tools by enabling stable and regulated gene silencing through programming of endogenous microRNA pathways. Since requirements for efficient shRNA biogenesis and target suppression are largely unknown, many predicted shRNAs fail to efficiently suppress their target. To overcome this barrier, we developed a “Sensor assay” that enables the biological identification of effective shRNAs at large scale. By constructing and evaluating 20,000 RNAi reporters covering every possible target site in 9 mammalian transcripts, we show that our assay reliably identifies potent shRNAs that are surprisingly rare and predominantly missed by existing algorithms. Our unbiased analyses reveal that potent shRNAs share various predicted and previously unknown features associated with specific microRNA processing steps, and suggest a new model for competitive strand selection. Together, our study establishes a powerful tool for large-scale identification of highly potent shRNAs and provides new insights into sequence requirements of effective RNAi.
Sequencing of multiple related species followed by comparative genomics analysis constitutes a powerful approach for the systematic understanding of any genome. Here, we use the genomes of 12 Drosophila species for the de novo discovery of functional elements in the fly. Each type of functional element shows characteristic patterns of change, or ‘evolutionary signatures’, dictated by its precise selective constraints. Such signatures enable recognition of new protein-coding genes and exons, spurious and incorrect gene annotations, and numerous unusual gene structures, including abundant stop-codon readthrough. Similarly, we predict non-protein-coding RNA genes and structures, and new microRNA (miRNA) genes. We provide evidence of miRNA processing and functionality from both hairpin arms and both DNA strands. We identify several classes of pre- and post-transcriptional regulatory motifs, and predict individual motif instances with high confidence. We also study how discovery power scales with the divergence and number of species compared, and we provide general guidelines for comparative studies.
Next-generation sequencing technologies generate vast catalogs of short RNA sequences from which to mine microRNAs. However, such data must be vetted to appropriately categorize microRNA precursors and interpret their evolution. A recent study annotated hundreds of microRNAs in three Drosophila species on the basis of singleton reads of heterogeneous length1. Our multi-million read datasets indicated that most of these were not substrates of RNAse III cleavage, and comprised many mRNA degradation fragments. We instead identified a distinct and smaller set of novel microRNAs supported by confident cloning signatures, including a high proportion of evolutionarily nascent mirtrons. Our data support a much lower rate in the emergence of lineage-specific microRNAs than previously inferred1, with a net flux of ~1 microRNA/million years of Drosophilid evolution.
Primary open angle glaucoma (POAG) is a genetically and phenotypically complex disease that is a leading cause of blindness worldwide. Previously we completed a genome-wide scan for early-onset POAG that identified a locus on 9q22 (GLC1J). To identify potential causative variants underlying GLC1J, we used targeted DNA capture followed by high throughput sequencing of individuals from four GLC1J pedigrees, followed by Sanger sequencing to screen candidate variants in additional pedigrees. A mutation likely to cause early-onset glaucoma was not identified, however COL15A1 variants were found in the youngest affected members of 7 of 15 pedigrees with variable disease onset. In addition, the most common COL15A1 variant, R163H, influenced the age of onset in adult POAG cases. RNA in situ hybridization of mouse eyes shows that Col15a1 is expressed in the multiple ocular structures including ciliary body, astrocytes of the optic nerve and cells in the ganglion cell layer. Sanger sequencing of COL18A1, a related multiplexin collagen, identified a rare variant, A1381T, in members of three additional pedigrees with early-onset disease. These results suggest genetic variation in COL15A1 and COL18A1 can modify the age of onset of both early and late onset POAG.
MiRNAs bear an increasing number of functions throughout development and in the aging adult. Here we address their role in establishing sexually dimorphic traits and sexual identity in male and female Drosophila. Our survey of miRNA populations in each sex identifies sets of miRNAs differentially expressed in male and female tissues across various stages of development. The pervasive sex-biased expression of miRNAs generally increases with the complexity and sexual dimorphism of tissues, gonads revealing the most striking biases. We find that the male-specific regulation of the X chromosome is relevant to miRNA expression on two levels. First, in the male gonad, testis-biased miRNAs tend to reside on the X chromosome. Second, in the soma, X-linked miRNAs do not systematically rely on dosage compensation. We set out to address the importance of a sex-biased expression of miRNAs in establishing sexually dimorphic traits. Our study of the conserved let-7-C miRNA cluster controlled by the sex-biased hormone ecdysone places let-7 as a primary modulator of the sex-determination hierarchy. Flies with modified let-7 levels present doublesex-related phenotypes and express sex-determination genes normally restricted to the opposite sex. In testes and ovaries, alterations of the ecdysone-induced let-7 result in aberrant gonadal somatic cell behavior and non-cell-autonomous defects in early germline differentiation. Gonadal defects as well as aberrant expression of sex-determination genes persist in aging adults under hormonal control. Together, our findings place ecdysone and let-7 as modulators of a somatic systemic signal that helps establish and sustain sexual identity in males and females and differentiation in gonads. This work establishes the foundation for a role of miRNAs in sexual dimorphism and demonstrates that similar to vertebrate hormonal control of cellular sexual identity exists in Drosophila.
