scholarly journals Cooperative recruitment of RDR6 by SGS3 and SDE5 during small interfering RNA amplification in Arabidopsis

2021 ◽  
Vol 118 (34) ◽  
pp. e2102885118
Author(s):  
Manabu Yoshikawa ◽  
Yong-Woon Han ◽  
Hirofumi Fujii ◽  
Shu Aizawa ◽  
Tatsuya Nishino ◽  
...  
Keyword(s):  
Recognition System ◽  
Small Interfering Rnas ◽  
In Vitro System ◽  
Double Stranded Rna ◽  
Cleavage Fragment ◽  
Secondary Sirnas ◽  
Secondary Sirna ◽  
Interfering Rna ◽  
Target Rna

Small interfering RNAs (siRNAs) are often amplified from transcripts cleaved by RNA-induced silencing complexes (RISCs) containing a small RNA (sRNA) and an Argonaute protein. Amplified siRNAs, termed secondary siRNAs, are important for reinforcement of target repression. In plants, target cleavage by RISCs containing 22-nucleotide (nt) sRNA and Argonaute 1 (AGO1) triggers siRNA amplification. In this pathway, the cleavage fragment is converted into double-stranded RNA (dsRNA) by RNA-dependent RNA polymerase 6 (RDR6), and the dsRNA is processed into siRNAs by Dicer-like proteins. Because nonspecific RDR6 recruitment causes nontarget siRNA production, it is critical that RDR6 is specifically recruited to the target RNA that serves as a template for dsRNA formation. Previous studies showed that Suppressor of Gene Silencing 3 (SGS3) binds and stabilizes 22-nt sRNA–containing AGO1 RISCs associated with cleaved target, but how RDR6 is recruited to targets cleaved by 22-nt sRNA–containing AGO1 RISCs remains unknown. Here, using cell-free extracts prepared from suspension-cultured Arabidopsis thaliana cells, we established an in vitro system for secondary siRNA production in which 22-nt siRNA–containing AGO1-RISCs but not 21-nt siRNA–containing AGO1-RISCs induce secondary siRNA production. In this system, addition of recombinant Silencing Defective 5 (SDE5) protein remarkably enhances secondary siRNA production. We show that RDR6 is recruited to a cleavage fragment by 22-nt siRNA–containing AGO1-RISCs in coordination with SGS3 and SDE5. The SGS3–SDE5–RDR6 multicomponent recognition system and the poly(A) tail inhibition may contribute to securing specificity of siRNA amplification.

scholarly journals Trafficking of siRNA precursors by the dsRBD protein Blanks in Drosophila

2020 ◽  
Vol 48 (7) ◽  
pp. 3906-3921 ◽  
Author(s):  
Volker Nitschko ◽  
Stefan Kunzelmann ◽  
Thomas Fröhlich ◽  
Georg J Arnold ◽  
Klaus Förstemann
Keyword(s):  
Small Rnas ◽  
Rna Binding ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
Convergent Transcription ◽  
Dsrna Binding ◽  
Functional Screens

Abstract RNA interference targets aberrant transcripts with cognate small interfering RNAs, which derive from double-stranded RNA precursors. Several functional screens have identified Drosophila blanks/lump (CG10630) as a facilitator of RNAi, yet its molecular function has remained unknown. The protein carries two dsRNA binding domains (dsRBD) and blanks mutant males have a spermatogenesis defect. We demonstrate that blanks selectively boosts RNAi triggered by dsRNA of nuclear origin. Blanks binds dsRNA via its second dsRBD in vitro, shuttles between nucleus and cytoplasm and the abundance of siRNAs arising at many sites of convergent transcription is reduced in blanks mutants. Since features of nascent RNAs - such as introns and transcription beyond the polyA site – contribute to the small RNA pool, we propose that Blanks binds dsRNA formed by cognate nascent RNAs in the nucleus and fosters its export to the cytoplasm for dicing. We refer to the resulting small RNAs as blanks exported siRNAs (bepsiRNAs). While bepsiRNAs were fully dependent on RNA binding to the second dsRBD of blanks in transgenic flies, male fertility was not. This is consistent with a previous report that linked fertility to the first dsRBD of Blanks. The role of blanks in spermatogenesis appears thus unrelated to its role in dsRNA export.

