Small RNAs, big impact: small RNA pathways in transposon control and their effect on the host stress response

SummarySmall RNA pathways defend the germlines of animals against selfish genetic elements and help to maintain genomic integrity. At the same time, their activity needs to be well-controlled to prevent silencing of ‘self’ genes. Here, we reveal a proteolytic mechanism that controls endogenous small interfering (22G) RNA activity in the Caenorhabditis elegans germline to protect genome integrity and maintain fertility. We find that WAGO-1 and WAGO-3 Argonaute (Ago) proteins are matured through proteolytic processing of their unusually proline-rich N-termini. In the absence of DPF-3, a P-granule-localized N-terminal dipeptidase orthologous to mammalian DPP8/9, processing fails, causing a change of identity of 22G RNAs bound to these WAGO proteins. Desilencing of repeat- and transposon-derived transcripts, DNA damage and acute sterility ensue. These phenotypes are recapitulated when WAGO-1 and WAGO-3 are rendered resistant to DFP-3-mediated processing, identifying them as critical substrates of DPF-3. We conclude that N-terminal processing of Ago proteins regulates their activity and promotes discrimination of self from non-self by ensuring association with the proper complement of small RNAs.Graphical Abstract: The role of DPF-3 in the fertility of the animalsIn wild type animals, the WAGO-1 and WAGO-3 Argonaute proteins are produced as immature pro-proteins with N-termini (N) that are unusually rich in prolines (P). N-terminal processing by DPF-3 is required for loading of the proper small RNA cargo and stabilization of WAGO-3. Accordingly, loss of this processing activity causes desilencing of transposable elements (TE), cell death and sterility.

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Gene Expression Changes Induced byTrypanosoma cruziShed Microvesicles in Mammalian Host Cells: Relevance of tRNA-Derived Halves

BioMed Research International ◽

10.1155/2014/305239 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11 ◽

Cited By ~ 52

Author(s):

Maria R. Garcia-Silva ◽

Florencia Cabrera-Cabrera ◽

Roberta Ferreira Cura das Neves ◽

Thaís Souto-Padrón ◽

Wanderley de Souza ◽

...

Keyword(s):

Gene Expression ◽

Host Cell ◽

Extracellular Vesicles ◽

Small Rna ◽

Hela Cells ◽

Small Rnas ◽

Host Cells ◽

Mammalian Host ◽

Large Set ◽

Rna Pathways

At present, noncoding small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, canonical small RNA pathways seem to be lost or excessively simplified in some unicellular organisms includingTrypanosoma cruziwhich lack functional RNAi pathways. Recently, we reported the presence of alternate small RNA pathways inT. cruzimainly represented by homogeneous populations of tRNA- and rRNA-derived small RNAs, which are secreted to the extracellular medium included in extracellular vesicles. Extracellular vesicle cargo could be delivered to other parasites and to mammalian susceptible cells promoting metacyclogenesis and conferring susceptibility to infection, respectively. Here we analyzed the changes in gene expression of host HeLa cells induced by extracellular vesicles fromT. cruzi. As assessed by microarray assays a large set of genes in HeLa cells were differentially expressed upon incorporation ofT. cruzi-derived extracellular vesicles. The elicited response modified mainly host cell cytoskeleton, extracellular matrix, and immune responses pathways. Some genes were also modified by the most abundant tRNA-derived small RNAs included in extracellular vesicles. These data suggest that microvesicles secreted byT. cruzicould be relevant players in early events of theT. cruzihost cell interplay.

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

10.1101/271270 ◽

2018 ◽

Cited By ~ 1

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.

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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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The RNA phosphatase PIR-1 regulates endogenous small RNA pathways in C. elegans

10.1101/2020.08.03.235143 ◽

2020 ◽

Cited By ~ 1

Author(s):

Daniel A Chaves ◽

Hui Dai ◽

Lichao Li ◽

James J Moresco ◽

Myung Eun Oh ◽

...

