Publications by authors named "Craig C Mello"

68 Publications

Germ granule dysfunction is a hallmark and mirror of Piwi mutant sterility.

Nat Commun 2021 03 3;12(1):1420. Epub 2021 Mar 3.

Department of Genetics, University of North Carolina, Chapel Hill, NC, USA.

In several species, Piwi/piRNA genome silencing defects cause immediate sterility that correlates with transposon expression and transposon-induced genomic instability. In C. elegans, mutations in the Piwi-related gene (prg-1) and other piRNA deficient mutants cause a transgenerational decline in fertility over a period of several generations. Here we show that the sterility of late generation piRNA mutants correlates poorly with increases in DNA damage signaling. Instead, sterile individuals consistently exhibit altered perinuclear germ granules. We show that disruption of germ granules does not activate transposon expression but induces multiple phenotypes found in sterile prg-1 pathway mutants. Furthermore, loss of the germ granule component pgl-1 enhances prg-1 mutant infertility. Environmental restoration of germ granule function for sterile pgl-1 mutants restores their fertility. We propose that Piwi mutant sterility is a reproductive arrest phenotype that is characterized by perturbed germ granule structure and is phenocopied by germ granule dysfunction, independent of genomic instability.
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http://dx.doi.org/10.1038/s41467-021-21635-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930041PMC
March 2021

The RNA phosphatase PIR-1 regulates endogenous small RNA pathways in C. elegans.

Mol Cell 2021 02 29;81(3):546-557.e5. Epub 2020 Dec 29.

Department of Molecular, Cell and Systems Biology, University of California, Riverside, Riverside, CA, USA. Electronic address:

Eukaryotic cells regulate 5'-triphosphorylated RNAs (ppp-RNAs) to promote cellular functions and prevent recognition by antiviral RNA sensors. For example, RNA capping enzymes possess triphosphatase domains that remove the γ phosphates of ppp-RNAs during RNA capping. Members of the closely related PIR-1 (phosphatase that interacts with RNA and ribonucleoprotein particle 1) family of RNA polyphosphatases remove both the β and γ phosphates from ppp-RNAs. Here, we show that C. elegans PIR-1 dephosphorylates ppp-RNAs made by cellular RNA-dependent RNA polymerases (RdRPs) and is required for the maturation of 26G-RNAs, Dicer-dependent small RNAs that regulate thousands of genes during spermatogenesis and embryogenesis. PIR-1 also regulates the CSR-1 22G-RNA pathway and has critical functions in both somatic and germline development. Our findings suggest that PIR-1 modulates both Dicer-dependent and Dicer-independent Argonaute pathways and provide insight into how cells and viruses use a conserved RNA phosphatase to regulate and respond to ppp-RNA species.
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http://dx.doi.org/10.1016/j.molcel.2020.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867642PMC
February 2021

Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in .

Genetics 2020 11 22;216(3):643-650. Epub 2020 Sep 22.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605.

CRISPR genome editing has revolutionized genetics in many organisms. In the nematode , one injection into each of the two gonad arms of an adult hermaphrodite exposes hundreds of meiotic germ cells to editing mixtures, permitting the recovery of multiple indels or small precision edits from each successfully injected animal. Unfortunately, particularly for long insertions, editing efficiencies can vary widely, necessitating multiple injections, and often requiring coselection strategies. Here, we show that melting double-stranded DNA (dsDNA) donor molecules prior to injection increases the frequency of precise homology-directed repair (HDR) by several fold for longer edits. We describe troubleshooting strategies that enable consistently high editing efficiencies resulting, for example, in up to 100 independent GFP knock-ins from a single injected animal. These efficiencies make by far the easiest metazoan to genome edit, removing barriers to the use and adoption of this facile system as a model for understanding animal biology.
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http://dx.doi.org/10.1534/genetics.120.303564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648581PMC
November 2020

GCNA Interacts with Spartan and Topoisomerase II to Regulate Genome Stability.

