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

2019 ◽  
Vol 47 (11) ◽  
pp. 5603-5616 ◽  
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.

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

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.

scholarly journals Small RNAs in parasitic nematodes – forms and functions

Parasitology ◽  
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.

scholarly journals GTSF-1 is required for the formation of a functional RNA-dependent RNA Polymerase complex in C. elegans

2018 ◽  
Author(s):  
Miguel Vasconcelos Almeida ◽  
Sabrina Dietz ◽  
Stefan Redl ◽  
Emil Karaulanov ◽  
Andrea Hildebrandt ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Gene Silencing ◽  
Small Rna ◽  
Small Rnas ◽  
Zinc Fingers ◽  
Specific Factor ◽  
Rna Dependent Rna Polymerase ◽  
C Elegans ◽  
Argonaute Proteins ◽  
Rna Pathways

AbstractIn every domain of life, Argonaute proteins and their associated small RNAs regulate gene expression. Despite great conservation of Argonaute proteins throughout evolution, many proteins acting in small RNA pathways are not widely conserved. Gametocyte-specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured, acidic C-terminal tail, are conserved in animals and act in small RNA pathways. In fly and mouse, they are required for fertility and have been shown to interact with Piwi clade Argonautes. We identified T06A10.3 as the Caenorhabditis elegans Gtsf1 homolog and named it gtsf-1. Given its conserved nature and roles in Piwi-mediated gene silencing, we sought out to characterize GTSF-1 in the context of the small RNA pathways of C. elegans. Like its homologs, GTSF-1 is required for normal fertility. Surprisingly, we report that GTSF-1 is not required for Piwi-mediated gene silencing. Instead, gtsf-1 mutants show strong depletion of a class of endogenous small RNAs, known as 26G-RNAs, and fully phenocopy mutants lacking RRF-3, the RNA-dependent RNA Polymerase that synthesizes 26G-RNAs. We show, both in vivo and in vitro, that GTSF-1 specifically and robustly interacts with RRF-3 via its tandem CHHC zinc fingers. Furthermore, we demonstrate that GTSF-1 is required for the assembly of a larger RRF-3 and DCR-1-containing complex, also known as ERIC, thereby allowing for 26G-RNA generation. We propose that GTSF-1 homologs may similarly act to drive the assembly of larger complexes that subsequently act in small RNA production and/or in imposing small RNA-mediated silencing activities.

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

2018 ◽  
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.

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

2019 ◽  
Vol 53 (1) ◽  
pp. 289-311 ◽  
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.

scholarly journals Modeling small RNA competition in C. elegans

2015 ◽  
Author(s):  
Joshua Elkington
Keyword(s):  
Small Rna ◽  
Random Sampling ◽  
Small Rnas ◽  
Small Interfering Rnas ◽  
System Of Differential Equations ◽  
Cell Dynamics ◽  
Rna Dynamics ◽  
C Elegans ◽  
Normal Behavior ◽  
Rna Pathways

Small RNAs have been determined to have an essential role in gene regulation. However, competition between small RNAs is a poorly understood aspect of small RNA dynamics. Recent evidence has suggested that competition between small RNA pathways arises from a scarcity of common resources essential for small RNA activity. In order to understand how competition affects small RNAs in C. elegans, a system of differential equations was used. The model recreates normal behavior of small RNAs and uses random sampling in order to determine the coefficients of competition for each small RNA class. The model includes endogenous small-interfering RNAs (endo-siRNA), exogenous small-interfering RNAs (exo-siRNA), and microRNAs (miRNA). The model predicts that exo-siRNAs is dominated by competition between endo-siRNAs and miRNAs. Furthermore, the model predicts that competition is required for normal levels of endogenous small RNAs to be maintained. Although the model makes several assumptions about cell dynamics, the model is still useful in order to understand competition between small RNA pathways.

scholarly journals Widespread occurrence of microRNA-mediated target cleavage on membrane-bound polysomes

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xiaoyu Yang ◽  
Chenjiang You ◽  
Xufeng Wang ◽  
Lei Gao ◽  
Beixin Mo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Gene Silencing ◽  
High Frequency ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Noncoding Rnas ◽  
Small Interfering Rnas ◽  
Widespread Occurrence ◽  
Membrane Bound ◽  
Different Tissues

