Nicolas J. Lehrbach

piRNAs Can Trigger a Multigenerational Epigenetic Memory in the Germline of C. elegans

Summary Transgenerational effects have wide-ranging implications for human health, biological adaptation, and evolution; however, their mechanisms and biology remain poorly understood. Here, we demonstrate that a germline nuclear small RNA/chromatin pathway can maintain stable inheritance for many generations when triggered by a piRNA-dependent foreign RNA response in C. elegans. Using forward genetic screens and candidate approaches, we find that a core set of nuclear RNAi and chromatin factors is required for multigenerational inheritance of environmental RNAi and piRNA silencing. These include a germline-specific nuclear Argonaute HRDE1/WAGO-9, a HP1 ortholog HPL-2, and two putative histone methyltransferases, SET-25 and SET-32. piRNAs can trigger highly stable long-term silencing lasting at least 20 generations. Once established, this long-term memory becomes independent of the piRNA trigger but remains dependent on the nuclear RNAi/chromatin pathway. Our data present a multigenerational epigenetic inheritance mechanism induced by piRNAs.

Piwi and piRNAs Act Upstream of an Endogenous siRNA Pathway to Suppress Tc3 Transposon Mobility in the Caenorhabditis elegans Germline

The Piwi proteins of the Argonaute superfamily are required for normal germline development in Drosophila, zebrafish, and mice and associate with 24-30 nucleotide RNAs termed piRNAs. We identify a class of 21 nucleotide RNAs, previously named 21U-RNAs, as the piRNAs of C. elegans. Piwi and piRNA expression is restricted to the male and female germline and independent of many proteins in other small-RNA pathways, including DCR-1. We show that Piwi is specifically required to silence Tc3, but not other Tc/mariner DNA transposons. Tc3 excision rates in the germline are increased at least 100-fold in piwi mutants as compared to wild-type. We find no evidence for a Ping-Pong model for piRNA amplification in C. elegans. Instead, we demonstrate that Piwi acts upstream of an endogenous siRNA pathway in Tc3 silencing. These data might suggest a link between piRNA and siRNA function.

LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans

The let-7 microRNA (miRNA) is an ultraconserved regulator of stem cell differentiation and developmental timing and a candidate tumor suppressor. Here we show that LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 processing in Caenorhabditis elegans. We demonstrate that lin-28 is necessary and sufficient to block let-7 activity in vivo; LIN-28 directly binds let-7 pre-miRNA to prevent Dicer processing. Moreover, we have identified a poly(U) polymerase, PUP-2, which regulates the stability of LIN-28–blockaded let-7 pre-miRNA and contributes to LIN-28–dependent regulation of let-7 during development. We show that PUP-2 and LIN-28 interact directly, and that LIN-28 stimulates uridylation of let-7 pre-miRNA by PUP-2 in vitro. Our results demonstrate that LIN-28 and let-7 form an ancient regulatory switch, conserved from nematodes to humans, and provide insight into the mechanism of LIN-28 action in vivo. Uridylation by a PUP-2 ortholog might regulate let-7 and additional miRNAs in other species. Given the roles of Lin28 and let-7 in stem cell and cancer biology, we propose that such poly(U) polymerases are potential therapeutic targets.

