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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► Epigenetic silencing triggered by piRNA-mediated recognition of nonself RNA ► piRNAs scan using imperfect base pairing to initiate gene silencing ► Maintenance of silencing requires chromatin factors and RdRP-generated small RNAs ► Activating and silencing signals may compete in self versus nonself discrimination
Evidence for a provocative theory that places piRNAs at the center of a “self” versus “nonself” sensor, with some piRNAs functioning to silence foreign DNA and others as part of a mechanism that protects endogenous germline-expressed genes from aberrant silencing.
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.
•C. elegans develops an adaptive memory of expressed/self-mRNAs•The CSR-1 Argonaute mediates RNA-induced epigenetic gene activation (RNAa)•RNAa counteracts Piwi-Argonaute-dependent epigenetic silencing (RNAe)•Multigenerational exposure to RNAa adapts an RNAe allele for independent expression
Seth et al. show that the C. elegans Argonaute CSR-1 protects cognate genes from Piwi-Argonaute-mediated silencing. Their findings suggest how Argonaute pathways function together in the germline to monitor the flow of transgenerational information, ensuring that progeny will express only those genes that are also expressed in their parents.
Argonaute/small RNA pathways and heterochromatin work together to propagate transgenerational gene silencing, but the mechanisms behind their interaction are not well understood. Here, we show that ...induction of heterochromatin silencing in C. elegans by RNAi or by artificially tethering pathway components to target RNA causes co-localization of target alleles in pachytene nuclei. Tethering the nuclear Argonaute WAGO-9/HRDE-1 induces heterochromatin formation and independently induces small RNA amplification. Consistent with this finding, HRDE-1, while predominantly nuclear, also localizes to peri-nuclear nuage domains, where amplification is thought to occur. Tethering a heterochromatin-silencing factor, NRDE-2, induces heterochromatin formation, which subsequently causes de novo synthesis of HRDE-1 guide RNAs. HRDE-1 then acts to further amplify small RNAs that load on downstream Argonautes. These findings suggest that HRDE-1 plays a dual role, acting upstream to initiate heterochromatin silencing and downstream to stimulate a new cycle of small RNA amplification, thus establishing a self-enforcing mechanism that propagates gene silencing to future generations.
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•Nuclear Argonaute HRDE-1 separately induces heterochromatin and small RNA production•HRDE-1 induces target alleles to merge into a single focus of heterochromatin•Transcription within heterochromatin feeds de novo loading of HRDE-1 with small RNAs•HRDE-1 shuttles to nuage and promotes small RNA production via its N-terminal domain
Ding and colleagues investigate inherited silencing in C. elegans. They demonstrate that the nuclear Argonaute HRDE-1 induces subnuclear-co-localization of target genes in heterochromatin. Heterochromatin formation subsequently triggers de novo HRDE-1 guide RNA loading. Finally, HRDE-1 enters nuage and activates small RNA amplification. Thus, HRDE-1 effects multiple steps of a self-enforcing transgenerational silencing process.
In Caenorhabditis elegans, targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of C. elegans germline editing is attributed to the syncytial ...nature of the pachytene ovary which is easily accessed by microinjection. This protocol describes the step-by-step details and troubleshooting tips for the entire CRISPR-Cas genome editing procedure, including gRNA design and microinjection of ribonucleoprotein complexes, followed by screening and genotyping in C. elegans, to help accessing this powerful genetic animal system.
For complete details on the use and execution of this protocol, please refer to Ghanta and Mello (2020).
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•Step-by-step microinjection protocol for CRISPR-Cas genome editing in C. elegans•Tips to troubleshoot microinjection and genome editing protocols•Melting dsDNA donor molecules potentiates precision genome editing•A single injected hermaphrodite can produce dozens of precisely edited progenies
In Caenorhabditis elegans, targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of C. elegans germline editing is attributed to the syncytial nature of the pachytene ovary which is easily accessed by microinjection. This protocol describes the step-by-step details and troubleshooting tips for the entire CRISPR-Cas genome editing procedure, including gRNA design and microinjection of ribonucleoprotein complexes, followed by screening and genotyping in C. elegans, to help accessing this powerful genetic animal system.
Although single-gene loss-of-function analyses can identify components of particular processes, important molecules are missed owing to the robustness of biological systems. Here we show that ...large-scale RNAi screening for suppression interactions with functionally related mutants greatly expands the repertoire of genes known to act in a shared process and reveals a new layer of functional relationships. We performed RNAi screens for 17 Caenorhabditis elegans cell polarity mutants, generating the most comprehensive polarity network in a metazoan, connecting 184 genes. Of these, 72% were not previously linked to cell polarity and 80% have human homologues. We experimentally confirmed functional roles predicted by the network and characterized through biophysical analyses eight myosin regulators. In addition, we discovered functional redundancy between two unknown polarity genes. Similar systematic genetic interaction screens for other biological processes will help uncover the inventory of relevant genes and their patterns of interactions.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The adenomatous polyposis coli gene (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. Here the APC gene product is shown to bind through its armadillo repeat ...domain to a Rac-specific guanine nucleotide exchange factor (GEF), termed Asef. Endogenous APC colocalized with Asef in mouse colon epithelial cells and neuronal cells. Furthermore, APC enhanced the GEF activity of Asef and stimulated Asef-mediated cell flattening, membrane ruffling, and lamellipodia formation in MDCK cells. These results suggest that the APC-Asef complex may regulate the actin cytoskeletal network, cell morphology and migration, and neuronal function.
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.
At the onset of embryogenesis, key developmental regulators called determinants are activated asymmetrically to specify the body axes and tissue layers. In
C. elegans, this process is regulated in ...part by a conserved family of CCCH-type zinc finger proteins that specify the fates of early embryonic cells. The asymmetric localization of these and other determinants is regulated in early embryos through motor-dependent physical translocation as well as selective proteolysis.
We show here that the CCCH-type zinc finger protein OMA-1 serves as a nexus for signals that regulate the transition from oogenesis to embryogenesis. While OMA-1 promotes oocyte maturation during meiosis, destruction of OMA-1 is needed during the first cell division for the initiation of ZIF-1-dependent proteolysis of cell-fate determinants. Mutations in four conserved protein kinase genes—
mbk-2/Dyrk,
kin-19/CK1α,
gsk-3, and
cdk-1/CDC2—cause stabilization of OMA-1 protein, and their phenotypes are partially suppressed by an
oma-1 loss-of-function mutation. OMA-1 proteolysis also depends on Cyclin B3 and on a ZIF-1-independent CUL-2-based E3 ubiquitin ligase complex, as well as the CUL-2-interacting protein ZYG-11 and the Skp1-related proteins SKR-1 and SKR-2.
Our findings suggest that a CDK1/Cyclin B3-dependent activity links OMA-1 proteolysis to completion of the first cell cycle and support a model in which OMA-1 functions to prevent the premature activation of cell-fate determinants until after they are asymmetrically partitioned during the first mitosis.
Over the past 15 years, the free-living nematode,
has become an important model system for exploring eukaryotic innate immunity to bacterial and fungal pathogens. More recently, infection models ...using either natural or non-natural nematode viruses have also been established in
. These models offer new opportunities to use the nematode to understand eukaryotic antiviral defense mechanisms. Here we report protocols for the infection of
with a non-natural viral pathogen, vesicular stomatitis virus (VSV) through microinjection. We also describe how recombinant VSV strains encoding fluorescent or luciferase reporter genes can be used in conjunction with simple fluorescence-, survival-, and luminescence-based assays to identify host genetic backgrounds with differential susceptibilities to virus infection.
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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