Dev Cell 2020 01 12;52(1):53-68.e6. Epub 2019 Dec 12.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Whitehead Institute, 455 Main Street, Cambridge, MA 02142, USA; Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. Electronic address:

GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan (DVC-1) and Wss1 resolve DNA-protein crosslinks (DPCs), including Topoisomerase-DNA adducts, during DNA replication. Here, we show that GCNA mutants in mouse and C. elegans display defects in genome maintenance including DNA damage, aberrant chromosome condensation, and crossover defects in mouse spermatocytes and spontaneous genomic rearrangements in C. elegans. We show that GCNA and topoisomerase II (TOP2) physically interact in both mice and worms and colocalize on condensed chromosomes during mitosis in C. elegans embryos. Moreover, C. elegans gcna-1 mutants are hypersensitive to TOP2 poison. Together, our findings support a model in which GCNA provides genome maintenance functions in the germline and may do so, in part, by promoting the resolution of TOP2 DPCs.
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http://dx.doi.org/10.1016/j.devcel.2019.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227305PMC
January 2020

Robust Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in .

Genetics 2018 11 13;210(3):781-787. Epub 2018 Sep 13.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605

CRISPR-based genome editing using ribonucleoprotein complexes and synthetic single-stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs-such as green fluorescent protein tags remains challenging in many systems. Here, we describe a streamlined and optimized editing protocol for the nematode We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging 14 Argonaute proteins in using ssODN donors. In addition, we describe a novel PCR-based, partially single-stranded, "hybrid" donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci, achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems, and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.
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http://dx.doi.org/10.1534/genetics.118.301532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218216PMC
November 2018

ZNFX-1 Functions within Perinuclear Nuage to Balance Epigenetic Signals.

Mol Cell 2018 05;70(4):639-649.e6

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Howard Hughes Medical Institute, Worcester, MA 01605, USA. Electronic address:

Animal cells have a remarkable capacity to adopt durable and heritable gene expression programs or epigenetic states that define the physical properties and diversity of somatic cell types. The maintenance of epigenetic programs depends on poorly understood pathways that prevent gain or loss of inherited signals. In the germline, epigenetic factors are enriched in liquid-like perinuclear condensates called nuage. Here, we identify the deeply conserved helicase-domain protein, ZNFX-1, as an epigenetic regulator and component of nuage that interacts with Argonaute systems to balance epigenetic inheritance. Our findings suggest that ZNFX-1 promotes the 3' recruitment of machinery that propagates the small RNA epigenetic signal and thus counteracts a tendency for Argonaute targeting to shift 5' along the mRNA. These functional insights support the idea that recently identified subdomains of nuage, including ZNFX-1 granules or "Z-granules," may define spatial and temporal zones of molecular activity during epigenetic regulation.
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http://dx.doi.org/10.1016/j.molcel.2018.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5994929PMC
May 2018

A Sex Chromosome piRNA Promotes Robust Dosage Compensation and Sex Determination in C. elegans.

Dev Cell 2018 03 15;44(6):762-770.e3. Epub 2018 Feb 15.

RNA Therapeutics Institute, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

In metazoans, Piwi-related Argonaute proteins engage piRNAs (Piwi-interacting small RNAs) to defend the genome against invasive nucleic acids, such as transposable elements. Yet many organisms-including worms and humans-express thousands of piRNAs that do not target transposons, suggesting that piRNA function extends beyond genome defense. Here, we show that the X chromosome-derived piRNA 21ux-1 downregulates XOL-1 (XO Lethal), a master regulator of X chromosome dosage compensation and sex determination in Caenorhabditis elegans. Mutations in 21ux-1 and several Piwi-pathway components sensitize hermaphrodites to dosage compensation and sex determination defects. We show that the piRNA pathway also targets xol-1 in C. briggsae, a nematode species related to C. elegans. Our findings reveal physiologically important piRNA-mRNA interactions, raising the possibility that piRNAs function broadly to ensure robust gene expression and germline development.
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http://dx.doi.org/10.1016/j.devcel.2018.01.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5991626PMC
March 2018

The Coding Regions of Germline mRNAs Confer Sensitivity to Argonaute Regulation in C. elegans.

Cell Rep 2018 02 15;22(9):2254-2264. Epub 2018 Feb 15.

