H3K9me3 is required for inheritance of small RNAs that target a unique subset of newly evolved genes

In Caenorhabditis elegans, RNA interference (RNAi) responses can transmit across generations via small RNAs. RNAi inheritance is associated with Histone-3-Lysine-9 tri-methylation (H3K9me3) of the targeted genes. In other organisms, maintenance of silencing requires a feed-forward loop between H3K9me3 and small RNAs. Here, we show that in C. elegans not only is H3K9me3 unnecessary for inheritance, the modification’s function depends on the identity of the RNAi-targeted gene. We found an asymmetry in the requirement for H3K9me3 and the main worm H3K9me3 methyltransferases, SET-25 and SET-32. Both methyltransferases promote heritable silencing of the foreign gene gfp, but are dispensable for silencing of the endogenous gene oma-1. Genome-wide examination of heritable endogenous small interfering RNAs (endo-siRNAs) revealed that endo-siRNAs that depend on SET-25 and SET-32 target newly acquired and highly H3K9me3 marked genes. Thus, ‘repressive’ chromatin marks could be important specifically for heritable silencing of genes which are flagged as ‘foreign’, such as gfp.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

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H3K9me3 is Required for Inheritance of Small RNAs that Target a Unique Subset of Newly Evolved Genes

10.1101/338582 ◽

2018 ◽

Cited By ~ 5

Author(s):

Itamar Lev ◽

Hila Gingold ◽

Oded Rechavi

Keyword(s):

Rna Interference ◽

Caenorhabditis Elegans ◽

Small Rnas ◽

Foreign Gene ◽

Small Interfering Rnas ◽

Endogenous Gene ◽

C Elegans ◽

Histone 3 ◽

Repressive Chromatin ◽

Genome Wide

AbstractIn Caenorhabditis elegans, RNA interference (RNAi) responses can transmit across generations via small RNAs. RNAi inheritance is associated with Histone-3-Lysine-9 tri-methylation (H3K9me3) of the targeted genes. In other organisms, maintenance of silencing requires a feed-forward loop between H3K9me3 and small RNAs. Here we show that in C. elegans not only is H3K9me3 unnecessary for inheritance, the modification’s function depends on the identity of the RNAi-targeted gene. We found an asymmetry in the requirement for H3K9me3 and the main worm H3K9me3 methyltransferases, SET-25 and SET-32. Both methyltransferases promote heritable silencing of the foreign gene gfp, but are dispensable for silencing of the endogenous gene oma-1. Genome-wide examination of heritable endogenous small interfering RNAs (endo-siRNAs) revealed that the SET-25-dependent heritable endo-siRNAs target newly acquired and highly H3K9me3 marked genes. Thus, “repressive” chromatin marks could be important specifically for heritable silencing of genes which are flagged as “foreign”, such as gfp.

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Suppression of endogenous gene silencing by bidirectional cytoplasmic RNA decay in Arabidopsis

Science ◽

10.1126/science.aaa2618 ◽

2015 ◽

Vol 348 (6230) ◽

pp. 120-123 ◽

Cited By ~ 76

Author(s):

Xinyan Zhang ◽

Ying Zhu ◽

Xiaodan Liu ◽

Xinyu Hong ◽

Yang Xu ◽

...

Keyword(s):

Gene Silencing ◽

Plant Immunity ◽

Rna Decay ◽

Foreign Gene ◽

Small Interfering Rnas ◽

Developmental Defects ◽

Posttranscriptional Gene Silencing ◽

Endogenous Gene ◽

Cytoplasmic Rna ◽

Cognate Gene

Plant immunity against foreign gene invasion takes advantage of posttranscriptional gene silencing (PTGS). How plants elaborately avert inappropriate PTGS of endogenous coding genes remains unclear. We demonstrate in Arabidopsis that both 5′-3′ and 3′-5′ cytoplasmic RNA decay pathways act as repressors of transgene and endogenous PTGS. Disruption of bidirectional cytoplasmic RNA decay leads to pleiotropic developmental defects and drastic transcriptomic alterations, which are substantially rescued by PTGS mutants. Upon dysfunction of bidirectional RNA decay, a large number of 21- to 22-nucleotide endogenous small interfering RNAs are produced from coding transcripts, including multiple microRNA targets, which could interfere with their cognate gene expression and functions. This study highlights the risk of unwanted PTGS and identifies cytoplasmic RNA decay pathways as safeguards of plant transcriptome and development.

