One Loop to Rule Them All: The Ping-Pong Cycle and piRNA-Guided Silencing.

The PIWI-interacting RNA (piRNA) pathway is a conserved defense mechanism that protects the genetic information of animal germ cells from the deleterious effects of molecular parasites, such as transposons. Discovered nearly a decade ago, this small RNA silencing system comprises PIWI-clade Argonaute proteins and their associated RNA-binding partners, the piRNAs. In this review, we highlight recent work that has advanced our understanding of how piRNAs preserve genome integrity across generations. We discuss the mechanism of piRNA biogenesis, give an overview of common themes as well as differences in piRNA-mediated silencing between species, and end by highlighting known and emerging functions of piRNAs

lncRNAs in development and disease: from functions to mechanisms

Differential expression of long non-coding RNAs (lncRNAs) during differentiation and their misregulation in cancer highlight their potential as cell fate regulators. While some example lncRNAs have been characterized in great detail, the functional in vivo relevance of others has been called into question. Finding functional lncRNAs will most probably require a combination of complementary approaches that will greatly vary depending on their mode of action. In this review, we discuss the different tools available to dissect genetically lncRNA requirements and how each is best suited to studies in particular contexts. Moreover, we review different strategies used to select candidate lncRNAs and give an overview of lncRNAs described to regulate development and cancer through different mechanisms.M.J.D. was funded by a PhD fellowship from Boehringer Ingelheim Fonds, a graduate studies fellowship from ‘la Caixa’ Foundation and by the Watson School of Biological Sciences. G.J.H. is a Wellcome Trust Investigator, was an investigator of the Howard Hughes Medical Institute, and is supported by Cancer Research UK, The Royal Society (Wolfson Professorship) and a generous gift from Kathryn W. Davis

Compressed Genotyping

Significant volumes of knowledge have been accumulated in recent years linking subtle genetic variations to a wide variety of medical disorders from Cystic Fibrosis to mental retardation. Nevertheless, there are still great challenges in applying this knowledge routinely in the clinic, largely due to the relatively tedious and expensive process of DNA sequencing. Since the genetic polymorphisms that underlie these disorders are relatively rare in the human population, the presence or absence of a disease-linked polymorphism can be thought of as a sparse signal. Using methods and ideas from compressed sensing and group testing, we have developed a cost-effective genotyping protocol. In particular, we have adapted our scheme to a recently developed class of high throughput DNA sequencing technologies, and assembled a mathematical framework that has some important distinctions from 'traditional' compressed sensing ideas in order to address different biological and technical constraints.Comment: Submitted to IEEE Transaction on Information Theory - Special Issue on Molecular Biology and Neuroscienc

An epigenetic memory of pregnancy in the mouse mammary gland.

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.We thank Antoine Molaro for helpful discussions. This work was performed with assistance from the CSHL Flow Cytometry Shared Resource and from the CSHL Histology Shared Resource, which are supported by Cancer Center Support Grant 5P30CA045508. This work was supported by the NIH Grand Opportunity award #1 RC2 CA148507 (G.J.H.), P01 award # 2P01CA013106 (G.J.H.), and NIH grant R01 H6005238 (A.D.S.). G.J.H. is an investigator of the Howard Hughes Medical Institute.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.celrep.2015.04.01

Oncogenic transformation of Drosophila somatic cells induces a functional piRNA pathway.

Germline genes often become re-expressed in soma-derived human cancers as "cancer/testis antigens" (CTAs), and piRNA (PIWI-interacting RNA) pathway proteins are found among CTAs. However, whether and how the piRNA pathway contributes to oncogenesis in human neoplasms remain poorly understood. We found that oncogenic Ras combined with loss of the Hippo tumor suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from annotated generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also altered expression of some protein-coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that, at least in this context, the piRNA pathway may play a functional role in cancer.We thank the Cold Spring Harbor Laboratory Microscopy Shared Resources for assistance, which are funded in part by Cancer Center Support Grant 5P30CA045508. This work was supported in part by a grant from the STARR Cancer Consortium, grants from the National Institutes of Health (NIH MERIT Award, R37GM062534 to G. J.H.), and a generous gift from Kathryn W. Davis to G.J.H. N.P. and G.J.H. are or were Investigators of the Howard Hughes Medical Institute. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. Cell lines have been deposited by A.S. at the Drosophila Genomics Resource Center (NIH 2P40OD010949-10A1). G.J.H. is supported by Cancer Research UK and is a Wellcome Trust Investigator.This is the final version of the article. It first appeared from Cold Spring Harbor Press at http://dx.doi.org/10.1101/gad.284927.116