Function, Targets, and Evolution of Caenorhabditis elegans piRNAs

Secondary Endogenous Small and Interfering In many eukaryotes, Piwi proteins bind small noncoding Piwi-interacting RNAs (piRNAs) that function to silence transposons in the germ line and protect the germ line from transposable element–driven recombination and mutation. Bagijn et al. (p. 574, published online 14 June; see the Perspective by Xiol and Pillai) show that in the nematode, Caenorhabditis elegans, a messenger RNA (mRNA) that contains a piRNA target sequence gives rise to a second, downstream class of small RNAs known as secondary endogenous small interfering RNAs, or endo siRNAs. These endo siRNAs map to the vicinity of the piRNA complementary sequence in the mRNA target and depend on both Piwi and on factors involved in the related RNA interference pathway for their genesis, but not on the Piwi slicer activity. Mapping the endo siRNAs reveals that piRNAs can target imperfectly matched targets and that piRNAs target a subset of both transposons and endogenous genes for silencing. Piwi-bound piwi-interacting RNAs recruit endogenous small interfering RNAs to silence mobile genetic elements. Piwi-interacting RNAs (piRNAs) are small RNAs required to maintain germline integrity and fertility, but their mechanism of action is poorly understood. Here we demonstrate that Caenorhabditis elegans piRNAs silence transcripts in trans through imperfectly complementary sites. Target silencing is independent of Piwi endonuclease activity or “slicing.” Instead, piRNAs initiate a localized secondary endogenous small interfering RNA (endo-siRNA) response. Endogenous protein-coding gene and transposon transcripts exhibit Piwi-dependent endo-siRNAs at sites complementary to piRNAs and are derepressed in Piwi mutants. Genomic loci of piRNA biogenesis are depleted of protein-coding genes and tend to overlap the start and end of transposons in sense and antisense, respectively. Our data suggest that nematode piRNA clusters are evolving to generate piRNAs against active mobile elements. Thus, piRNAs provide heritable, sequence-specific triggers for RNA interference in C. elegans.

A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans

Efficient experimental strategies are needed to validate computationally predicted microRNA (miRNA) target genes. Here we present a large-scale targeted proteomics approach to validate predicted miRNA targets in Caenorhabditis elegans. Using selected reaction monitoring (SRM), we quantified 161 proteins of interest in extracts from wild-type and let-7 mutant worms. We demonstrate by independent experimental downstream analyses such as genetic interaction, as well as polysomal profiling and luciferase assays, that validation by targeted proteomics substantially enriched for biologically relevant let-7 interactors. For example, we found that the zinc finger protein ZTF-7 was a bona fide let-7 miRNA target. We also validated predicted miR-58 targets, demonstrating that this approach is adaptable to other miRNAs. We propose that targeted mass spectrometry can be applied generally to validate candidate lists generated by computational methods or in large-scale experiments, and that the described strategy should be readily adaptable to other organisms.

A deletion polymorphism in the Caenorhabditis elegans RIG-I homolog disables viral RNA dicing and antiviral immunity

RNA interference defends against viral infection in plant and animal cells. The nematode Caenorhabditis elegans and its natural pathogen, the positive-strand RNA virus Orsay, have recently emerged as a new animal model of host-virus interaction. Using a genome-wide association study in C. elegans wild populations and quantitative trait locus mapping, we identify a 159 base-pair deletion in the conserved drh-1 gene (encoding a RIG-I-like helicase) as a major determinant of viral sensitivity. We show that DRH-1 is required for the initiation of an antiviral RNAi pathway and the generation of virus-derived siRNAs (viRNAs). In mammals, RIG-I-domain containing proteins trigger an interferon-based innate immunity pathway in response to RNA virus infection. Our work in C. elegans demonstrates that the RIG-I domain has an ancient role in viral recognition. We propose that RIG-I acts as modular viral recognition factor that couples viral recognition to different effector pathways including RNAi and interferon responses. DOI: http://dx.doi.org/10.7554/eLife.00994.001

The Conserved miR-51 microRNA Family Is Redundantly Required for Embryonic Development and Pharynx Attachment in Caenorhabditis elegans

microRNAs (miRNAs) are ∼22-nucleotide small RNAs that act as endogenous regulators of gene expression by base-pairing with target mRNAs. Here we analyze the function of the six members of the Caenorhabditis elegans miR-51 family of miRNAs (miR-51, miR-52, miR-53, miR-54, miR-55, miR-56). miR-51 family miRNAs are broadly expressed from mid-embryogenesis onward. The miR-51 family is redundantly required for embryonic development. mir-51 family mutants display a highly penetrant pharynx unattached (Pun) phenotype, where the pharyngeal muscle, the food pump of C. elegans, is not attached to the mouth. Unusually, the Pun phenotype in mir-51 family mutants is not due to a failure to attach, but instead a failure to maintain attachment during late embryogenesis. Expression of the miR-51 family in the mouth is sufficient to maintain attachment. The Fat cadherin ortholog CDH-3 is expressed in the mouth and is a direct target of the miR-51 family miRNAs. Genetic analysis reveals that miR-51 family miRNAs might act in part through CDH-3 to regulate pharynx attachment. This study is the first to assign a function to the miR-51/miR-100 miRNA family in any organism.

