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.
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.
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.
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.
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.
DNA methylation is of pivotal importance during development. Previous genome-wide studies identified numerous differentially methylated regions upon differentiation of stem cells, many of them associated with transcriptional start sites.
We present the first genome-wide, single-base-resolution view into DNA methylation dynamics during differentiation of a mammalian epithelial stem cell: the mouse small intestinal Lgr5+ stem cell. Very little change was observed at transcriptional start sites and our data suggest that differentiation-related genes are already primed for expression in the stem cell. Genome-wide, only 50 differentially methylated regions were identified. Almost all of these loci represent enhancers driving gene expression in the differentiated part of the small intestine. Finally, we show that binding of the transcription factor Tcf4 correlates with hypo-methylation and demonstrate that Tcf4 is one of the factors contributing to formation of differentially methylated regions.
Our results reveal limited DNA methylation dynamics during small intestine stem cell differentiation and an impact of transcription factor binding on shaping the DNA methylation landscape during differentiation of stem cells in vivo.
Adult stem cells; Differentiation; DNA Methylation; Methylome; Enhancer; Tcf4
RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double-stranded (ds)RNA precursors by Dicer and guide the RNA-induced silencing complex (RISC) to targets. Although principles governing small RNA sorting into RISC have been uncovered, the spectrum of RNA species that can be targeted by Dicer proteins, particularly the viral RNAs present during an infection, are poorly understood. Dicer-2 potently restricts viral infection in insects by generating virus-derived siRNAs from viral RNA. To better characterize the substrates of Dicer-2, we examined the virus-derived siRNAs produced during the Drosophila antiviral RNAi response to four different viruses using high-throughput sequencing. We found that each virus was uniquely targeted by the RNAi pathway; dicing substrates included dsRNA replication intermediates and intramolecular RNA stem loops. For instance, a putative intergenic RNA hairpin encoded by Rift Valley Fever virus generates abundant small RNAs in both Drosophila and mosquito cells, while repetitive sequences within the genomic termini of Vaccinia virus, which give rise to abundant small RNAs in Drosophila, were found to be transcribed in both insect and mammalian cells. Moreover, we provide evidence that the RNA species targeted by Dicer-2 can be modulated by the presence of a viral suppressor of RNAi. This study uncovered several novel, heavily targeted features within viral genomes, offering insight into viral replication, viral immune evasion strategies, and the mechanism of antiviral RNAi.
MicroRNAs (miRNAs) regulate mRNA targets through perfect pairing with their seed region (position 2-7). Recently, a precise genome-wide map of miRNA interaction sites in mouse brain was generated by high-throughput sequencing of clusters of ~50 nucleotide RNA tags associated with Argonaute (Ago HITS-CLIP). By analyzing Ago HITS-CLIP “orphan clusters” – Ago binding regions from HITS-CLIP that cannot be explained by canonical seed matches – we have identified an alternative binding mode used by miRNAs. Specifically, G-bulge sites (position 5-6) are often bound and regulated by miR-124 in brain. More generally, bulged sites comprise ≥ 15% (≥ 1441 sites) of all Ago-miRNA interactions in mouse brain and are evolutionally conserved. We have termed position 6 the “pivot” nucleotide and suggest a model in which a transitional “nucleation-bulge” leads to functional bulge mRNA-miRNA interactions, expanding the number of potential miRNA regulatory sites.
RNA interference (RNAi) enables sequence-specific, experimentally induced silencing of almost any gene by tapping into innate regulatory mechanisms that are conserved among virtually all eukaryotes. In a typical RNAi experiment, an artificial silencing trigger directs the RNAi pathway toward a target that it would not normally recognize. This is most often an endogenous protein-coding gene, although some noncoding RNAs can also be silenced effectively. The artificial silencing trigger varies; this protocol uses synthetic small interfering RNAs (siRNAs). Lipofectamine 2000 is used to deliver the siRNAs into HEK293 cells. This lipid reagent has proven to be effective for many different cultured mammalian cell lines.
The challenge of understanding the widespread biological roles of animal microRNAs (miRNAs) has prompted the development of genetic and functional genomics technologies for miRNA loss-of-function studies. However, tools for exploring the functions of entire miRNA families are still limited. We developed a method that enables antagonism of miRNA function using seed-targeting 8-mer locked nucleic acid (LNA) oligonucleotides, termed tiny LNAs. Transfection of tiny LNAs into cells resulted in simultaneous inhibition of miRNAs within families sharing the same seed with concomitant upregulation of direct targets. In addition, systemically delivered, unconjugated tiny LNAs showed uptake in many normal tissues and in breast tumors in mice, coinciding with long-term miRNA silencing. Transcriptional and proteomic profiling suggested that tiny LNAs have negligible off-target effects, not significantly altering the output from mRNAs with perfect tiny LNA complementary sites. Considered together, these data support the utility of tiny LNAs in elucidating the functions of miRNA families in vivo.
Argonaute proteins lie at the heart of the RNA-induced silencing complex (RISC), wherein they use small RNA guides to recognize targets. Initial insight into the architecture of Argonautes came from studies of prokaryotic proteins, revealing a crescent-shaped base made up of the amino-terminal, PAZ, middle, and PIWI domains. The recently reported crystal structure of human Argonaute-2 (hAgo2), the “slicer” in RNA interference, in complex with a mixed population of RNAs derived from insect cells provides insight into the architecture of a eukaryotic Argonaute protein with defined biochemical and biological functions. Here, we report the structure of human Ago2 bound to a physiologically relevant microRNA, microRNA-20a, at 2.2 Å resolution. The miRNA is anchored at both ends by the Mid and PAZ domains and makes several kinks and turns along the binding groove. Interestingly, miRNA binding confers remarkable stability on hAgo2, locking this otherwise flexible enzyme into a stable conformation.