scholarly journals Arginine methylation promotes siRNA-binding specificity for a spermatogenesis-specific isoform of the Argonaute protein CSR-1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dieu An H. Nguyen ◽  
Carolyn M. Phillips
Keyword(s):  
Small Rnas ◽  
Rna Binding ◽  
Regulatory Mechanism ◽  
Expression Patterns ◽  
Arginine Methylation ◽  
Argonaute Protein ◽  
C Elegans ◽  
Argonaute Proteins ◽  
Preferential Binding ◽  
Temporal And Spatial

AbstractCSR-1 is an essential Argonaute protein that binds to a subclass of 22G-RNAs targeting most germline-expressed genes. Here we show that the two isoforms of CSR-1 have distinct expression patterns; CSR-1B is ubiquitously expressed throughout the germline and during all stages of development while CSR-1A expression is restricted to germ cells undergoing spermatogenesis. Furthermore, CSR-1A associates preferentially with 22G-RNAs mapping to spermatogenesis-specific genes whereas CSR-1B-bound small RNAs map predominantly to oogenesis-specific genes. Interestingly, the exon unique to CSR-1A contains multiple dimethylarginine modifications, which are necessary for the preferential binding of CSR-1A to spermatogenesis-specific 22G-RNAs. Thus, we have discovered a regulatory mechanism for C. elegans Argonaute proteins that allows for specificity of small RNA binding between similar Argonaute proteins with overlapping temporal and spatial localization.

scholarly journals Arginine methylation promotes siRNA-binding specificity for a spermatogenesis-specific isoform of the Argonaute protein CSR-1

2020 ◽  
Author(s):  
Dieu An H. Nguyen ◽  
Carolyn M. Phillips
Keyword(s):  
Small Rnas ◽  
Rna Binding ◽  
Regulatory Mechanism ◽  
Expression Patterns ◽  
Arginine Methylation ◽  
Argonaute Protein ◽  
C Elegans ◽  
Argonaute Proteins ◽  
Preferential Binding ◽  
Temporal And Spatial

SummaryCSR-1 is an essential Argonaute protein that binds to a subclass of 22G-RNAs targeting most germline-expressed genes. Here we show that the two isoforms of CSR-1 have distinct expression patterns; CSR-1B is ubiquitously expressed throughout the germline and during all stages of development while CSR-1A expression is restricted to germ cells undergoing spermatogenesis. Furthermore, CSR-1A associates preferentially with 22G-RNAs mapping to spermatogenesis-specific genes whereas CSR-1B-bound small RNAs map predominantly to oogenesis-specific genes. Interestingly, the exon unique to CSR-1A contains multiple dimethylarginine modifications, which are necessary for the preferential binding of CSR-1A to spermatogenesis-specific 22G-RNAs. Thus, we have discovered a regulatory mechanism for C. elegans Argonaute proteins that allows for specificity of small RNA binding between similar Argonaute proteins with overlapping temporal and spatial localization.

scholarly journals Two isoforms of the essential C. elegans Argonaute CSR-1 differentially regulate sperm and oocyte fertility through distinct small RNA classes

2020 ◽  
Author(s):  
Amanda G. Charlesworth ◽  
Nicolas J. Lehrbach ◽  
Uri Seroussi ◽  
Mathias S. Renaud ◽  
Ruxandra I. Molnar ◽  
...  
Keyword(s):  
Small Rna ◽  
Regulatory Network ◽  
Small Rnas ◽  
Male Fertility ◽  
Expression Patterns ◽  
C Elegans ◽  
Argonaute Proteins ◽  
Germline Genes ◽  
Somatic Tissues ◽  
Normal Laboratory

