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 The IDR-containing protein PID-2 affects Z granules and is required for piRNA-induced silencing in the embryo

2020 ◽  
Author(s):  
Maria Placentino ◽  
António Miguel de Jesus Domingues ◽  
Jan Schreier ◽  
Sabrina Dietz ◽  
Svenja Hellmann ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Small Rna ◽  
Rna Silencing ◽  
Rna Dependent Rna Polymerase ◽  
Prolyl Aminopeptidase ◽  
C Elegans

AbstractIn Caenorhabditis elegans, the piRNA (21U RNA) pathway is required to establish proper gene regulation and an immortal germline. To achieve this, PRG-1-bound 21U RNAs trigger silencing mechanisms mediated by RNA-dependent RNA polymerase (RdRP)-synthetized 22G RNAs. This silencing can become PRG-1-independent, and heritable over many generations. This state is named RNAe. It is unknown how and when RNAe is established, and how it is maintained. We show that maternally provided 21U RNAs can be sufficient to trigger RNAe in embryos. Additionally, we identify the IDR-containing protein PID-2, as a factor required to establish and maintain RNAe. PID-2 interacts with two novel, partially redundant, eTudor domain proteins, PID-4 and PID-5. Additionally, PID-5 has a domain related to the X-prolyl aminopeptidase protein APP-1, and binds APP-1, implicating N-terminal proteolysis in RNAe. All three proteins are required for germline immortality, localize to perinuclear foci, affect Z granules, and are required for balancing of 22G RNA populations. Overall, our study identifies three new proteins with crucial functions in the C. elegans small RNA silencing network.

scholarly journals Maternal and zygotic gene regulatory effects of endogenous RNAi pathways

2018 ◽  
Author(s):  
Miguel Vasconcelos Almeida ◽  
António Miguel de Jesus Domingues ◽  
René F. Ketting
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Gene Silencing ◽  
Small Rnas ◽  
Self Regulation ◽  
Fine Tuning ◽  
Specific Gene ◽  
Next Generation ◽  
C Elegans ◽  
Argonaute Proteins

AbstractEndogenous small RNAs (sRNAs) and Argonaute proteins are ubiquitous regulators of gene expression in germline and somatic tissues. sRNA-Argonaute complexes are often expressed in gametes and are consequently inherited by the next generation upon fertilization. In Caenorhabditis elegans, 26G-RNAs are primary endogenous sRNAs that trigger the expression of downstream secondary sRNAs. Two subpopulations of 26G-RNAs exist, each of which displaying strongly compartmentalized expression: one is expressed in the spermatogenic gonad and associates with the Argonautes ALG-3/4; plus another expressed in oocytes and in embryos, which associates with the Argonaute ERGO-1. The determinants and dynamics of gene silencing elicited by 26G-RNAs are largely unknown. Here, we provide diverse new insights into these endogenous sRNA pathways of C. elegans. Using genetics and deep sequencing, we dissect a maternal effect of the ERGO-1 branch sRNA pathway. We find that maternal primary sRNAs can trigger the production of zygotic secondary sRNAs that are able to silence targets, even in the absence of zygotic primary triggers. Thus, the interaction of maternal and zygotic sRNA populations, assures target gene silencing throughout animal development. Furthermore, we find that sRNA abundance, the pattern of origin of sRNA and 3’ UTR length are predictors of the regulatory outcome by the Argonautes ALG-3/4. Lastly, we discovered that ALG-3- and ALG-4-bound 26G-RNAs are dampening the expression of their own mRNAs, revealing a negative feedback loop. Altogether, we provide several new regulatory insights on the dynamics, target regulation and self-regulation of the endogenous RNAi pathways of C. elegans.Author SummarySmall RNAs (sRNAs) and their partner Argonaute proteins regulate the expression of target RNAs. When sperm and egg meet upon fertilization, a diverse set of proteins and RNA, including sRNA-Argonaute complexes, is passed on to the developing progeny. Thus, these two players are important to initiate specific gene expression programs in the next generation. The nematode Caenorhabditis elegans expresses several classes of sRNAs. 26G-RNAs are a particular class of sRNAs that are divided into two subpopulations: one expressed in the spermatogenic gonad and another expressed in oocytes and in embryos. In this work, we describe the dynamics whereby oogenic 26G-RNAs setup gene silencing in the next generation. We also show several ways that spermatogenic 26G-RNAs and their partner Argonautes, ALG-3 and ALG-4, use to regulate their targets. Finally, we show that ALG-3 and ALG-4 are fine-tuning their own expression, a rare role of Argonaute proteins. Overall, we provide new insights into how sRNAs and Argonautes are regulating gene expression.