RNA Therapeutics Institute, UMass Medical School, 368 Plantation Street, Worcester, MA 01605, USA; Howard Hughes Medical Institute. Electronic address:

Protein-coding genes undergo a wide array of regulatory interactions with factors that engage non-coding regions. Open reading frames (ORFs), in contrast, are thought to be constrained by coding function, precluding a major role in gene regulation. Here, we explore Piwi-interacting (pi)RNA-mediated transgene silencing in C. elegans and show that marked differences in the sensitivity to piRNA silencing map to the endogenous sequences within transgene ORFs. Artificially increasing piRNA targeting within the ORF of a resistant transgene can lead to a partial yet stable reduction in expression, revealing that piRNAs not only silence but can also "tune" gene expression. Our findings support a model that involves a temporal element to mRNA regulation by germline Argonautes, likely prior to translation, and suggest that piRNAs afford incremental control of germline mRNA expression by targeting the body of the mRNA, including the coding region.
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http://dx.doi.org/10.1016/j.celrep.2018.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5918280PMC
February 2018

Identification of piRNA Binding Sites Reveals the Argonaute Regulatory Landscape of the C. elegans Germline.

Cell 2018 02 15;172(5):937-951.e18. Epub 2018 Feb 15.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Howard Hughes Medical Institute. Electronic address:

piRNAs (Piwi-interacting small RNAs) engage Piwi Argonautes to silence transposons and promote fertility in animal germlines. Genetic and computational studies have suggested that C. elegans piRNAs tolerate mismatched pairing and in principle could target every transcript. Here we employ in vivo cross-linking to identify transcriptome-wide interactions between piRNAs and target RNAs. We show that piRNAs engage all germline mRNAs and that piRNA binding follows microRNA-like pairing rules. Targeting correlates better with binding energy than with piRNA abundance, suggesting that piRNA concentration does not limit targeting. In mRNAs silenced by piRNAs, secondary small RNAs accumulate at the center and ends of piRNA binding sites. In germline-expressed mRNAs, however, targeting by the CSR-1 Argonaute correlates with reduced piRNA binding density and suppression of piRNA-associated secondary small RNAs. Our findings reveal physiologically important and nuanced regulation of individual piRNA targets and provide evidence for a comprehensive post-transcriptional regulatory step in germline gene expression.
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http://dx.doi.org/10.1016/j.cell.2018.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905434PMC
February 2018

The Antiviral RNA Interference Response Provides Resistance to Lethal Arbovirus Infection and Vertical Transmission in Caenorhabditis elegans.

Curr Biol 2017 Mar 2;27(6):795-806. Epub 2017 Mar 2.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

The recent discovery of the positive-sense single-stranded RNA (ssRNA) Orsay virus (OV) as a natural pathogen of the nematode Caenorhabditis elegans has stimulated interest in exploring virus-nematode interactions. However, OV infection is restricted to a small number of intestinal cells, even in nematodes defective in their antiviral RNA interference (RNAi) response, and is neither lethal nor vertically transmitted. Using a fluorescent reporter strain of the negative-sense ssRNA vesicular stomatitis virus (VSV), we show that microinjection of VSV particles leads to a dose-dependent, muscle tissue-tropic, lethal infection in C. elegans. Furthermore, we find nematodes deficient for components of the antiviral RNAi pathway, such as Dicer-related helicase 1 (DRH-1), to display hypersusceptibility to VSV infection as evidenced by elevated infection rates, virus replication in multiple tissue types, and earlier mortality. Strikingly, infection of oocytes and embryos could also be observed in drh-1 mutants. Our results suggest that the antiviral RNAi response not only inhibits vertical VSV transmission but also promotes transgenerational inheritance of antiviral immunity. Our study introduces a new, in vivo virus-host model system for exploring arbovirus pathogenesis and provides the first evidence for vertical pathogen transmission in C. elegans.
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http://dx.doi.org/10.1016/j.cub.2017.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446062PMC
March 2017

A widely employed germ cell marker is an ancient disordered protein with reproductive functions in diverse eukaryotes.

Elife 2016 10 8;5. Epub 2016 Oct 8.

