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

DEPS-1 is required for piRNA-dependent silencing and PIWI condensate organisation in Caenorhabditis elegans

Abstract: Membraneless organelles are sites for RNA biology including small non-coding RNA (ncRNA) mediated gene silencing. How small ncRNAs utilise phase separated environments for their function is unclear. We investigated how the PIWI-interacting RNA (piRNA) pathway engages with the membraneless organelle P granule in Caenorhabditis elegans. Proteomic analysis of the PIWI protein PRG-1 reveals an interaction with the constitutive P granule protein DEPS-1. DEPS-1 is not required for piRNA biogenesis but piRNA-dependent silencing: deps-1 mutants fail to produce the secondary endo-siRNAs required for the silencing of piRNA targets. We identify a motif on DEPS-1 which mediates a direct interaction with PRG-1. DEPS-1 and PRG-1 form intertwining clusters to build elongated condensates in vivo which are dependent on the Piwi-interacting motif of DEPS-1. Additionally, we identify EDG-1 as an interactor of DEPS-1 and PRG-1. Our study reveals how specific protein-protein interactions drive the spatial organisation and piRNA-dependent silencing within membraneless organelles

A team of heterochromatin factors collaborates with small RNA pathways to combat repetitive elements and germline stress.

Repetitive sequences derived from transposons make up a large fraction of eukaryotic genomes and must be silenced to protect genome integrity. Repetitive elements are often found in heterochromatin; however, the roles and interactions of heterochromatin proteins in repeat regulation are poorly understood. Here we show that a diverse set of C. elegans heterochromatin proteins act together with the piRNA and nuclear RNAi pathways to silence repetitive elements and prevent genotoxic stress in the germ line. Mutants in genes encoding HPL-2/HP1, LIN-13, LIN-61, LET-418/Mi-2, and H3K9me2 histone methyltransferase MET-2/SETDB1 also show functionally redundant sterility, increased germline apoptosis, DNA repair defects, and interactions with small RNA pathways. Remarkably, fertility of heterochromatin mutants could be partially restored by inhibiting cep-1/p53, endogenous meiotic double strand breaks, or the expression of MIRAGE1 DNA transposons. Functional redundancy among factors and pathways underlies the importance of safeguarding the genome through multiple means

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

SUMO-modified Sp3 represses transcription by provoking local heterochromatic gene silencing

Modification of many transcription factors including Sp3 and steroidogenic factor 1 with the small ubiquitin-like modifier (SUMO) is associated with transcriptional repression. Here, we show that SUMOylation of transcription factors bound to DNA provokes the establishment of compacted repressive chromatin with characteristics of heterochromatin. Chromatin immunoprecipitation experiments revealed SUMO-dependent recruitment of the chromatin remodeller Mi-2, MBT-domain proteins, heterochromatic protein 1, and the histone methyltransferases SETDB1 and SUV4-20H, concomitant with the establishment of histone modifications associated with repressed genes, including H3K9 and H4K20 trimethylation. These results indicate that SUMOylation has a crucial role in regulating gene expression by initiating chromatin structure changes that render DNA inaccessible to the transcription machinery

RNA uridyl transferases TUT4/7 differentially regulate miRNA variants depending on the cancer cell type.

The human terminal uridyl transferases TUT4 and TUT7 (TUT4/7) catalyze the additions of uridines at the 3' end of RNAs, including the precursors of the tumor suppressor miRNA let-7 upon recruitment by the oncoprotein LIN28A. As a consequence, let-7 family miRNAs are down-regulated. Disruption of this TUT4/7 activity inhibits tumorigenesis. Hence, targeting TUT4/7 could be a potential anticancer therapy. In this study, we investigate TUT4/7-mediated RNA regulation in two cancer cell lines by establishing catalytic knockout models. Upon TUT4/7 mutation, we observe a significant reduction in miRNA uridylation, which results in defects in cancer cell properties such as cell proliferation and migration. With the loss of TUT4/7-mediated miRNA uridylation, the uridylated miRNA variants are replaced by adenylated isomiRs. Changes in miRNA modification profiles are accompanied by deregulation of expression levels in specific cases. Unlike let-7s, most miRNAs do not depend on LIN28A for TUT4/7-mediated regulation. Additionally, we identify TUT4/7-regulated cell-type-specific miRNA clusters and deregulation in their corresponding mRNA targets. Expression levels of miR-200c-3p and miR-141-3p are regulated by TUT4/7 in a cancer cell-type-specific manner. Subsequently, BCL2, which is a well-established target of miR-200c is up-regulated. Therefore, TUT4/7 loss causes deregulation of miRNA-mRNA networks in a cell-type-specific manner. Understanding of the underlying biology of such cell-type-specific deregulation will be an important aspect of targeting TUT4/7 for potential cancer therapies

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

Nuclear RNAi is necessary to initiate synthesis of tertiary siRNAs.

<p>A) The principle of the <i>operon</i> transgene expression. Straight line represents the DNA locus (bottom), curved lines pre-mRNA and mRNA (before and after nuclear export). Diamonds and snowflakes are mCherry protein (red) and GFP protein (green), respectively (top). B) Schematic representation of the transgenes in the <i>operon; piRNA sensor</i> strain. The <i>piRNA sensor</i> (bottom) generates 22G-RNAs (black lines) against GFP that can confer trans-silencing of the <i>operon</i>-derived GFP. In case a spreading of 22G-RNAs occurs in the nucleus, silencing of mCherry would be expected. C) 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), silenced wild type <i>operon; piRNA sensor</i> animals (middle) and de-silenced <i>operon; piRNA sensor; hrde-1</i> mutant (right) animals. D) Small RNA high-throughput sequencing reads with unique matches antisense to the <i>operon</i> from animals as indicated in C). 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.</p