scholarly journals Protease-mediated processing of Argonaute proteins controls small RNA association

2020 ◽  
Author(s):  
Rajani Kanth Gudipati ◽  
Kathrin Braun ◽  
Foivos Gypas ◽  
Daniel Hess ◽  
Jan Schreier ◽  
...  
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Proteolytic Processing ◽  
Genome Integrity ◽  
Wild Type ◽  
Selfish Genetic Elements ◽  
Argonaute Proteins ◽  
Processing Activity ◽  
Rna Pathways

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.

scholarly journals Translation and codon usage regulate Argonaute slicer activity to trigger small RNA biogenesis

2020 ◽  
Author(s):  
Meetali Singh ◽  
Eric Cornes ◽  
Blaise Li ◽  
Piergiuseppe Quarato ◽  
Loan Bourdon ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Embryonic Development ◽  
Rna Polymerase ◽  
Codon Usage ◽  
Small Rna ◽  
Small Rnas ◽  
Rna Dependent Rna Polymerase ◽  
Coding Sequences ◽  
Germ Granules ◽  
Rna Biogenesis

In the Caenorhabditis elegans germline, thousands of mRNAs are concomitantly expressed with antisense 22G-RNAs, which are loaded into the Argonaute CSR-1. Despite their essential functions for animal fertility and embryonic development, how CSR-1 22G-RNAs are produced remains unknown. Here, we show that CSR-1 slicer activity is primarily involved in triggering the synthesis of small RNAs on the coding sequences of germline mRNAs and post-transcriptionally regulates a fraction of targets. CSR-1-cleaved mRNAs prime the RNA-dependent RNA polymerase, EGO-1, to synthesize 22G-RNAs in phase with ribosome translation in the cytoplasm, in contrast to other 22G-RNAs mostly synthesized in germ granules. Moreover, codon optimality and efficient translation antagonize CSR-1 slicing and 22G-RNAs biogenesis. We propose that codon usage differences encoded into mRNA sequences might be a conserved strategy in eukaryotes to regulate small RNA biogenesis and Argonaute targeting.

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.

Assembly and function of small RNA – Argonaute protein complexes

2014 ◽  
Vol 395 (6) ◽  
pp. 611-629 ◽  
Author(s):  
Anne Dueck ◽  
Gunter Meister
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
Small Rna ◽  
Small Rnas ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Protein Family ◽  
Argonaute Protein ◽  
Argonaute Proteins ◽  
And Function

Abstract Small RNAs such as microRNAs (miRNAs), short interfering RNAs (siRNAs) or Piwi-interacting RNAs (piRNAs) are important regulators of gene expression in various organisms. Small RNAs bind to a member of the Argonaute protein family and are incorporated into larger structures that mediate diverse gene silencing events. The loading of Argonaute proteins with small RNAs is aided by a number of auxiliary factors as well as ATP hydrolysis. This review will focus on the mechanisms of Argonaute loading in different organisms. Furthermore, we highlight the versatile functions of small RNA-Argonaute protein complexes in organisms from all three kingdoms of life.

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

2020 ◽  
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.

scholarly journals Translation and codon usage regulate Argonaute slicer activity to trigger small RNA biogenesis

2020 ◽  
Author(s):  
Germano Cecere ◽  
Meetali Singh ◽  
Eric Cornes ◽  
Blaise Li ◽  
Piergiuseppe Quarato ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Embryonic Development ◽  
Rna Polymerase ◽  
Codon Usage ◽  
Small Rna ◽  
Small Rnas ◽  
Rna Dependent Rna Polymerase ◽  
Coding Sequences ◽  
Germ Granules ◽  
Rna Biogenesis

Abstract In the Caenorhabditis elegans germline, thousands of mRNAs are concomitantly expressed with antisense 22G-RNAs, which are loaded into the Argonaute CSR-1. Despite their essential functions for animal fertility and embryonic development, how CSR-1 22G-RNAs are produced remains unknown. Here, we show that CSR-1 slicer activity is primarily involved in triggering the synthesis of small RNAs on the coding sequences of germline mRNAs and post-transcriptionally regulates a fraction of targets. CSR-1-cleaved mRNAs prime the RNA-dependent RNA polymerase, EGO-1, to synthesize 22G-RNAs in phase with ribosome translation in the cytoplasm, in contrast to other 22G-RNAs mostly synthesized in germ granules. Moreover, codon optimality and efficient translation antagonize CSR-1 slicing and 22G-RNAs biogenesis. We propose that codon usage differences encoded into mRNA sequences might be a conserved strategy in eukaryotes to regulate small RNA biogenesis and Argonaute targeting.

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.

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