Whitehead Institute, Cambridge, United States.

The advent of sexual reproduction and the evolution of a dedicated germline in multicellular organisms are critical landmarks in eukaryotic evolution. We report an ancient family of GCNA (germ cell nuclear antigen) proteins that arose in the earliest eukaryotes, and feature a rapidly evolving intrinsically disordered region (IDR). Phylogenetic analysis reveals that GCNA proteins emerged before the major eukaryotic lineages diverged; GCNA predates the origin of a dedicated germline by a billion years. gene expression is enriched in reproductive cells across eukarya - either just prior to or during meiosis in single-celled eukaryotes, and in stem cells and germ cells of diverse multicellular animals. Studies of mutant and mice indicate that GCNA has functioned in reproduction for at least 600 million years. Homology to IDR-containing proteins implicated in DNA damage repair suggests that GCNA proteins may protect the genomic integrity of cells carrying a heritable genome.
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http://dx.doi.org/10.7554/eLife.19993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5098910PMC
October 2016

The RNase PARN-1 Trims piRNA 3' Ends to Promote Transcriptome Surveillance in C. elegans.

Cell 2016 Feb;164(5):974-84

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Howard Hughes Medical Institute. Electronic address:

Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and are essential for fertility in diverse organisms. An interesting feature of piRNAs is that, while piRNA lengths are stereotypical within a species, they can differ widely between species. For example, piRNAs are mainly 29 and 30 nucleotides in humans, 24 to 30 nucleotides in D. melanogaster, and uniformly 21 nucleotides in C. elegans. However, how piRNA length is determined and whether length impacts function remains unknown. Here, we show that C. elegans deficient for PARN-1, a conserved RNase, accumulate untrimmed piRNAs with 3' extensions. Surprisingly, these longer piRNAs are stable and associate with the Piwi protein PRG-1 but fail to robustly recruit downstream silencing factors. Our findings identify PARN-1 as a key regulator of piRNA length in C. elegans and suggest that length is regulated to promote efficient transcriptome surveillance.
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http://dx.doi.org/10.1016/j.cell.2016.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785802PMC
February 2016

Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals.

Science 2016 Jan 31;351(6271):391-396. Epub 2015 Dec 31.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Several recent studies link parental environments to phenotypes in subsequent generations. In this work, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA (sRNA) levels in mature sperm, with decreased let-7 levels and increased amounts of 5' fragments of glycine transfer RNAs (tRNAs). In testicular sperm, tRNA fragments are scarce but increase in abundance as sperm mature in the epididymis. Epididymosomes (vesicles that fuse with sperm during epididymal transit) carry RNA payloads matching those of mature sperm and can deliver RNAs to immature sperm in vitro. Functionally, tRNA-glycine-GCC fragments repress genes associated with the endogenous retroelement MERVL, in both embryonic stem cells and embryos. Our results shed light on sRNA biogenesis and its dietary regulation during posttesticular sperm maturation, and they also link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo.
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http://dx.doi.org/10.1126/science.aad6780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888079PMC
January 2016

Influenza A virus preferentially snatches noncoding RNA caps.

RNA 2015 Dec 1;21(12):2067-75. Epub 2015 Oct 1.

Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

Influenza A virus (IAV) lacks the enzyme for adding 5' caps to its RNAs and snatches the 5' ends of host capped RNAs to prime transcription. Neither the preference of the host RNA sequences snatched nor the effect of cap-snatching on host processes is completely defined. Previous studies of influenza cap-snatching used poly(A)-selected RNAs from infected cells or relied on annotated host genes to define the snatched host RNAs, and thus lack details on many noncoding host RNAs including snRNAs, snoRNAs, and promoter-associated capped small (cs)RNAs, which are made by "paused" Pol II during transcription initiation. In this study, we used a nonbiased technique, CapSeq, to identify host and viral-capped RNAs including nonpolyadenylated RNAs in the same samples, and investigated the substrate-product correlation between the host RNAs and the viral RNAs. We demonstrated that noncoding host RNAs, particularly U1 and U2, are the preferred cap-snatching source over mRNAs or pre-mRNAs. We also found that csRNAs are highly snatched by IAV. Because the functions of csRNAs remain mostly unknown, especially in somatic cells, our finding reveals that csRNAs at least play roles in the process of IAV infection. Our findings support a model where nascent RNAs including csRNAs are the preferred targets for cap-snatching by IAV and raise questions about how IAV might use snatching preferences to modulate host-mRNA splicing and transcription.
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http://dx.doi.org/10.1261/rna.054221.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4647461PMC
December 2015