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Small RNAs in parasitic nematodes – forms and functions

Parasitology ◽

10.1017/s0031182019001689 ◽

2019 ◽

Vol 147 (8) ◽

pp. 855-864

Author(s):

Collette Britton ◽

Roz Laing ◽

Eileen Devaney

Keyword(s):

Gene Expression ◽

Small Rna ◽

Small Rnas ◽

Target Genes ◽

Parasitic Nematodes ◽

Small Rna Sequencing ◽

Small Interfering Rnas ◽

Rna Sequences ◽

C Elegans

AbstractSmall RNAs are important regulators of gene expression. They were first identified in Caenorhabditis elegans, but it is now apparent that the main small RNA silencing pathways are functionally conserved across diverse organisms. Availability of genome data for an increasing number of parasitic nematodes has enabled bioinformatic identification of small RNA sequences. Expression of these in different lifecycle stages is revealed by small RNA sequencing and microarray analysis. In this review we describe what is known of the three main small RNA classes in parasitic nematodes – microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) – and their proposed functions. miRNAs regulate development in C. elegans and the temporal expression of parasitic nematode miRNAs suggest modulation of target gene levels as parasites develop within the host. miRNAs are also present in extracellular vesicles released by nematodes in vitro, and in plasma from infected hosts, suggesting potential regulation of host gene expression. Roles of piRNAs and siRNAs in suppressing target genes, including transposable elements, are also reviewed. Recent successes in RNAi-mediated gene silencing, and application of small RNA inhibitors and mimics will continue to advance understanding of small RNA functions within the parasite and at the host–parasite interface.

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The interplay between small RNA pathways shapes chromatin landscapes in C. elegans

10.1101/320713 ◽

2018 ◽

Author(s):

Ekaterina Gushchanskaia ◽

Ruben Esse ◽

Qicheng Ma ◽

Nelson Lau ◽

Alla Grishok

Keyword(s):

Small Rnas ◽

Target Genes ◽

Noncoding Rnas ◽

Small Interfering Rnas ◽

Loss Of Function ◽

Partial Loss ◽

Rna Dependent Rna Polymerase ◽

C Elegans ◽

Highly Expressed Genes ◽

Rna Pathways

ABSTRACTThe nematode C. elegans contains several types of endogenous small interfering RNAs (endo-siRNAs) produced by RNA-dependent RNA polymerase (RdRP) complexes. Both “silencing” siRNAs bound by Worm-specific Argonautes (WAGO) and “activating” siRNAs bound by the CSR-1 Argonaute require the DRH-3 helicase, an RdRP component. Here we show that, in the drh-3(ne4253) mutant deficient in RdRP-produced secondary endo-siRNAs, the silencing histone mark H3K9me3 is largely depleted, whereas in the csr-1 partial loss-of-function mutant this mark is ectopically deposited on CSR-1 target genes. Moreover, we observe ectopic H3K9me3 at enhancer elements in both drh-3 and csr-1 partial loss-of-function mutants and describe small RNAs matching enhancers. Finally, we detect accumulation of H3K27me3 at highly expressed genes in the drh-3(ne4253) mutant, which correlates with their reduced transcription. Our study shows that when abundant RdRP-produced siRNAs are depleted, there is ectopic elevation of noncoding RNAs linked to increase in silencing chromatin marks. Moreover, our results suggest that enhancer small RNAs may guide local H3K9 methylation.

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Unusual predominance of maintenance DNA methylation in Spirodela polyrhiza

10.1101/2020.12.03.410332 ◽

2020 ◽

Author(s):

Alex Harkess ◽

Adam J. Bewick ◽

Zefu Lu ◽

Paul Fourounjian ◽

Joachim Messing ◽

...

Keyword(s):

Dna Methylation ◽

Plant Species ◽

Repetitive Dna ◽

Small Rnas ◽

Small Interfering Rnas ◽

Gene Body Methylation ◽

Spirodela Polyrhiza ◽

Genome Wide ◽

Low Levels ◽

Maintenance Methylation

Abstract5-methylcytosine (5mC) is a modified base often described as necessary for the proper regulation of genes and transposons and for the maintenance of genome integrity in plants. However, the extent of this dogma is limited by the current phylogenetic sampling of land plant species diversity. Here, we report that a monocot plant, Spirodela polyrhiza, has lost CG gene body methylation, genome-wide CHH methylation, and the presence or expression of several genes in the highly conserved RNA-directed DNA methylation (RdDM) pathway. It has also lost the CHH methyltransferase CHROMOMETHYLASE 2. Consequently, the transcriptome is depleted of 24-nucleotide, heterochromatic, small interfering RNAs that act as guides for the deposition of 5mC to RdDM-targeted loci in all other currently sampled angiosperm genomes. Although the genome displays low levels of genome-wide 5mC primarily at LTR retrotransposons, CG maintenance methylation is still functional. In contrast, CHG methylation is weakly maintained even though H3K9me2 is present at loci dispersed throughout the euchromatin and highly enriched at regions likely demarcating pericentromeric regions. Collectively, these results illustrate that S. polyrhiza is maintaining CG and CHG methylation mostly at repeats in the absence of small RNAs. S. polyrhiza reproduces rapidly through clonal propagation in aquatic environments, which we hypothesize may enable low levels of maintenance methylation to persist in large populations.Significance StatementDNA methylation is a widespread chromatin modification that is regularly found in all plant species. By examining one aquatic duckweed species, Spirodela polyrhiza, we find that it has lost highly conserved genes involved in methylation of DNA at sites often associated with repetitive DNA, and within genes, however DNA methylation and heterochromatin is maintained during cell division at other sites. Consequently, small RNAs that normally guide methylation to silence repetitive DNA like retrotransposons are diminished. Despite the loss of a highly conserved methylation pathway, and the reduction of small RNAs that normally target repetitive DNA, transposons have not proliferated in the genome, perhaps due in part to the rapid, clonal growth lifestyle of duckweeds.