Tertiary siRNAs mediate paramutation in C. elegans.

In the nematode Caenorhabditis elegans, different small RNA-dependent gene silencing mechanisms act in the germline to initiate transgenerational gene silencing. Piwi-interacting RNAs (piRNAs) can initiate transposon and gene silencing by acting upstream of endogenous short interfering RNAs (siRNAs), which engage a nuclear RNA interference (RNAi) pathway to trigger transcriptional gene silencing. Once gene silencing has been established, it can be stably maintained over multiple generations without the requirement of the initial trigger and is also referred to as RNAe or paramutation. This heritable silencing depends on the integrity of the nuclear RNAi pathway. However, the exact mechanism by which silencing is maintained across generations is not understood. Here we demonstrate that silencing of piRNA targets involves the production of two distinct classes of small RNAs with different genetic requirements. The first class, secondary siRNAs, are localized close to the direct target site for piRNAs. Nuclear import of the secondary siRNAs by the Argonaute HRDE-1 leads to the production of a distinct class of small RNAs that map throughout the transcript, which we term tertiary siRNAs. Both classes of small RNAs are necessary for full repression of the target gene and can be maintained independently of the initial piRNA trigger. Consistently, we observed a form of paramutation associated with tertiary siRNAs. Once paramutated, a tertiary siRNA generating allele confers dominant silencing in the progeny regardless of its own transmission, suggesting germline-transmitted siRNAs are sufficient for multigenerational silencing. This work uncovers a multi-step siRNA amplification pathway that promotes germline integrity via epigenetic silencing of endogenous and invading genetic elements. In addition, the same pathway can be engaged in environmentally induced heritable gene silencing and could therefore promote the inheritance of acquired traits.

A LIN28-Dependent Structural Change in pre-let-7g Directly Inhibits Dicer Processing

Several recent studies have provided evidence that LIN28, a cytoplasmic RNA-binding protein, inhibits the biogenesis of members of the let-7 microRNA family at the Dicer step in both mammals and Caenorhabditis elegans. However, the precise mechanism of inhibition is still poorly understood. Here we report on an in vitro study, which combined RNase footprinting, gel shift binding assays, and processing assays, to investigate the molecular basis and function of the interaction between the native let-7g precursor (pre-let-7g) and LIN28. We have mapped the structure of pre-let-7g and identified some regions of the terminal loop of pre-let-7g that physically interact with LIN28. We have also identified a conformational change upon LIN28 binding that results in the unwinding of an otherwise double-stranded region at the Dicer processing site of pre-let-7g. Furthermore, we showed that a mutant pre-let-7g that displays an open upper stem inhibited pre-let-7g Dicer processing to the same extent as LIN28. The data support a mechanism by which LIN28 can directly inhibit let-7g biogenesis at the Dicer processing step.

Post-developmental microRNA expression is required for normal physiology, and regulates aging in parallel to insulin/IGF-1 signaling in C. elegans

Regulation of gene expression by microRNAs (miRNAs) is essential for normal development, but the roles of miRNAs in the physiology of adult animals are poorly understood. We have isolated a conditional allele of DGCR8/pash-1, which allows reversible and rapid inactivation of miRNA synthesis in vivo in Caenorhabditis elegans. This is a powerful new tool that allows dissection of post-developmental miRNA functions. We demonstrate that continuous synthesis of miRNAs is dispensable for cellular viability but critical for the physiology of adult animals. Loss of miRNA synthesis in the adult reduces lifespan and results in rapid aging. The insulin/IGF-1 signaling pathway is a critical determinant of lifespan, and is modulated by miRNAs. We find that although miRNA expression is required for some mechanisms of lifespan extension, it is not essential for the longevity of animals lacking insulin/IGF-1 signaling. Further, misregulated insulin/IGF-1 signaling cannot account for the reduced lifespan caused by disruption of miRNA synthesis. We show that miRNAs act in parallel with insulin/IGF-1 signaling to regulate a shared set of downstream genes important for physiological processes that determine lifespan. We conclude that coordinated transcriptional and post-transcriptional regulation of gene expression promotes longevity.