POS-1 Promotes Endo-mesoderm Development by Inhibiting the Cytoplasmic Polyadenylation of neg-1 mRNA.

Dev Cell 2015 Jul 18;34(1):108-18. Epub 2015 Jun 18.

Program in Molecular Medicine, RNA Therapeutics Institute and Howard Hughes Medical Institute, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01605, USA. Electronic address:

The regulation of mRNA translation is of fundamental importance in biological mechanisms ranging from embryonic axis specification to the formation of long-term memory. POS-1 is one of several CCCH zinc-finger RNA-binding proteins that regulate cell fate specification during C. elegans embryogenesis. Paradoxically, pos-1 mutants exhibit striking defects in endo-mesoderm development but have wild-type distributions of SKN-1, a key determinant of endo-mesoderm fates. RNAi screens for pos-1 suppressors identified genes encoding the cytoplasmic poly(A)-polymerase homolog GLD-2, the Bicaudal-C homolog GLD-3, and the protein NEG-1. We show that NEG-1 localizes in anterior nuclei, where it negatively regulates endo-mesoderm fates. In posterior cells, POS-1 binds the neg-1 3' UTR to oppose GLD-2 and GLD-3 activities that promote NEG-1 expression and cytoplasmic lengthening of the neg-1 mRNA poly(A) tail. Our findings uncover an intricate series of post-transcriptional regulatory interactions that, together, achieve precise spatial expression of endo-mesoderm fates in C. elegans embryos.
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http://dx.doi.org/10.1016/j.devcel.2015.05.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507413PMC
July 2015

RNA interference-mediated antiviral defense in insects.

Curr Opin Insect Sci 2015 Apr;8:111-120

RNA Therapeutics Institute, University of Massachusetts Medical School, USA ; Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Small interfering RNA (siRNA)-mediated RNA interference (RNAi) pathways are critical for the detection and inhibition of RNA virus replication in insects. Recent work has also implicated RNAi pathways in the establishment of persistent virus infections and in the control of DNA virus replication. Accumulating evidence suggests that diverse double-stranded RNAs produced by RNA and DNA viruses can trigger RNAi responses yet many viruses have evolved mechanisms to inhibit RNAi defenses. Therefore, an evolutionary arms race exists between host RNAi pathways and invading viral pathogens. Here we review recent advances in our knowledge of how insect RNAi pathways are elicited upon infection, the strategies used by viruses to counter these defenses, and discuss recent evidence implicating Piwi-interacting RNAs in antiviral defense.
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http://dx.doi.org/10.1016/j.cois.2015.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4448697PMC
April 2015

Suppression of pervasive noncoding transcription in embryonic stem cells by esBAF.

Genes Dev 2015 Feb;29(4):362-78

Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA; Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA;

Approximately 75% of the human genome is transcribed, the majority of which does not encode protein. However, many noncoding RNAs (ncRNAs) are rapidly degraded after transcription, and relatively few have established functions, questioning the significance of this observation. Here we show that esBAF, a SWI/SNF family nucleosome remodeling factor, suppresses transcription of ncRNAs from ∼57,000 nucleosome-depleted regions (NDRs) throughout the genome of mouse embryonic stem cells (ESCs). We show that esBAF functions to both keep NDRs nucleosome-free and promote elevated nucleosome occupancy adjacent to NDRs. Reduction of adjacent nucleosome occupancy upon esBAF depletion is strongly correlated with ncRNA expression, suggesting that flanking nucleosomes form a barrier to pervasive transcription. Upon forcing nucleosome occupancy near two NDRs using a nucleosome-positioning sequence, we found that esBAF is no longer required to silence transcription. Therefore, esBAF's function to enforce nucleosome occupancy adjacent to NDRs, and not its function to maintain NDRs in a nucleosome-free state, is necessary for silencing transcription over ncDNA. Finally, we show that the ability of a strongly positioned nucleosome to repress ncRNA depends on its translational positioning. These data reveal a novel role for esBAF in suppressing pervasive transcription from open chromatin regions in ESCs.
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http://dx.doi.org/10.1101/gad.253534.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335293PMC
February 2015

A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi.