scholarly journals Functional specialization of monocot DCL3 and DCL5 proteins through the evolution of the PAZ domain

2021 ◽  
Author(s):  
Shirui Chen ◽  
Wei Liu ◽  
Masahiro Naganuma ◽  
Yukihide Tomari ◽  
Hiro-oki Iwakawa
Keyword(s):  
Small Rnas ◽  
Rna Silencing ◽  
Functional Differentiation ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Secondary Sirnas ◽  
Fixed End ◽  
Paz Domain ◽  
Reproductive Processes

Monocot DICER-LIKE3 (DCL3) and DCL5 produce distinct 24-nt heterochromatic small interfering RNAs (hc-siRNAs) and phased secondary siRNAs (phasiRNAs). The former small RNAs are linked to plant heterochromatin, and the latter to reproductive processes. It is assumed that these DCLs evolved from an ancient "eudicot-type" DCL3 ancestor, which may have produced both types of siRNAs. However, how functional differentiation was achieved after gene duplication remains elusive. Here, we find that monocot DCL3 and DCL5 exhibit biochemically distinct preferences for 3′ overhangs and 5′ phosphates, consistent with the structural properties of their in vivo double-stranded RNA substrates. Importantly, these distinct substrate specificities are determined by the PAZ domains of DCL3 and DCL5 which have accumulated mutations during the course of evolution. These data explain the mechanism by which these DCLs cleave their cognate substrates from a fixed end, ensuring the production of functional siRNAs. Our study also indicates how plants have diversified and optimized RNA silencing mechanisms during evolution.

scholarly journals Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus

2007 ◽  
Vol 81 (11) ◽  
pp. 5449-5459 ◽  
Author(s):  
Beau J. Fenner ◽  
Winnie Goh ◽  
Jimmy Kwang
Keyword(s):  
Rna Silencing ◽  
Rna Binding ◽  
Amino Acid Residues ◽  
Alanine Scanning Mutagenesis ◽  
Marine Aquaculture ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Fish Cells ◽  
Interfering Rna

ABSTRACT Betanodaviruses are small RNA viruses that infect teleost fish and pose a considerable threat to marine aquaculture production. These viruses possess a small protein, termed B2, which binds to and protects double-stranded RNA. This prevents cleavage of virus-derived double-stranded RNAs (dsRNAs) by Dicer and subsequent production of small interfering RNA (siRNA), which would otherwise induce an RNA-silencing response against the virus. In this work, we have performed charged-to-alanine scanning mutagenesis of the B2 protein in order to identify residues required for dsRNA binding and protection. While the majority of the 19 mutated B2 residues were required for maximal dsRNA binding and protection in vitro, residues R53 and R60 were essential for both activities. Subsequent experiments in fish cells confirmed these findings by showing that mutations in these residues abolished accumulation of both the RNA1 and RNA2 components of the viral genome, in addition to preventing any significant induction of the host interferon gene, Mx. Moreover, an obvious positive correlation was found between dsRNA binding and protection in vitro and RNA1, RNA2, and Mx accumulation in fish cells, further validating the importance of the selected amino acid residues. The same trend was also demonstrated using an RNA silencing system in HeLa cells, with residues R53 and R60 being essential for suppression of RNA silencing. Importantly, we found that siRNA-mediated knockdown of Dicer dramatically enhanced the accumulation of a B2 mutant. In addition, we found that B2 is able to induce apoptosis in fish cells but that this was not the result of dsRNA binding.

scholarly journals Cytotoxicity assessment, inflammatory properties, and cellular uptake of Neutraplex lipid-based nanoparticles in THP-1 monocyte-derived macrophages

