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.
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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•PARN-1 is required for piRNA 3′ end trimming in C. elegans•PARN-1 is expressed in germline nuage and acts as a 3′ to 5′ exo-RNase•3′ trimming of piRNAs is important for both fertility and gene silencing
In C. elegans, piRNA length is determined by the RNase PARN-1 and is important for piRNA-directed gene silencing.
Metazoans guard their germlines against transposons and other foreign transcripts with PIWI-interacting RNAs (piRNAs). Due to the robust heritability of the silencing initiated by piRNAs in ...Caenorhabditis elegans (C. elegans), previous screens using C. elegans were strongly biased to uncover members of this pathway in the maintenance process but not in the initiation process. To identify novel piRNA pathway members, we have utilized a sensitized reporter strain which detects defects in initiation, amplification, or regulation of piRNA silencing. Using our reporter, we have identified Integrator complex subunits, nuclear pore components, protein import components, and pre-mRNA splicing factors as essential for piRNA-mediated gene silencing. We found the small nuclear processing cellular machine termed the Integrator complex is required for both type I and type II piRNA production. Notably, we identified a role for nuclear pore and nucleolar components NPP-1/Nup54, NPP-6/Nup160, NPP-7/Nup153, and FIB-1 in promoting the perinuclear localization of anti-silencing CSR-1 Argonaute, as well as a role for Importin factor IMA-3 in nuclear localization of silencing Argonaute HRDE-1. Together, we have shown that piRNA silencing in C. elegans is dependent on evolutionarily ancient RNA processing machinery that has been co-opted to function in the piRNA-mediated genome surveillance pathway.
P granules are phase-separated liquid droplets that play important roles in the maintenance of germ cell fate in
Both the localization and formation of P granules are highly dynamic, but mechanisms ...that regulate such processes remain poorly understood. Here, we show evidence that the VASA-like germline RNA helicase GLH-1 couples distinct steps of its ATPase hydrolysis cycle to control the formation and disassembly of P granules. In addition, we found that the phenylalanine-glycine-glycine repeats in GLH-1 promote its localization at the perinucleus. Proteomic analyses of the GLH-1 complex with a GLH-1 mutation that interferes with P granule disassembly revealed transient interactions of GLH-1 with several Argonautes and RNA-binding proteins. Finally, we found that defects in recruiting the P granule component PRG-1 to perinuclear foci in the adult germline correlate with the fertility defects observed in various GLH-1 mutants. Together, our results highlight the versatile roles of an RNA helicase in controlling the formation of liquid droplets in space and time.
Piwi Argonautes and Piwi-interacting RNAs (piRNAs) mediate genome defense by targeting transposons. However, many piRNA species lack obvious sequence complementarity to transposons or other loci; ...only one C. elegans transposon is a known piRNA target. Here, we show that, in mutants lacking the Piwi Argonaute PRG-1 (and consequently its associated piRNAs/21U-RNAs), many silent loci in the germline exhibit increased levels of mRNA expression with a concomitant depletion of RNA-dependent RNA polymerase (RdRP)-derived secondary small RNAs termed 22G-RNAs. Sequences depleted of 22G-RNAs are proximal to potential target sites that base pair imperfectly but extensively to 21U-RNAs. We show that PRG-1 is required to initiate, but not to maintain, silencing of transgenes engineered to contain complementarity to endogenous 21U-RNAs. Our findings support a model in which C. elegans piRNAs utilize their enormous repertoire of targeting capacity to scan the germline transcriptome for foreign sequences, while endogenous germline-expressed genes are actively protected from piRNA-induced silencing.
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► Transcriptome-wide surveillance of germline transcripts by C. elegans piRNAs ► C. elegans piRNAs use imperfect base pairing to initiate silencing ► Maintenance of silencing depends on the WAGO/RdRP pathway ► mRNAs targeted by the CSR-1 Argonaute appear to be protected from silencing
piRNA-mediated silencing persists for dozens of generations in worms, and a hand-off from piRNA-binding proteins to the nuclear WAGO Argonaute transcriptional silencing pathway means that the initial trigger does not need to be present to maintain the transgenerational effect.
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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► A new enzymatic method enriches 5′ ends of Pol II transcripts ► Identification of transcription start sites for most C. elegans Pol II genes ► Capped small RNAs of ∼26 nt are expressed from Pol II promoters genome wide ► csRNAs, but not longer capped RNAs, are the piRNA precursors in C. elegans
A new method for genome-wide cloning of RNA polymerase II transcripts reveals capped 26-nucleotide RNAs as the elusive precursors for C. elegans piRNAs, which silence foreign nucleic acids.