Cell 2015 Jan;160(3):407-19

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering RNAs (siRNAs) are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However, in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3' uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3' uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal.
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http://dx.doi.org/10.1016/j.cell.2015.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318647PMC
January 2015

RNA Interference in Caenorhabditis elegans.

Curr Protoc Mol Biol 2015 Jan 5;109:26.3.1-26.3.30. Epub 2015 Jan 5.

RNA Therapeutics Institute and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts.

RNAi has become an essential tool in C. elegans research. This unit describes procedures for RNAi in C. elegans by microinjecting with dsRNA, feeding with bacteria expressing dsRNA, and soaking in dsRNA solution, as well as high-throughput methods for RNAi-based screens.
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http://dx.doi.org/10.1002/0471142727.mb2603s109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5396541PMC
January 2015

A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection.

Elife 2014 Jun 25;3. Epub 2014 Jun 25.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States.

Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), are completely restricted in their replication after entry into Lepidopteran cells. This restriction is overcome when cells are co-infected with vaccinia virus (VACV), a vertebrate DNA virus. Using RNAi screening, we show that Lepidopteran RNAi, Nuclear Factor-κB, and ubiquitin-proteasome pathways restrict RNA virus infection. Surprisingly, a highly conserved, uncharacterized VACV protein, A51R, can partially overcome this virus restriction. We show that A51R is also critical for VACV replication in vertebrate cells and for pathogenesis in mice. Interestingly, A51R colocalizes with, and stabilizes, host microtubules and also associates with ubiquitin. We show that A51R promotes viral protein stability, possibly by preventing ubiquitin-dependent targeting of viral proteins for destruction. Importantly, our studies reveal exciting new opportunities to study virus-host interactions in experimentally-tractable Lepidopteran systems.
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http://dx.doi.org/10.7554/eLife.02910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112549PMC
June 2014

A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans.

Genetics 2014 Aug 30;197(4):1069-80. Epub 2014 May 30.

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605 Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605

Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.
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http://dx.doi.org/10.1534/genetics.114.166389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4125384PMC
August 2014

The Vasa Homolog RDE-12 engages target mRNA and multiple argonaute proteins to promote RNAi in C. elegans.

Curr Biol 2014 Apr 27;24(8):845-51. Epub 2014 Mar 27.

Program in Molecular Medicine, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01605, USA; RNA Therapeutics Institute, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01605, USA; Howard Hughes Medical Institute, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01605, USA. Electronic address:

Argonaute (AGO) proteins are key nuclease effectors of RNAi. Although purified AGOs can mediate a single round of target RNA cleavage in vitro, accessory factors are required for small interfering RNA (siRNA) loading and to achieve multiple-target turnover. To identify AGO cofactors, we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi, including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 colocalizes with WAGO-1 in germline P granules and in cytoplasmic and perinuclear foci in somatic cells. These findings and our genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs (RDE-1 and ERGO-1), where it promotes small RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing.
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http://dx.doi.org/10.1016/j.cub.2014.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4017897PMC
April 2014

Divide and differentiate: CDK/Cyclins and the art of development.

Cell Cycle 2014 26;13(9):1384-91. Epub 2014 Mar 26.

RNA Therapeutics Institute; University of Massachusetts Medical School; Worcester, MA USA; Howard Hughes Medical Institute; University of Massachusetts Medical School; Worcester, MA USA.