Keyword(s):

Small Rna ◽

Small Rnas ◽

Cellular Functions ◽

Germline Development ◽

C Elegans ◽

Rna Sensors ◽

Rna Pathways ◽

Rna Capping ◽

Capping Enzymes ◽

Insight Into

SUMMARYEukaryotic cells regulate 5’ triphosphorylated (ppp-) RNAs to promote cellular functions and prevent recognition by antiviral RNA sensors. For example, RNA capping enzymes possess triphosphatase domains that remove the γ phosphates of ppp-RNAs during RNA capping. Members of the closely related PIR1 family of RNA polyphosphatases remove both the β and γ phosphates from ppp-RNAs. Here we show that C. elegans PIR-1 dephosphorylates ppp-RNAs made by cellular RdRPs and is required for the maturation of 26G-RNAs, Dicer-dependent small RNAs that regulate thousands of genes during spermatogenesis and embryogenesis. PIR-1 also regulates the CSR-1 22G-RNA pathway and has critical functions in both somatic and germline development. Our findings suggest that PIR-1 modulates both Dicer-dependent and - independent Argonaute pathways, and provide insight into how cells and viruses use a conserved RNA phosphatase to regulate and respond to ppp-RNA species.

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Fidelity in RNA-based recognition of transposable elements

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2018.0168 ◽

2018 ◽

Vol 373 (1762) ◽

pp. 20180168 ◽

Cited By ~ 4

Author(s):

Ilaria Ugolini ◽

Mario Halic

Keyword(s):

Transposable Elements ◽

Small Rna ◽

Small Rnas ◽

Rna Degradation ◽

Current Data ◽

Theme Issue ◽

Rna Pathways ◽

Transposon Insertions

Genomes are under constant threat of invasion by transposable elements and other genomic parasites. How can host genomes recognize these elements and target them for degradation? This requires a system that is highly adaptable, and at the same time highly specific. Current data suggest that perturbation of transcription patterns by transposon insertions could be detected by the RNAi surveillance pathway. Multiple transposon insertions might generate sufficient amounts of primal small RNAs to initiate generation of secondary small RNAs and silencing. At the same time primal small RNAs need to be constantly degraded to reduce the level of noise small RNAs below the threshold required for initiation of silencing. Failure in RNA degradation results in loss of fidelity of small RNA pathways and silencing of ectopic targets. This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.

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The Aquarius/EMB-4 helicase licenses co-transcriptional gene silencing

10.1101/089763 ◽

2016 ◽

Cited By ~ 1

Author(s):

Alper Akay ◽

Tomas Di Domenico ◽

Kin M. Suen ◽

Amena Nabih ◽

Guillermo E. Parada ◽

...

Keyword(s):

Transposable Elements ◽

Gene Silencing ◽

Small Rna ◽

Small Rnas ◽

Transcriptional Gene Silencing ◽

Helicase Activity ◽

Rna Transcripts ◽

Nascent Rna ◽

Rna Pathways ◽

Overlapping Sets

SUMMARYSmall RNAs (sRNAs) play an ancient role in genome defence against transposable elements. In animals, plants and fungi small RNAs guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. In animals the link between small RNA pathways and the transcriptional machinery remains unclear. Here we show that the Caenorhabditis elegans germline Argonaute HRDE-1 physically interacts with the conserved RNA helicase Aquarius/EMB-4. We demonstrate that the Aquarius/EMB-4 helicase activity is required to initiate small RNA-induced co-transcriptional gene silencing. HRDE-1 and Aquarius/EMB-4 are required to silence the transcription of overlapping sets of transposable elements. Surprisingly, removal of introns from a small RNA pathway target abolishes the requirement for Aquarius/EMB-4, but not HRDE-1, for gene silencing. We conclude that the Aquarius/EMB-4 helicase activity allows HRDE-1/sRNA complexes to efficiently engage nascent RNA transcripts - in competition with the general RNA processing machinery. We postulate that Aquarius/EMB-4 facilitates the surveillance of the nascent transcriptome to detect and silence transposable elements through small RNA pathways.

