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.This study was supported by funding from: Cancer Research UK (http://www.cancerresearchuk. org), grant RG57329 to EAM; European Research Council (erc.europa.eu/) Framework Programme 7, grant RG58558 to EAM; Gonville and Caius College fellowship to PS; Career Development Award from the Medical Research Council (http://www.mrc.ac.uk/) to PS. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).This is the final published version. It first appeared at http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1005078

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

SummaryTransgenerational 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.Graphical AbstractHighlights► Multigenerational inheritance and piRNAs converge on same nuclear silencing pathway ► HRDE1/WAGO-9 and chromatin factors required for inheritance of piRNA silencing ► piRNAs can induce multigenerational silencing for more than 20 generations. ► Long-term memory independent of piRNA triggers but remains dependent on nuclear pathwayMultigenerational inheritance and piRNAs converge on same silencing pathway, in which both nuclear WAGOs and chromatin factors are required. The piRNA trigger can be lost, but the nuclear silencing pathway maintains the silencing for more than 20 generations

ϒ production in p–Pb collisions at √sNN=8.16 TeV

ϒ production in p–Pb interactions is studied at the centre-of-mass energy per nucleon–nucleon collision √sNN = 8.16 TeV with the ALICE detector at the CERN LHC. The measurement is performed reconstructing bottomonium resonances via their dimuon decay channel, in the centre-of-mass rapidity intervals 2.03 < ycms < 3.53 and −4.46 < ycms < −2.96, down to zero transverse momentum. In this work, results on the ϒ(1S) production cross section as a function of rapidity and transverse momentum are presented. The corresponding nuclear modification factor shows a suppression of the ϒ(1S) yields with respect to pp collisions, both at forward and backward rapidity. This suppression is stronger in the low transverse momentum region and shows no significant dependence on the centrality of the interactions. Furthermore, the ϒ(2S) nuclear modification factor is evaluated, suggesting a suppression similar to that of the ϒ(1S). A first measurement of the ϒ(3S) has also been performed. Finally, results are compared with previous ALICE measurements in p–Pb collisions at √sNN = 5.02 TeV and with theoretical calculations.publishedVersio

Proteasome dysfunction triggers activation of SKN-1A/Nrf1 by the aspartic protease DDI-1

Proteasomes are essential for protein homeostasis in eukaryotes. To preserve cellular function, transcription of proteasome subunit genes is induced in response to proteasome dysfunction caused by pathogen attacks or proteasome inhibitor drugs. In Caenorhabditis elegans, this response requires SKN-1, a transcription factor related to mammalian Nrf1/2. Here, we use comprehensive genetic analyses to identify the pathway required for C. elegans to detect proteasome dysfunction and activate SKN-1. Genes required for SKN-1 activation encode regulators of ER traffic, a peptide N-glycanase, and DDI-1, a conserved aspartic protease. DDI-1 expression is induced by proteasome dysfunction, and we show that DDI-1 is required to cleave and activate an ER-associated isoform of SKN-1. Mammalian Nrf1 is also ER-associated and subject to proteolytic cleavage, suggesting a conserved mechanism of proteasome surveillance. Targeting mammalian DDI1 protease could mitigate effects of proteasome dysfunction in aging and protein aggregation disorders, or increase effectiveness of proteasome inhibitor cancer chemotherapies. DOI: http://dx.doi.org/10.7554/eLife.17721.00

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

Model of multigenerational target gene silencing by piRNAs and downstream 22G-RNAs.

<p>Left: piRNAs against target sites (blue) initiate localized 22G-RNA (blue) production that involves RNA-dependent RNA Polymerases (RdRPs) and Mutator proteins (Muts). 3′ to 5′ spreading of 22G-RNAs along the target gene (e.g. GFP, green) requires the nuclear RNAi pathway. This induces gene silencing that can be maintained over subsequent generations. Middle: Tertiary 22G-RNAs against a target (e.g. GFP, green) are able to silence genes with sequence similarity <i>in trans</i>. This leads to further generation of tertiary 22G-RNAs along the trans-silenced target (e.g. the <i>operon</i>/mCherry, red) by nuclear RNAi factors. Silencing by tertiary 22G-RNAs can become trigger-independent. Right: Paramutation by tertiary 22G-RNAs against mCherry (red) can be stably maintained in the absence of the original trigger(s).</p

Tertiary 22G-RNAs mediate paramutation.