The elegant choreography of metazoan development demands exquisite regulation of cell-division timing, orientation, and asymmetry. In this review, we discuss studies in Drosophila and C. elegans that reveal how the cell cycle machinery, comprised of cyclin-dependent kinase (CDK) and cyclins functions as a master regulator of development. We provide examples of how CDK/cyclins: (1) regulate the asymmetric localization and timely destruction of cell fate determinants; (2) couple signaling to the control of cell division orientation; and (3) maintain mitotic zones for stem cell proliferation. These studies illustrate how the core cell cycle machinery should be viewed not merely as an engine that drives the cell cycle forward, but rather as a dynamic regulator that integrates the cell-division cycle with cellular differentiation, ensuring the coherent and faithful execution of developmental programs.
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http://dx.doi.org/10.4161/cc.28656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050136PMC
April 2016

The C. elegans CSR-1 argonaute pathway counteracts epigenetic silencing to promote germline gene expression.

Dev Cell 2013 Dec;27(6):656-63

Organisms can develop adaptive sequence-specific immunity by reexpressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piwi-interacting RNA (piRNA) pathway recruits RNA-dependent RNA polymerase (RdRP) to foreign sequences to amplify a transgenerational small-RNA-induced epigenetic silencing signal (termed RNAe). Here, we provide evidence that, in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self-mRNAs. We refer to this mechanism, which can prevent or reverse RNAe, as RNA-induced epigenetic gene activation (RNAa). We show that CSR-1, which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. We show that a transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. Our findings suggest that C. elegans adaptively acquires and maintains a transgenerational CSR-1 memory that recognizes and protects self-mRNAs, allowing piRNAs to recognize foreign sequences innately, without the need for prior exposure
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http://dx.doi.org/10.1016/j.devcel.2013.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3954781PMC
December 2013

Argonautes promote male fertility and provide a paternal memory of germline gene expression in C. elegans.

Cell 2013 Dec;155(7):1532-44

RNA Therapeutics Institute and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA; Howard Hughes Medical Institute, Worcester, MA 01605, USA. Electronic address:

During each life cycle, germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute proteins are expressed during male gametogenesis and promote male fertility. Here, we show that the CSR-1 Argonaute functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small RNA signal that represents an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. Together, these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic binary signals in the form of Argonaute/small RNA complexes that constitute a memory of gene expression in preceding generations.
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http://dx.doi.org/10.1016/j.cell.2013.11.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3924572PMC
December 2013

Wnt and CDK-1 regulate cortical release of WRM-1/β-catenin to control cell division orientation in early Caenorhabditis elegans embryos.

Proc Natl Acad Sci U S A 2013 Mar 19;110(10):E918-27. Epub 2013 Feb 19.

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.

In early Caenorhabditis elegans embryos, the Wingless/int (Wnt)- and Src-signaling pathways function in parallel to induce both the division orientation of the endomesoderm (EMS) blastomere and the endoderm fate of the posterior EMS daughter cell, called E. Here, we show that, in addition to its role in endoderm specification, the β-catenin-related protein Worm armadillo 1 (WRM-1) also plays a role in controlling EMS division orientation. WRM-1 localizes to the cortex of cells in both embryos and larvae and is released from the cortex in a Wnt-responsive manner. We show that WRM-1 cortical release is disrupted in a hypomorphic cyclin-dependent protein kinase 1 (cdk-1) mutant and that WRM-1 lacking potential CDK-1 phosphoacceptor sites is retained at the cortex. In both cases, cortical WRM-1 interferes with EMS spindle rotation without affecting endoderm specification. Finally, we show that removal of WRM-1 from the cortex can restore WT division orientation, even when both Wnt- and Src-signaling pathways are compromised. Our findings are consistent with a model in which Wnt signaling and CDK-1 modify WRM-1 in a temporal and spatial manner to unmask an intrinsic polarity cue required for proper orientation of the EMS cell division axis.
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http://dx.doi.org/10.1073/pnas.1300769110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593879PMC
March 2013

CapSeq and CIP-TAP identify Pol II start sites and reveal capped small RNAs as C. elegans piRNA precursors.

Cell 2012 Dec;151(7):1488-500

Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA.