Abstract Background Small RNAs (sRNAs) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) serve as core players in gene silencing at transcriptional and post-transcriptional levels in plants, but their subcellular localization has not yet been well studied, thus limiting our mechanistic understanding of sRNA action. Results We investigate the cytoplasmic partitioning of sRNAs and their targets globally in maize (Zea mays, inbred line “B73”) and rice (Oryza sativa, cv. “Nipponbare”) by high-throughput sequencing of polysome-associated sRNAs and 3′ cleavage fragments, and find that both miRNAs and a subset of 21-nucleotide (nt)/22-nt siRNAs are enriched on membrane-bound polysomes (MBPs) relative to total polysomes (TPs) across different tissues. Most of the siRNAs are generated from transposable elements (TEs), and retrotransposons positively contributed to MBP overaccumulation of 22-nt TE-derived siRNAs (TE-siRNAs) as opposed to DNA transposons. Widespread occurrence of miRNA-mediated target cleavage is observed on MBPs, and a large proportion of these cleavage events are MBP-unique. Reproductive 21PHAS (21-nt phasiRNA-generating) and 24PHAS (24-nt phasiRNA-generating) precursors, which were commonly considered as noncoding RNAs, are bound by polysomes, and high-frequency cleavage of 21PHAS precursors by miR2118 and 24PHAS precursors by miR2275 is further detected on MBPs. Reproductive 21-nt phasiRNAs are enriched on MBPs as opposed to TPs, whereas 24-nt phasiRNAs are nearly completely devoid of polysome occupancy. Conclusions MBP overaccumulation is a conserved pattern for cytoplasmic partitioning of sRNAs, and endoplasmic reticulum (ER)-bound ribosomes function as an independent regulatory layer for miRNA-induced gene silencing and reproductive phasiRNA biosynthesis in maize and rice.

lag-1, a gene required for lin-12 and glp-1 signaling in Caenorhabditis elegans, is homologous to human CBF1 and Drosophila Su(H)

Development ◽  
1996 ◽  
Vol 122 (5) ◽  
pp. 1373-1383 ◽  
Author(s):  
S. Christensen ◽  
V. Kodoyianni ◽  
M. Bosenberg ◽  
L. Friedman ◽  
J. Kimble
Keyword(s):  
Caenorhabditis Elegans ◽  
Binding Sites ◽  
Feedback Loop ◽  
Target Genes ◽  
Sequence Similarity ◽  
Nematode Caenorhabditis Elegans ◽  
C Elegans ◽  
Downstream Target ◽  
Binding Factor ◽  
Flanking Regions

The homologous receptors LIN-12 and GLP-1 mediate diverse cell-signaling events during development of the nematode Caenorhabditis elegans. These two receptors appear to be functionally interchangeable and have sequence similarity to Drosophila Notch. Here we focus on a molecular analysis of the lag-1 gene (lin-12 -and glp-1), which plays a central role in LIN-12 and GLP-1-mediated signal transduction. We find that the predicted LAG-1 protein is homologous to two DNA-binding proteins: human C Promoter Binding Factor (CBF1) and Drosophila Suppressor of Hairless (Su(H)). Furthermore, we show that LAG-1 binds specifically to the DNA sequence RTGGGAA, previously identified as a CBF-1/Su(H)-binding site. Finally, we report that the 5′ flanking regions and first introns of the lin-12, glp-1 and lag-1 genes are enriched for potential LAG-1-binding sites. We propose that LAG-1 is a transcriptional regulator that serves as a primary link between the LIN-12 and GLP-1 receptors and downstream target genes in C. elegans. In addition, we propose that LAG-1 may be a key component of a positive feedback loop that amplifies activity of the LIN-12/GLP-1 pathway.

scholarly journals Diversification of small RNA pathways underlies germline RNAi incompetence in wild C. elegans strains

2021 ◽  
Author(s):  
Han Ting Chou ◽  
Francisco Valencia ◽  
Jacqueline C. Alexander ◽  
Avery Davis Bell ◽  
Diptodip Deb ◽  
...  
Keyword(s):  
Target Genes ◽  
Biological Significance ◽  
Laboratory Strain ◽  
Biological Processes ◽  
C Elegans ◽  
Genetics Research ◽  
Rna Pathways ◽  
Increased Activity ◽  
Rnai Response

ABSTRACTThe discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, by both uncovering essential biological processes and creating new tools for developmental geneticists. However, wild-type C. elegans strains vary dramatically in their response to exogenous RNAi, challenging our characterization of RNAi in the lab relative to its activity and significance in nature. Here, we investigate why some strains fail to mount a robust RNAi response to germline targets. We observe diversity in mechanism: in some strains, the response is stochastic, either on or off among individuals, while in others the response is consistent but delayed. Increased activity of the Argonaute PPW-1, which is required for germline RNAi in the laboratory strain N2, rescues the response in some strains, but dampens it further in others. Across strains, we observe variability in expression of known RNAi genes and strain-specific instances of pseudogenization and allelic divergence. Our results support the conclusions that Argonautes share overlapping functions, that germline RNAi incompetence is strain-specific but likely caused by genetic variants at common genes, and that RNAi pathways are evolving rapidly and dynamically. This work expands our understanding of RNAi by identifying conserved and variable pathway components, and it offers new access into characterizing gene function, identifying pathway interactions, and elucidating the biological significance of RNAi.

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

2018 ◽  
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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