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The Influence of Competition Among C. elegans Small RNA Pathways on Development

Genes ◽

10.3390/genes3040671 ◽

2012 ◽

Vol 3 (4) ◽

pp. 671-685 ◽

Cited By ~ 15

Author(s):

Jimmy J. Zhuang ◽

Craig P. Hunter

Keyword(s):

Rna Interference ◽

Caenorhabditis Elegans ◽

Small Rna ◽

Small Rnas ◽

Wild Type ◽

C Elegans ◽

Exogenous Rna ◽

Rna Pathways ◽

Sirna Pathway ◽

The Relationship

Small RNAs play a variety of regulatory roles, including highly conserved developmental functions. Caenorhabditis elegans not only possesses most known small RNA pathways, it is also an easy system to study their roles and interactions during development. It has been proposed that in C. elegans, some small RNA pathways compete for access to common limiting resources. The strongest evidence supporting this model is that disrupting the production or stability of endogenous short interfering RNAs (endo-siRNAs) enhances sensitivity to experimentally induced exogenous RNA interference (exo-RNAi). Here, we examine the relationship between the endo-siRNA and microRNA (miRNA) pathways, and find that, consistent with competition among these endogenous small RNA pathways, endo-siRNA pathway mutants may enhance miRNA efficacy. Furthermore, we show that exo-RNAi may also compete with both endo-siRNAs and miRNAs. Our data thus provide support that all known Dicer-dependent small RNA pathways may compete for limiting common resources. Finally, we observed that both endo-siRNA mutants and animals experiencing exo-RNAi have increased expression of miRNA-regulated stage-specific developmental genes. These observations suggest that perturbing the small RNA flux and/or the induction of exo-RNAi, even in wild-type animals, may impact development via effects on the endo-RNAi and microRNA pathways.

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Modeling small RNA competition in C. elegans

10.1101/021576 ◽

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.

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Nematode Small RNA Pathways in the Absence of piRNAs

10.1101/2021.07.23.453445 ◽

2021 ◽

Author(s):

Maxim Zagoskin ◽

Jianbin Wang ◽

Ashley T. Neff ◽

Giovana M.B. Veronezi ◽

Richard E. Davis

Keyword(s):

Small Rna ◽

Small Rnas ◽

Parasitic Nematode ◽

Repetitive Sequences ◽

C Elegans ◽

Early Embryos ◽

Specific Mrnas ◽

Rna Pathways ◽

Pirna Pathway ◽

Mature Mrnas

Small RNA pathways play diverse regulatory roles in the nematode C. elegans. However, our understanding of small RNA pathways, their conservation, and their roles in other nematodes is limited. Here, we analyzed small RNA pathways in the parasitic nematode Ascaris. Ascaris has ten Argonautes with five worm-specific Argonautes (WAGOs) that are associated with secondary 5'-triphosphate small RNAs (22-24G-RNAs). These Ascaris WAGOs and their small RNAs target repetitive sequences (WAGO-1, WAGO-2, WAGO-3, and NRDE-3) or mature mRNAs (CSR-1, NRDE-3, and WAGO-3) and are similar to the C. elegans mutator, nuclear, and CSR-1 small RNA pathways. Ascaris CSR-1 likely functions to "license" gene expression in the absence of an Ascaris piRNA pathway. Ascaris ALG-4 and its associated 26G-RNAs target and appear to repress specific mRNAs during meiosis in the testes. Notably, Ascaris WAGOs (WAGO-3 and NRDE-3) small RNAs change their targets between repetitive sequences and mRNAs during spermatogenesis or in early embryos illustrating target plasticity of these WAGOs. We provide a unique and comprehensive view of mRNA and small RNA expression throughout nematode spermatogenesis that illustrates the dynamics and flexibility of small RNA pathways. Overall, our study provides key insights into the conservation and divergence of nematode small RNA pathways.

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