SUMMARYThe C. elegans genome encodes nineteen functional Argonaute proteins that utilize 22G-RNAs, 26G-RNAs, miRNAs, or piRNAs to regulate their target transcripts. Only one of these proteins is essential under normal laboratory conditions: CSR-1. While CSR-1 has been studied in various developmental and functional contexts, nearly all studies investigating CSR-1 have overlooked the fact that the csr-1 locus encodes two isoforms. These isoforms differ by an additional 163 amino acids present in the N-terminus of CSR-1a. Using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG epitopes into the long (CSR-1a) and short (CSR-1b) isoforms of CSR-1, we identified differential expression patterns for the two isoforms. CSR-1a is expressed specifically during spermatogenesis and in select somatic tissues, including the intestine. In contrast, CSR-1b, is expressed constitutively in the germline. Essential functions of csr-1 described in the literature coincide with CSR-1b. In contrast, CSR-1a plays tissue specific functions during spermatogenesis, where it integrates into a spermatogenesis sRNA regulatory network including ALG-3, ALG-4, and WAGO-10 that is necessary for male fertility. CSR-1a is also required in the intestine for the silencing of repetitive transgenes. Sequencing of small RNAs associated with each CSR-1 isoform reveals that CSR-1a engages with 22G- and 26G-RNAs, while CSR-1b interacts with only 22G-RNAs to regulate distinct groups of germline genes and regulate both sperm and oocyte-mediated fertility.

scholarly journals Temporal and spatial expression patterns of the small heat shock (hsp16) genes in transgenic Caenorhabditis elegans.

1992 ◽  
Vol 3 (2) ◽  
pp. 221-233 ◽  
Author(s):  
E G Stringham ◽  
D K Dixon ◽  
D Jones ◽  
E P Candido
Keyword(s):  
Caenorhabditis Elegans ◽  
Heat Shock ◽  
Gene Pair ◽  
Expression Patterns ◽  
Developmentally Regulated ◽  
Noncoding Sequences ◽  
C Elegans ◽  
Gene Pairs ◽  
Temporal And Spatial ◽  
Beta Galactosidase

The expression of the hsp16 gene family in Caenorhabditis elegans has been examined by introducing hsp16-lacZ fusions into the nematode by transformation. Transcription of the hsp16-lacZ transgenes was totally heat-shock dependent and resulted in the rapid synthesis of detectable levels of beta-galactosidase. Although the two hsp16 gene pairs of C. elegans are highly similar within both their coding and noncoding sequences, quantitative and qualitative differences in the spatial pattern of expression between gene pairs were observed. The hsp16-48 promoter was shown to direct greater expression of beta-galactosidase in muscle and hypodermis, whereas the hsp16-41 promoter was more efficient in intestine and pharyngeal tissue. Transgenes that eliminated one promoter from a gene pair were expressed at reduced levels, particularly in postembryonic stages, suggesting that the heat shock elements in the intergenic region of an hsp16 gene pair may act cooperatively to achieve high levels of expression of both genes. Although the hsp16 gene pairs are never constitutively expressed, their heat inducibility is developmentally restricted; they are not heat inducible during gametogenesis or early embryogenesis. The hsp16 genes represent the first fully inducible system in C. elegans to be characterized in detail at the molecular level, and the promoters of these genes should find wide applicability in studies of tissue- and developmentally regulated genes in this experimental organism.

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 Chromatin Compaction by Small RNAs and the Nuclear RNAi Machinery in C. elegans

2018 ◽  
Author(s):  
Brandon D. Fields ◽  
Scott Kennedy
Keyword(s):  
Small Rnas ◽  
Rna Binding ◽  
Higher Order ◽  
Post Translational Modification ◽  
Rnai Machinery ◽  
Regulate Gene Expression ◽  
Chromatin Compaction ◽  
C Elegans ◽  
Regulation Mechanisms ◽  
Nuclear Rnai

AbstractDNA is organized and compacted into higher-order structures in order to fit within nuclei and to facilitate proper gene regulation. Mechanisms by which higher order chromatin structures are established and maintained are poorly understood. In C. elegans, nuclear-localized small RNAs engage the nuclear RNAi machinery to regulate gene expression and direct the post-translational modification of histone proteins. Here we confirm a recent report suggesting that nuclear small RNAs are required to initiate or maintain chromatin compaction states in C. elegans germ cells. Additionally, we show that experimentally provided small RNAs are sufficient to direct chromatin compaction and that this compaction requires the small RNA-binding Argonaute NRDE-3, the pre-mRNA associated factor NRDE-2, and the HP1-like protein HPL-2. Our results show that small RNAs, acting via the nuclear RNAi machinery and an HP1-like protein, are capable of driving chromatin compaction in C. elegans.