2017 ◽  
Vol 4 ◽  
pp. 184954351774625 ◽  
Author(s):  
Eric Berger ◽  
Dalibor Breznan ◽  
Sandra Stals ◽  
Viraj J Jasinghe ◽  
David Gonçalves ◽  
...  
Keyword(s):  
Targeted Delivery ◽  
Small Interfering Rna ◽  
Human Monocyte ◽  
Antiretroviral Drugs ◽  
Small Interfering Rnas ◽  
Lactate Dehydrogenase Release ◽  
Drug Concentrations ◽  
Interfering Rna ◽  
Hiv 1

Current antiretroviral drugs used to prevent or treat human immunodeficiency virus type 1 (HIV-1) infection are not able to eliminate the virus within tissues or cells where HIV establishes reservoirs. Hence, there is an urgent need to develop targeted delivery systems to enhance drug concentrations in these viral sanctuary sites. Macrophages are key players in HIV infection and contribute significantly to the cellular reservoirs of HIV because the virus can survive for prolonged periods in these cells. In the present work, we investigated the potential of the lipid-based Neutraplex nanosystem to deliver anti-HIV therapeutics in human macrophages using the human monocyte/macrophage cell line THP-1. Neutraplex nanoparticles as well as cationic and anionic Neutraplex nanolipoplexes (Neutraplex/small interfering RNA) were prepared and characterized by dynamic light scattering. Neutraplex nanoparticles showed low cytotoxicity in CellTiter-Blue reduction and lactate dehydrogenase release assays and were not found to have pro-inflammatory effects. In addition, confocal studies showed that the Neutraplex nanoparticles and nanolipoplexes are rapidly internalized into THP-1 macrophages and that they can escape the late endosome/lysosome compartment allowing the delivery of small interfering RNAs in the cytoplasm. Furthermore, HIV replication was inhibited in the in vitro TZM-bl infectivity assay when small interfering RNAs targeting CXCR4 co-receptor was delivered by Neutraplex nanoparticles compared to a random small interfering RNA sequence. This study demonstrates that the Neutraplex nanosystem has potential for further development as a delivery strategy to efficiently and safely enhance the transport of therapeutic molecules into human monocyte-derived macrophages in the aim of targeting HIV-1 in this cellular reservoir.

scholarly journals Role of the Trypanosoma brucei HEN1 Family Methyltransferase in Small Interfering RNA Modification

2013 ◽  
Vol 13 (1) ◽  
pp. 77-86 ◽  
Author(s):  
Huafang Shi ◽  
Rebecca L. Barnes ◽  
Nicholas Carriero ◽  
Vanessa D. Atayde ◽  
Christian Tschudi ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Small Interfering Rna ◽  
Leishmania Major ◽  
Rna Modification ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Content Type ◽  
Interfering Rna ◽  
Core Genes

ABSTRACT Parasitic protozoa of the flagellate order Kinetoplastida represent one of the deepest branches of the eukaryotic tree. Among this group of organisms, the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser degree in Leishmania ( Viannia ) spp. The pathway is triggered by long double-stranded RNA (dsRNA) and in T. brucei requires a set of five core genes, including a single Argonaute (AGO) protein, T. brucei AGO1 ( Tb AGO1). The five genes are conserved in Leishmania ( Viannia ) spp. but are absent in other major kinetoplastid species, such as Trypanosoma cruzi and Leishmania major . In T. brucei small interfering RNAs (siRNAs) are methylated at the 3′ end, whereas Leishmania ( Viannia ) sp. siRNAs are not. Here we report that T. brucei HEN1, an ortholog of the metazoan HEN1 2′- O -methyltransferases, is required for methylation of siRNAs. Loss of Tb HEN1 causes a reduction in the length of siRNAs. The shorter siRNAs in hen1 −/− parasites are single stranded and associated with Tb AGO1, and a subset carry a nontemplated uridine at the 3′ end. These findings support a model wherein Tb HEN1 methylates siRNA 3′ ends after they are loaded into Tb AGO1 and this methylation protects siRNAs from uridylation and 3′ trimming. Moreover, expression of Tb HEN1 in Leishmania ( Viannia ) panamensis did not result in siRNA 3′ end methylation, further emphasizing mechanistic differences in the trypanosome and Leishmania RNAi mechanisms.