miRNA; sex determination; ecdysteroid; gonad; development; Drosophila; genetics of sex
RNA interference (RNAi) enables sequence-specific, experimentally induced silencing of virtually any gene by tapping into innate regulatory mechanisms that are conserved among most eukaryotes. The principles that enable transgenic RNAi in cell lines can also be used to create transgenic animals, which express short-hairpin RNAs (shRNAs) in a regulated or tissue-specific fashion. However, RNAi in transgenic animals is somewhat more challenging than RNAi in cultured cells. The activities of promoters that are commonly used for shRNA expression in cell culture can vary enormously in different tissues, and founder lines also typically vary in transgene expression due to the effects of their single integration sites. There are many ways to produce mice carrying shRNA transgenes and the method described here uses recombinase-mediated cassette exchange (RMCE). RMCE permits insertion of the shRNA transgene into a well-characterized locus that gives reproducible and predictable expression in each founder and enhances the probability of potent expression in many cell types. This procedure is more involved and complex than simple pronuclear injection, but if even a few shRNA mice are envisioned, for example, to probe the functions of several genes, the effort of setting up the processes outlined below are well worthwhile. Note that when creating a transgenic mouse, one should take care to use the most potent shRNA possible. As a rule of thumb, the sequence chosen should provide >90% knockdown when introduced into cultured cells at single copy (e.g., on retroviral infection at a multiplicity of ≤0.3).
Epigenetic control of gene expression is a critical component of transcriptional regulation. Remarkably, the deposition of epigenetic modifications is often guided by noncoding RNAs. Although noncoding RNAs have been most often implicated in posttranscriptional gene silencing, these molecules are now emerging as critical regulators of gene expression and genomic stability at the transcriptional level. Here, we review recent efforts to understand the mechanisms by which RNA controls the expression or content of DNA. We discuss the role of both small RNAs and long noncoding RNAs in directing chromatin changes through histone modifications and DNA methylation. Furthermore, we highlight the function of RNA in mediating DNA cleavage during genome rearrangements and pathogen defense. In understanding the mechanisms of RNA control over DNA, the power of RNA may one day be harnessed to impact gene expression in a therapeutic setting.
A large fraction of our genome consists of mobile genetic elements. Governing transposons in germ cells is critically important, and failure to do so compromises genome integrity, leading to sterility. In animals, the piRNA pathway is the key to transposon constraint, yet the precise molecular details of how piRNAs are formed and how the pathway represses mobile elements remain poorly understood. In an effort to identify general requirements for transposon control and novel components of the piRNA pathway, we carried out a genome-wide RNAi screen in Drosophila ovarian somatic sheet cells. We identified and validated 87 genes necessary for transposon silencing. Among these were several novel piRNA biogenesis factors. We also found CG3893 (asterix) to be essential for transposon silencing, most likely by contributing to the effector step of transcriptional repression. Asterix loss leads to decreases in H3K9me3 marks on certain transposons but has no effect on piRNA levels.
The Drosophila piRNA pathway provides an RNA-based immune system that defends the germline genome against selfish genetic elements. Two inter-related branches of the piRNA system exist: somatic cells that support oogenesis only employ Piwi, whereas germ cells utilize a more elaborated pathway centered on the three gonad-specific Argonaute proteins Piwi, Aubergine, and Argonaute3. While several key factors of each branch have been identified, our current knowledge is insufficient to explain the complex workings of the piRNA machinery. Here, we report a reverse genetic screen spanning the ovarian transcriptome in an attempt to uncover the full repertoire of genes required for piRNA-mediated transposon silencing in the female germline. Our screen reveals new key factors of piRNA-mediated transposon silencing, including the novel piRNA biogenesis factors, CG2183 (GASZ) and Deadlock. Last, our data uncovers a previously unanticipated set of factors preferentially required for repression of different transposons types.
Drosophila; RNAi; piRNA pathway; transposon silencing; germ cells
Motivation: Post-transcriptional and co-transcriptional regulation is a crucial link between genotype and phenotype. The central players are the RNA-binding proteins, and experimental technologies [such as cross-linking with immunoprecipitation- (CLIP-) and RIP-seq] for probing their activities have advanced rapidly over the course of the past decade. Statistically robust, flexible computational methods for binding site identification from high-throughput immunoprecipitation assays are largely lacking however.