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RppH can faithfully replace TAP to allow cloning of 5’-triphosphate carrying small RNAs

10.1101/283077 ◽

2018 ◽

Cited By ~ 1

Author(s):

Miguel Vasconcelos Almeida ◽

António Miguel de Jesus Domingues ◽

Hanna Lukas ◽

Maria Mendez-Lago ◽

René F. Ketting

Keyword(s):

Small Rna ◽

Small Rnas ◽

Small Rna Sequencing ◽

Cloning Efficiency ◽

Library Preparation ◽

Small Interfering Rnas ◽

Rdrp Activity ◽

C Elegans ◽

Rna Pathways ◽

Small Rna Species

AbstractRNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. Much like plants, but unlike Drosophila and mammals, worms have RNA-dependent RNA Polymerases (RdRPs) that directly synthesize small RNAs using other transcripts as a template. The very prominent secondary small interfering RNAs, also called 22G-RNAs, produced by the RdRPs RRF-1 and EGO-1 in C. elegans, maintain the 5’ triphosphate group, stemming from RdRP activity, also after loading into an Argonaute protein. This creates a technical issue, since 5’PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5’ pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison.

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Stress Resets Transgenerational Small RNA Inheritance

10.1101/669051 ◽

2019 ◽

Cited By ~ 2

Author(s):

Leah Houri-Ze’evi ◽

Guy Teichman ◽

Hila Gingold ◽

Oded Rechavi

Keyword(s):

Small Rna ◽

Small Rnas ◽

Memory Trace ◽

Mapk Pathway ◽

Transgenerational Inheritance ◽

Cultivation Conditions ◽

C Elegans ◽

Genome Wide ◽

A Genome ◽

Basic Concepts

AbstractTransgenerational inheritance of small RNAs is challenging basic concepts of heredity and achieving control over such responses is of great interest. InC. elegansnematodes, small RNAs are transmitted across generations to establish a transgenerational memory trace of ancestral environments and distinguish self from non-self genes. Inheritance of small RNAs is regulated by dedicated machinery and carryover of aberrant heritable small RNA responses was shown to be maladaptive and to induce sterility. Here we show that various types of stress (starvation, high temperatures, and high osmolarity) but not non-stressful changes in cultivation conditions, lead to resetting of small RNA inheritance. We found that stress leads to a genome-wide reduction in heritable small RNA levels and that mutants defective in different stress pathways exhibit irregular RNAi inheritance dynamics. Moreover, we discovered that resetting of heritable RNAi is orchestrated by MAPK pathway factors, the transcription factor SKN-1, and the MET-2 methyltransferase. Termination of small RNA inheritance, and the fact that this process depends on stress, could protect from run-on of environment-irrelevant heritable gene regulation.

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Multigenerational Regulation of the Caenorhabditis elegans Chromatin Landscape by Germline Small RNAs

Annual Review of Genetics ◽

10.1146/annurev-genet-112618-043505 ◽

2019 ◽

Vol 53 (1) ◽

pp. 289-311 ◽

Cited By ~ 10

Author(s):

Natasha E. Weiser ◽

John K. Kim

Keyword(s):

Gene Expression ◽

Caenorhabditis Elegans ◽

Small Rnas ◽

Model Organism ◽

Epigenetic Inheritance ◽

Small Interfering Rnas ◽

Transgenerational Epigenetic Inheritance ◽

C Elegans ◽

Small Noncoding Rnas ◽

Rna Pathways

In animals, small noncoding RNAs that are expressed in the germline and transmitted to progeny control gene expression to promote fertility. Germline-expressed small RNAs, including endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs), drive the repression of deleterious transcripts such as transposons, repetitive elements, and pseudogenes. Recent studies have highlighted an important role for small RNAs in transgenerational epigenetic inheritance via regulation of heritable chromatin marks; therefore, small RNAs are thought to convey an epigenetic memory of genomic self and nonself elements. Small RNA pathways are highly conserved in metazoans and have been best described for the model organism Caenorhabditis elegans. In this review, we describe the biogenesis, regulation, and function of C. elegans endo-siRNAs and piRNAs, along with recent insights into how these distinct pathways are integrated to collectively regulate germline gene expression, transgenerational epigenetic inheritance, and ultimately, animal fertility.