scholarly journals In Vitro Reassortment between Endemic Bluetongue Viruses Features Global Shifts in Segment Frequencies and Preferred Segment Combinations

2021 ◽  
Vol 9 (2) ◽  
pp. 405
Author(s):  
Jennifer Kopanke ◽  
Justin Lee ◽  
Mark Stenglein ◽  
Christie Mayo
Keyword(s):  
Bluetongue Virus ◽  
In Vitro Propagation ◽  
Severe Disease ◽  
Whole Genome ◽  
Biting Midges ◽  
Genetic Changes ◽  
In Vitro System ◽  
Double Stranded Rna ◽  
The Usa

Bluetongue virus (BTV) is an arthropod-borne pathogen that is associated with sometimes severe disease in both domestic and wild ruminants. Predominantly transmitted by Culicoides spp. biting midges, BTV is composed of a segmented, double-stranded RNA genome. Vector expansion and viral genetic changes, such as reassortment between BTV strains, have been implicated as potential drivers of ongoing BTV expansion into previously BTV-free regions. We used an in vitro system to investigate the extent and flexibility of reassortment that can occur between two BTV strains that are considered enzootic to the USA, BTV-2 and BTV-10. Whole genome sequencing (WGS) was coupled with plaque isolation and a novel, amplicon-based sequencing approach to quantitate the viral genetic diversity generated across multiple generations of in vitro propagation. We found that BTV-2 and BTV-10 were able to reassort across multiple segments, but that a preferred BTV-2 viral backbone emerged in later passages and that certain segments were more likely to be found in reassortant progeny. Our findings indicate that there may be preferred segment combinations that emerge during BTV reassortment. Moreover, our work demonstrates the usefulness of WGS and amplicon-based sequencing approaches to improve understanding of the dynamics of reassortment among segmented viruses such as BTV.

scholarly journals High-pressure sprayed siRNA triggers influence the efficiency but not the profile of transitive silencing

2021 ◽  
Author(s):  
Veli Vural Uslu ◽  
Athanasios Dalakouras ◽  
Victor A Steffens ◽  
Gabi Krczal ◽  
Michael Wassenegger
Keyword(s):  
High Pressure ◽  
Feedback Mechanism ◽  
Small Interfering Rnas ◽  
Primary Target ◽  
Endonucleolytic Cleavage ◽  
Argonaute Proteins ◽  
Target Sites ◽  
Secondary Sirnas ◽  
Positive Feedback Mechanism ◽  
Target Rna

ABSTRACTIn plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)-inducing molecules produced by the endonucleolytic cleavage of double stranded RNAs (dsRNAs). In order to ensure robust RNAi, the siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA-DIRECTED-RNA POLYMERASE 6 (RDR6)-mediated dsRNA synthesis using siRNA-targeted RNA. This secondary dsRNA is subsequently cleaved into secondary, mainly phased, siRNAs (phasiRNAs) by DICER-LIKE (DCL) endonucleases. As primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA-induced silencing complex (RISC) reinforcing cleavage of the target RNA. Although the molecular players underlying transitivity are well established, the mode of action of transitivity remains elusive. In this study, we investigated the influence of primary target sites on transgene silencing and transitivity using the GFP-expressing Nicotiana benthamiana 16C line, high pressure spraying protocol (HPSP), and synthetic 22-nucleotide (nt) long siRNAs. We found that the siRNA targeting the 3’ of the GFP transgene was less efficient in inducing silencing when compared to the siRNAs targeting the 5’ and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs are shaped by the occupancy of the primary siRNA triggers on the target RNA. Our findings suggest that RDR6-mediated dsRNA synthesis is not primed by primary siRNAs and that dsRNA synthesis appears to be generally initiated at the 3’ end of the target RNA.