Piwi-interacting (pi) RNAs are germline-expressed small RNAs linked to epigenetic programming. C. elegans piRNAs are thought to be transcribed as independent gene-like loci. To test this idea and to identify potential transcription start (TS) sites for piRNA precursors, we developed CapSeq, an efficient enzymatic method for 5' anchored RNA profiling. Using CapSeq, we identify candidate TS sites, defined by 70-90 nt sequence tags, for >50% of annotated Pol II loci. Surprisingly, however, these CapSeq tags failed to identify the overwhelming majority of piRNA loci. Instead, we show that the likely piRNA precursors are ∼26 nt capped small (cs) RNAs that initiate precisely 2 nt upstream of mature piRNAs and that piRNA processing or stability requires a U at the csRNA +3 position. Finally, we identify a heretofore unrecognized class of piRNAs processed from csRNAs that are expressed at promoters genome wide, nearly doubling the number of piRNAs available for genome surveillance.
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http://dx.doi.org/10.1016/j.cell.2012.11.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581324PMC
December 2012

Specific miRNA stabilization by Gld2-catalyzed monoadenylation.

Cell Rep 2012 Dec 29;2(6):1537-45. Epub 2012 Nov 29.

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.

MicroRNAs (miRNAs) are small, noncoding RNAs that inhibit translation and promote mRNA decay. The levels of mature miRNAs are the result of different rates of transcription, processing, and turnover. The noncanonical polymerase Gld2 has been implicated in the stabilization of miR-122, possibly through catalyzing 3' monoadenylation; however, there is little evidence that this relationship is one of cause and effect. Here, we biochemically characterize Gld2's involvement in miRNA monoadenylation and its effect on miRNA stability. We find that Gld2 directly monoadenylates and stabilizes specific miRNA populations in human fibroblasts and that sensitivity to monoadenylation-induced stability depends on nucleotides in the miRNA 3' end. These results establish a mechanism of miRNA stability and resulting posttranscriptional gene regulation.
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http://dx.doi.org/10.1016/j.celrep.2012.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3534910PMC
December 2012

The translin-TRAX complex (C3PO) is a ribonuclease in tRNA processing.

Nat Struct Mol Biol 2012 Aug 8;19(8):824-30. Epub 2012 Jul 8.

Department of Physiology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Conserved translin-TRAX complexes, also known as C3POs, have been implicated in many biological processes, but how they function remains unclear. Recently, C3PO was shown to be an endoRNase that promotes RNA interference (RNAi) in animal cells. Here, we show that C3PO does not play a significant role in RNAi in the filamentous fungus Neurospora crassa. Instead, the Neurospora C3PO functions as an RNase that removes the 5' pre-tRNA fragments after the processing of pre-tRNAs by RNase P. In addition, translin and trax mutants have elevated levels of tRNA and protein translation and are more resistant to a cell death-inducing agent. Finally, we show that C3PO is also involved in tRNA processing in mouse embryonic fibroblast cells. This study identifies the endogenous RNA substrates of C3PO and provides a potential explanation for its roles in apparently diverse biological processes.
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http://dx.doi.org/10.1038/nsmb.2337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3414638PMC
August 2012

piRNAs initiate an epigenetic memory of nonself RNA in the C. elegans germline.

Cell 2012 Jul 25;150(1):65-77. Epub 2012 Jun 25.

Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA.

Organisms employ a fascinating array of strategies to silence invasive nucleic acids such as transposons and viruses. Although evidence exists for several pathways that detect foreign sequences, including pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), in many cases, the mechanisms used to distinguish "self" from "nonself" nucleic acids remain mysterious. Here, we describe an RNA-induced epigenetic silencing pathway that permanently silences single-copy transgenes. We show that the Piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors initiate permanent silencing, and maintenance depends on chromatin factors and the WAGO Argonaute pathway. Our findings support a model in which PRG-1 scans for foreign sequences and two other Argonaute pathways serve as epigenetic memories of "self" and "nonself" RNAs. These findings suggest how organisms can utilize RNAi-related mechanisms to detect foreign sequences not by any molecular signature, but by comparing the foreign sequence to a memory of previous gene expression.
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http://dx.doi.org/10.1016/j.cell.2012.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597741PMC
July 2012