Results: We introduce a method for site identification which provides four key advantages over previous methods: (i) it can be applied on all variations of CLIP and RIP-seq technologies, (ii) it accurately models the underlying read-count distributions, (iii) it allows external covariates, such as transcript abundance (which we demonstrate is highly correlated with read count) to inform the site identification process and (iv) it allows for direct comparison of site usage across cell types or conditions.
Availability and implementation: We have implemented our method in a software tool called Piranha. Source code and binaries, licensed under the GNU General Public License (version 3) are freely available for download from http://smithlab.usc.edu.
Supplementary data available at Bioinformatics online.
Argonaute proteins lie at the heart of all RNAi-related effector complexes. Their PAZ domain recognizes small RNA targets; the PIWI domain is related to RNase H and responsible for target cleavage.
RNAi has existed at least since the divergence of prokaryotes and eukaryotes. This collection of pathways responds to a diversity of “abberant” RNAs and generally silences or eliminates genes sharing sequence content with the silencing trigger. In the canonical pathway, double-stranded RNAs are processed into small RNAs, which guide effector complexes to their targets by complementary base pairing. Many alternative routes from silencing trigger to small RNA are continuously being uncovered. Though the triggers of the pathway and the mechanisms of small RNA production are many, all RNAi-related mechanisms share Argonaute proteins as the heart of their effector complexes. These can act as self-contained silencing machines, binding directly to small RNAs, carrying out homology-based target recognition, and in some cases cleaving targets using an endogenous nuclease domain. Here, we discuss the diversity of Argonaute proteins from a structural and functional perspective.
Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1–4) only hAgo2 is an active slicer. We determined the structure of hAgo1 bound to endogenous copurified RNAs to 1.75 Å resolution and hAgo1 loaded with let-7 miRNA to 2.1 Å. Both structures are strikingly similar to the structures of hAgo2. A conserved catalytic tetrad within the PIWI domain of hAgo2 is required for its slicing activity. Completion of the tetrad combined with a mutation on a loop adjacent to the active site of hAgo1 results in slicer activity that is substantially enhanced by swapping in the N domain of hAgo2. hAgo3, with an intact tetrad, becomes an active slicer by swapping the N domain of hAgo2, without additional mutations. Intriguingly, the elements that make Argonaute an active slicer involve a sophisticated interplay between the active site and more distant regions of the enzyme.
Eukaryotic genomes harbor transposable elements and other repetitive sequences that must be silenced. Small RNA interference pathways play a major role in their repression. Here, we reveal another mechanism for silencing these sequences in Drosophila. Depleting the linker histone H1 in vivo leads to strong activation of these elements. H1-mediated silencing occurs in combination with the heterochromatin-specific histone H3 lysine 9 methyltransferase Su(var)3-9. H1 physically interacts with Su(var)3-9 and recruits it to chromatin in vitro, which promotes H3 methylation. We propose that H1 plays a key role in silencing by tethering Su(var)3-9 to heterochromatin. The tethering function of H1 adds to its established role as a regulator of chromatin compaction and accessibility.
RNA interference (RNAi) is an extremely effective tool for studying gene function in almost all metazoan and eukaryotic model systems. RNAi in mice, through the expression of short hairpin RNA s (shRNA s), offers something not easily achieved with traditional genetic approaches—inducible and reversible gene silencing. However, technical variability associated with the production of shRNA transgenic strains has so far limited their widespread use. Here we describe a pipeline for the generation of miR30-based shRNA transgenic mice that enables efficient and consistent targeting of doxycycline-regulated, fluorescence-linked shRNA s to the Col1a1 locus. Notably, the protocol details crucial steps in the design and testing of miR30-based shRNA s to maximize the potential for developing effective transgenic strains. In all, this 14-week procedure provides a fast and cost-effective way for any laboratory to investigate gene function in vivo in the mouse.
Small RNAs directly or indirectly impact nearly every biological process in eukaryotic cells. To perform their myriad roles, not only must precise small RNA species be generated, but they must also be loaded into specific effector complexes called RNA-induced silencing complexes (RISCs). Argonaute proteins form the core of RISCs and different members of this large family have specific expression patterns, protein binding partners and biochemical capabilities. In this Review, we explore the mechanisms that pair specific small RNA strands with their partner proteins, with an eye towards the substantial progress that has been recently made in understanding the sorting of the major small RNA classes — microRNAs (miRNAs) and small interfering RNAs (siRNAs) — in plants and animals.