scholarly journals Diverging Affinity of Tospovirus RNA Silencing Suppressor Proteins, NSs, for Various RNA Duplex Molecules

2010 ◽  
Vol 84 (21) ◽  
pp. 11542-11554 ◽  
Author(s):  
Esther Schnettler ◽  
Hans Hemmes ◽  
Rik Huismann ◽  
Rob Goldbach ◽  
Marcel Prins ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Fluorescent Protein ◽  
Micro Rna ◽  
Impatiens Necrotic Spot Virus ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Cell Extracts ◽  
Green Fluorescent

ABSTRACT The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

Small interfering RNA treatment can inhibit Cyprinid herpesvirus 3 associated cell death in vitro

2014 ◽  
Vol 17 (4) ◽  
pp. 733-735 ◽  
Author(s):  
M. Adamek ◽  
G. Rauch ◽  
G. Brogden ◽  
D. Steinhagen
Keyword(s):  
Cell Death ◽  
Rna Interference ◽  
Small Interfering Rna ◽  
Enzyme Synthesis ◽  
Small Interfering Rnas ◽  
Viral Dna ◽  
Cyprinid Herpesvirus 3 ◽  
Interfering Rna ◽  
Fibroblastic Cells

Abstract A Cyprinid herpesvirus 3 infection of carp induces a disease which causes substantial losses in carp culture. Here we present the use of a possible strategy for the management of the virus infection RNA interference based on small interfering RNAs. As a result of in vitro studies, we found that a mixture of short interfering RNAs specific for viral DNA enzyme synthesis and capsid proteins of the CyHV-3 can be a potential inhibitor of virus replication in fibroblastic cells. This gives the basis for the development of a combinatorial RNA interference strategy to treat CyHV-3 infections.

scholarly journals The p122 Subunit of Tobacco Mosaic Virus Replicase Is a Potent Silencing Suppressor and Compromises both Small Interfering RNA- and MicroRNA-Mediated Pathways

2007 ◽  
Vol 81 (21) ◽  
pp. 11768-11780 ◽  
Author(s):  
Tibor Csorba ◽  
Aurelie Bovi ◽  
Tamás Dalmay ◽  
József Burgyán
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Nuclear Export ◽  
Rna Silencing ◽  
Plant Viruses ◽  
Silencing Suppressor ◽  
Small Interfering Rnas ◽  
In Planta ◽  
Interfering Rna

ABSTRACT One of the functions of RNA silencing in plants is to defend against molecular parasites, such as viruses, retrotransposons, and transgenes. Plant viruses are inducers, as well as targets, of RNA silencing-based antiviral defense. Replication intermediates or folded viral RNAs activate RNA silencing, generating small interfering RNAs (siRNAs), which are the key players in the antiviral response. Viruses are able to counteract RNA silencing by expressing silencing-suppressor proteins. It has been shown that many of the identified silencing-suppressor proteins bind long double-stranded RNA or siRNAs and thereby prevent assembly of the silencing effector complexes. In this study, we show that the 122-kDa replicase subunit (p122) of crucifer-infecting Tobacco mosaic virus (cr-TMV) is a potent silencing-suppressor protein. We found that the p122 protein preferentially binds to double-stranded 21-nucleotide (nt) siRNA and microRNA (miRNA) intermediates with 2-nt 3′ overhangs inhibiting the incorporation of siRNA and miRNA into silencing-related complexes (e.g., RNA-induced silencing complex [RISC]) both in vitro and in planta but cannot interfere with previously programmed RISCs. In addition, our results also suggest that the virus infection and/or sequestration of the siRNA and miRNA molecules by p122 enhances miRNA accumulation despite preventing its methylation. However, the p122 silencing suppressor does not prevent the methylation of certain miRNAs in hst-15 mutants, in which the nuclear export of miRNAs is compromised.

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