Abstract
pirScan is a web-based tool for identifying C. elegans piRNA-targeting sites within a given mRNA or spliced DNA sequence. The purpose of our tool is to allow C. elegans researchers to ...predict piRNA targeting sites and to avoid the persistent germline silencing of transgenes that has rendered many constructs unusable. pirScan fulfills this purpose by first enumerating the predicted piRNA-targeting sites present in an input sequence. This prediction can be exported in a tabular or graphical format. Subsequently, pirScan suggests silent mutations that can be introduced to the input sequence that would allow the modified transgene to avoid piRNA targeting. The user can customize the piRNA targeting stringency and the silent mutations that he/she wants to introduce into the sequence. The modified sequences can be re-submitted to be certain that any previously present piRNA-targeting sites are now absent and no new piRNA-targeting sites are accidentally generated. This revised sequence can finally be downloaded as a text file and/or visualized in a graphical format. pirScan is freely available for academic use at http://cosbi4.ee.ncku.edu.tw/pirScan/.
Multiple‐stimuli‐responsive photoluminescence films based on a ZnII–organic framework, {Zn2(Htpim)(3,4‐pydc)2⋅4 DMF⋅4 H2O}n (1, Htpim=2,4,5‐tri(4‐pyridyl)imidazole, ...3,4‐H2pydc=3,4‐pyridinedicarboxylic acid), were fabricated. This compound consisted of a 2D corrugated layer, {Zn(3,4‐pydc)}n, which was further pillared using a Y‐shaped pillar N‐donor ligand (Htpim) to form a 3D‐pillared‐layer framework with 1D open channels. The rectangular channels in the as‐synthesized compound are fully occupied by guest DMF and H2O molecules. The framework exhibits instant and reversible thermochromic properties corresponding to the removal of different H2O and DMF guest molecules as temperature increases. The pale‐yellow crystal undergoes significant redshifting to a greenish emission centered at 530 nm. Compound 1 also showed remarkable solvatochromic effects in the presence of various organic solvents without affecting its structural integrity. In addition, polycrystalline MOF films were grown on an α‐Al2O3 support for switchable and fast‐response thermochromic and solvatochromic sensors.
Multiple‐stimuli‐responsive photoluminescence films based on a ZnII–organic framework were fabricated. The framework exhibits instant and reversible thermochromic properties corresponding to the removal of different H2O and DMF guest molecules as temperature increases. The pale‐yellow crystal undergoes significant redshifting to a greenish emission centered at 530 nm (see figure).
Abstract
piRNAs function as guardians of the genome by silencing non-self nucleic acids and transposable elements in animals. Many piRNA factors are enriched in perinuclear germ granules, but whether ...their localization is required for piRNA biogenesis or function is not known. Here we show that GLH/VASA helicase mutants exhibit defects in forming perinuclear condensates containing PIWI and other small RNA cofactors. These mutant animals produce largely normal levels of piRNA but are defective in triggering piRNA silencing. Strikingly, while many piRNA targets are activated in GLH mutants, we observe that hundreds of endogenous genes are aberrantly silenced by piRNAs. This defect in self versus non-self recognition is also observed in other mutants where perinuclear germ granules are disrupted. Together, our results argue that perinuclear germ granules function critically to promote the fidelity of piRNA-based transcriptome surveillance in
C. elegans
and preserve self versus non-self distinction.
RNA interference pathways use small RNAs to mediate gene silencing in eukaryotes. In addition to small interfering RNAs (siRNAs) and microRNAs, several types of endogenously produced small RNAs have ...important roles in gene regulation, germ cell maintenance and transposon silencing. The production of some of these RNAs requires the synthesis of aberrant RNAs (aRNAs) or pre-siRNAs, which are specifically recognized by RNA-dependent RNA polymerases to make double-stranded RNA. The mechanism for aRNA synthesis and recognition is largely unknown. Here we show that DNA damage induces the expression of the Argonaute protein QDE-2 and a new class of small RNAs in the filamentous fungus Neurospora crassa. This class of small RNAs, known as qiRNAs because of their interaction with QDE-2, are about 20-21 nucleotides long (several nucleotides shorter than Neurospora siRNAs), with a strong preference for uridine at the 5′ end, and originate mostly from the ribosomal DNA locus. The production of qiRNAs requires the RNA-dependent RNA polymerase QDE-1, the Werner and Bloom RecQ DNA helicase homologue QDE-3 and dicers. qiRNA biogenesis also requires DNA-damage-induced aRNAs as precursors, a process that is dependent on both QDE-1 and QDE-3. Notably, our results suggest that QDE-1 is the DNA-dependent RNA polymerase that produces aRNAs. Furthermore, the Neurospora RNA interference mutants show increased sensitivity to DNA damage, suggesting a role for qiRNAs in the DNA-damage response by inhibiting protein translation.
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