<p>A) Representative fluorescence images of somatic and germline GFP (top row) and mCherry (middle row) expression and DIC images (bottom row) of the parental <i>operon</i> strain (left) and the outcrossed, silenced wild type <i>operon</i> animals (right). B) Small RNA high-throughput sequencing reads with unique matches antisense to the <i>operon</i> from outcrossed <i>operon</i> animals. The values of the <i>y</i>-axes correspond to reads matching the <i>operon</i> normalised to reads matching Histone 2B (<i>his-58</i>). The x-axes represent the relative position of reads in the <i>operon</i> transgene with numbers representing nucleotides from the start codon (set as 0). The transgene structure is schematically represented at the bottom. Colour code: red = mCherry, dark grey = <i>gpd-2</i> trans-splicing linker (L), green = GFP, light grey = <i>par-5</i> 3′UTR. C) Fluorescence images of germline mCherry expression (top panels) and DIC images (bottom panels) of the parental <i>mCherry</i>::<i>H2A</i> (left) and the outcrossed, trans-silenced wild type <i>mCherry</i>::<i>H2A</i> strains (right). D) Small RNA high-throughput sequencing reads with unique matches antisense to the mCherry transgene from animals as indicated in C). The values of the <i>y</i>-axes correspond to reads matching the mCherry sequence normalised to reads matching Histone 2B (<i>his-58</i>). The x-axes represent the relative position of reads in the <i>mCherry</i>::<i>H2A</i> transgene with numbers representing nucleotides from the start codon (set as 0). E) Crossing schemes (top) to generate trans-silenced <i>mCherry</i>::<i>H2A</i> animals as indicated above the representative fluorescence (GFP top, mCherry middle) and DIC images (bottom). Left panels are from a control cross using the parental non-silenced <i>operon</i> strain. Trans-silencing occurs with or without transmission of a silenced <i>operon</i> transgene copy.</p

An endogenous target shows 3′ to 5′ spreading of 22G-RNAs.

<p>Small RNA high-throughput sequencing reads with unique matches antisense to Y48G1B8M.5 from wild type and mutant animals as indicated. The values of the <i>y</i>-axes are antisense 22G siRNA reads per million of total reads. The x-axes represent the relative position of reads in the target gene with numbers representing nucleotides from the start codon (set as 0). The transcript structure is schematically depicted at the bottom with light grey and dark grey boxes representing alternating exons.</p

22G-RNAs distal to piRNA target sites require the nuclear RNAi pathway.

<p>A) Small RNA high-throughput sequencing reads with unique matches antisense to the <i>piRNA sensor</i> from wild type and various mutant animals as indicated. The values of the <i>y</i>-axes correspond to reads matching the <i>piRNA sensor</i> normalised to reads matching Histone 2B (<i>his-58</i>). The x-axes represent the relative position of reads in the <i>piRNA sensor</i> transgene with numbers representing nucleotides from the start codon (set as 0). The transgene structure is schematically represented at the bottom. Colour code: green = GFP, grey = Histone 2B (<i>his-58</i>), dark blue = 21UR-1 target site plus/minus 50 bp, light blue = <i>tbb-2</i> 3′UTR. B) Left panel: Enrichment of small RNA high-throughput sequencing reads with matches antisense to the <i>piRNA sensor</i> in HRDE-1 Immunoprecipitation (IP). Displayed is the fold enrichment of reads found in anti-HRDE-1 IP from wild type versus <i>hrde-1</i> mutant animals. X-axis, schematic representation of the transgene and colour code as in A). Right panel: Western blot of anti-HRDE-1 Immunoprecipitation from wild type (left 3 lanes) and <i>hrde-1</i> mutant (right 3 lanes) animals. Antibodies used for western blot are anti-HRDE-1 and anti-PRG-1 as loading control. Inp. = 1% input, sup. = supernatant, IP = Immuoprecipitate. On the left, the relative migration of the 130 and 100 kDa bands of the PAGE Ruler Plus (MBI Fermentas) marker are indicated.</p