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Interplay between small RNA pathways shapes chromatin landscapes in C. elegans

Nucleic Acids Research ◽

10.1093/nar/gkz275 ◽

2019 ◽

Vol 47 (11) ◽

pp. 5603-5616 ◽

Cited By ~ 3

Author(s):

Ekaterina S Gushchanskaia ◽

Ruben Esse ◽

Qicheng Ma ◽

Nelson C Lau ◽

Alla Grishok

Keyword(s):

Small Rnas ◽

Target Genes ◽

Noncoding Rnas ◽

Null Mutant ◽

Small Interfering Rnas ◽

Nematode Caenorhabditis Elegans ◽

Rna Dependent Rna Polymerase ◽

C Elegans ◽

Highly Expressed Genes ◽

Rna Pathways

Abstract The nematode Caenorhabditis elegans contains several types of endogenous small interfering RNAs (endo-siRNAs) produced by RNA-dependent RNA polymerase (RdRP) complexes. Both ‘silencing’ siRNAs bound by Worm-specific Argonautes (WAGO) and ‘activating’ siRNAs bound by the CSR-1 Argonaute require the DRH-3 helicase, an RdRP component. Here, we show that, in the drh-3(ne4253) mutant deficient in RdRP-produced secondary endo-siRNAs, the silencing histone mark H3K9me3 is largely depleted, whereas in the csr-1 partially rescued null mutant strain (WM193), this mark is ectopically deposited on CSR-1 target genes. Moreover, we observe ectopic H3K9me3 at enhancer elements and an increased number of small RNAs that match enhancers in both drh-3 and csr-1 mutants. Finally, we detect accumulation of H3K27me3 at highly expressed genes in the drh-3(ne4253) mutant, which correlates with their reduced transcription. Our study shows that when abundant RdRP-produced siRNAs are depleted, there is ectopic elevation of noncoding RNAs linked to sites with increased silencing chromatin marks. Moreover, our results suggest that enhancer small RNAs may guide local H3K9 methylation.

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Mobile small RNAs regulate genome-wide DNA methylation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1515072113 ◽

2016 ◽

Vol 113 (6) ◽

pp. E801-E810 ◽

Cited By ~ 111

Author(s):

Mathew G. Lewsey ◽

Thomas J. Hardcastle ◽

Charles W. Melnyk ◽

Attila Molnar ◽

Adrián Valli ◽

...

Keyword(s):

Dna Methylation ◽

Small Rnas ◽

De Novo ◽

Dna Methyltransferases ◽

Transcriptional Gene Silencing ◽

Triple Mutant ◽

Root Cells ◽

Endogenous Gene ◽

Genome Wide ◽

Root Tissues

RNA silencing at the transcriptional and posttranscriptional levels regulates endogenous gene expression, controls invading transposable elements (TEs), and protects the cell against viruses. Key components of the mechanism are small RNAs (sRNAs) of 21–24 nt that guide the silencing machinery to their nucleic acid targets in a nucleotide sequence-specific manner. Transcriptional gene silencing is associated with 24-nt sRNAs and RNA-directed DNA methylation (RdDM) at cytosine residues in three DNA sequence contexts (CG, CHG, and CHH). We previously demonstrated that 24-nt sRNAs are mobile from shoot to root in Arabidopsis thaliana and confirmed that they mediate DNA methylation at three sites in recipient cells. In this study, we extend this finding by demonstrating that RdDM of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot and that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. Mobile sRNA-dependent non-CG methylation is largely dependent on the DOMAINS REARRANGED METHYLTRANSFERASES 1/2 (DRM1/DRM2) RdDM pathway but is independent of the CHROMOMETHYLASE (CMT)2/3 DNA methyltransferases. Specific superfamilies of TEs, including those typically found in gene-rich euchromatic regions, lose DNA methylation in a mutant lacking 22- to 24-nt sRNAs (dicer-like 2, 3, 4 triple mutant). Transcriptome analyses identified a small number of genes whose expression in roots is associated with mobile sRNAs and connected to DNA methylation directly or indirectly. Finally, we demonstrate that sRNAs from shoots of one accession move across a graft union and target DNA methylation de novo at normally unmethylated sites in the genomes of root cells from a different accession.

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