Dicer-2 Processes Diverse Viral RNA Species

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. © 2013 Sabin et al

Essential Role for endogenous siRNAs during meiosis in mouse oocytes.

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.This research was supported by the National Institutes of Health Grants HD022681 (to RMS), and R37 GM062534-14 (to GJH), National Human Genome Research Institute 5T32HG000046-13 (to FL) and by a kind gift from Kathryn W. Davis. GJH is an investigator of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final version of the article. It first appeared from PLoS via http://dx.doi.org/10.1371/journal.pgen.100501

Cytoplasmic Compartmentalization of the Fetal piRNA Pathway in Mice

Derepression of transposable elements (TEs) in the course of epigenetic reprogramming of the mouse embryonic germline necessitates the existence of a robust defense that is comprised of PIWI/piRNA pathway and de novo DNA methylation machinery. To gain further insight into biogenesis and function of piRNAs, we studied the intracellular localization of piRNA pathway components and used the combination of genetic, molecular, and cell biological approaches to examine the performance of the piRNA pathway in germ cells of mice lacking Maelstrom (MAEL), an evolutionarily conserved protein implicated in transposon silencing in fruit flies and mice. Here we show that principal components of the fetal piRNA pathway, MILI and MIWI2 proteins, localize to two distinct types of germinal cytoplasmic granules and exhibit differential association with components of the mRNA degradation/translational repression machinery. The first type of granules, pi-bodies, contains the MILI-TDRD1 module of the piRNA pathway and is likely equivalent to the enigmatic “cementing material” first described in electron micrographs of rat gonocytes over 35 years ago. The second type of granules, piP-bodies, harbors the MIWI2-TDRD9-MAEL module of the piRNA pathway and signature components of P-bodies, GW182, DCP1a, DDX6/p54, and XRN1 proteins. piP-bodies are found predominantly in the proximity of pi-bodies and the two frequently share mouse VASA homolog (MVH) protein, an RNA helicase. In Mael-mutant gonocytes, MIWI2, TDRD9, and MVH are lost from piP-bodies, whereas no effects on pi-body composition are observed. Further analysis revealed that MAEL appears to specifically facilitate MIWI2-dependent aspects of the piRNA pathway including biogenesis of secondary piRNAs, de novo DNA methylation, and efficient downregulation of TEs. Cumulatively, our data reveal elaborate cytoplasmic compartmentalization of the fetal piRNA pathway that relies on MAEL function

Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export.

The PIWI-interacting RNA (piRNA) pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilization. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use noncanonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by nuclear export factor 1 (Nxf1). Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila nuclear export factor family protein Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We found that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production

piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis.

MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells.This work was supported by the National Institutes of Health R37 grant GM062534-14 to G.J.H. iTRAQ was performed with assistance from the Cold Spring Harbor Laboratory Proteomics Shared Resource, which is supported by Cancer Center support grant 5P30CA045508. W.S.S.G. is a McClintock Fellow of the Watson School of Biological Sciences and is supported by the NSS Scholarship from the Agency for Science, Technology and Research, Singapore. O.H.T. is supported by a fellowship of the Human Frontier Science Program. R.B. is supported by the Starr Centennial Scholarship from the Watson School of Biological Sciences. G.J.H. is a Howard Hughes Medical Institute Investigator.This is the final version of the article. It first appeared from Cold Spring Harbor Laboratory Press via http://dx.doi.org/10.1101/gad.260455.11