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 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 Engineering a conserved RNA regulatory protein repurposes its biological function in vivo

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Vandita D Bhat ◽  
Kathleen L McCann ◽  
Yeming Wang ◽  
Dallas R Fonseca ◽  
Tarjani Shukla ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Rna Binding ◽  
Biological Function ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Regulatory Protein ◽  
Motif Length ◽  
C Elegans ◽  
Preferential Binding

PUF (PUmilio/FBF) RNA-binding proteins recognize distinct elements. In C. elegans, PUF-8 binds to an 8-nt motif and restricts proliferation in the germline. Conversely, FBF-2 recognizes a 9-nt element and promotes mitosis. To understand how motif divergence relates to biological function, we first determined a crystal structure of PUF-8. Comparison of this structure to that of FBF-2 revealed a major difference in a central repeat. We devised a modified yeast 3-hybrid screen to identify mutations that confer recognition of an 8-nt element to FBF-2. We identified several such mutants and validated structurally and biochemically their binding to 8-nt RNA elements. Using genome engineering, we generated a mutant animal with a substitution in FBF-2 that confers preferential binding to the PUF-8 element. The mutant largely rescued overproliferation in animals that spontaneously generate tumors in the absence of puf-8. This work highlights the critical role of motif length in the specification of biological function.

Secondary siRNAs Result from Unprimed RNA Synthesis and Form a Distinct Class

Science ◽  
2006 ◽  
Vol 315 (5809) ◽  
pp. 244-247 ◽  
Author(s):  
Titia Sijen ◽  
Florian A. Steiner ◽  
Karen L. Thijssen ◽  
Ronald H. A. Plasterk
Keyword(s):  
Rna Interference ◽  
Small Rnas ◽  
Rna Synthesis ◽  
Argonaute Protein ◽  
Single Nucleotide ◽  
Distinct Class ◽  
C Elegans ◽  
Secondary Sirnas ◽  
Secondary Sirna ◽  
Rnai Response

In Caenorhabditis elegans, an effective RNA interference (RNAi) response requires the production of secondary short interfering RNAs (siRNAs) by RNA-directed RNA polymerases (RdRPs). We cloned secondary siRNAs from transgenic C. elegans lines expressing a single 22-nucleotide primary siRNA. Several secondary siRNAs start a few nucleotides downstream of the primary siRNA, indicating that non–RISC (RNA-induced silencing complex)–cleaved mRNAs are substrates for secondary siRNA production. In lines expressing primary siRNAs with single-nucleotide mismatches, secondary siRNAs do not carry the mismatch but contain the nucleotide complementary to the mRNA. We infer that RdRPs perform unprimed RNA synthesis. Secondary siRNAs are only of antisense polarity, carry 5′ di- or triphosphates, and are only in the minority associated with RDE-1, the RNAi-specific Argonaute protein. Therefore, secondary siRNAs represent a distinct class of small RNAs. Their biogenesis depends on RdRPs, and we propose that each secondary siRNA is an individual RdRP product.

scholarly journals A branched heterochronic pathway directs juvenile-to-adult transition through two LIN-29 isoforms

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Chiara Azzi ◽  
Florian Aeschimann ◽  
Anca Neagu ◽  
Helge Großhans
Keyword(s):  
Small Rnas ◽  
Rna Binding ◽  
Expression Patterns ◽  
Functional Specialization ◽  
Adult Transition ◽  
Physiological Processes ◽  
Regulatory Cascade ◽  
Isoform Expression ◽  
The Individual ◽  
Organismal Development

Robust organismal development relies on temporal coordination of disparate physiological processes. In Caenorhabditis elegans, the heterochronic pathway controls a timely juvenile-to-adult (J/A) transition. This regulatory cascade of conserved proteins and small RNAs culminates in accumulation of the transcription factor LIN-29, which triggers coordinated execution of transition events. We report that two LIN-29 isoforms fulfill distinct functions. Functional specialization is a consequence of distinct isoform expression patterns, not protein sequence, and we propose that distinct LIN-29 dose sensitivities of the individual J/A transition events help to ensure their temporal ordering. We demonstrate that unique isoform expression patterns are generated by the activities of LIN-41 for lin-29a, and of HBL-1 for lin-29b, whereas the RNA-binding protein LIN-28 coordinates LIN-29 isoform activity, in part by regulating both hbl-1 and lin-41. Our findings reveal that coordinated transition from juvenile to adult involves branching of a linear pathway to achieve timely control of multiple events.

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