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.

Target site effects in the RNA interference and microRNA pathways

2008 ◽  
Vol 36 (6) ◽  
pp. 1216-1219 ◽  
Author(s):  
Gregor Obernosterer ◽  
Hakim Tafer ◽  
Javier Martinez
Keyword(s):  
Rna Interference ◽  
Site Effects ◽  
Initial Study ◽  
Target Site ◽  
Small Interfering Rnas ◽  
Endonucleolytic Cleavage ◽  
Downstream Effects ◽  
Rna Targets ◽  
Target Sites ◽  
Rnai Activity

In RNAi (RNA interference), siRNAs (small interfering RNAs) are loaded into the RISC (RNA-induced silencing complex), which then mediates endonucleolytic cleavage of complementary target RNAs. Although RNAi has become one of the most powerful tools in molecular biology to assess gene function, there remains a great number of ineffective siRNAs. It is already known that the assembly and activation of RISC is a crucial determinant of RNAi activity, but downstream effects such as target accessibility have not been analysed extensively. Therefore we assessed the effect of target site accessibility and found that it significantly improves the potency of siRNAs. Similarly, miRNAs (microRNAs) act by repressing protein synthesis through imperfect base-pairing to the 3′-UTR (untranslated region) of target mRNAs. We found that predicted target sites reside in regions of high accessibility and tested whether this criterion could be used in the search of functional miRNA targets. In addition, we performed reporter gene assays to test whether accessibility correlates with measured mRNA suppression levels. The results of our initial study suggest that secondary structures might add a so far underrepresented layer of complexity in the recognition of RNA targets by miRNAs.

scholarly journals Functional diversification of Argonautes in nematodes: an expanding universe

2013 ◽  
Vol 41 (4) ◽  
pp. 881-886 ◽  
Author(s):  
Amy H. Buck ◽  
Mark Blaxter
Keyword(s):  
Genome Stability ◽  
Rapid Evolution ◽  
Small Interfering Rnas ◽  
Epigenetic Memory ◽  
Functional Diversification ◽  
Nematode Caenorhabditis Elegans ◽  
Argonaute Proteins ◽  
Central Protein ◽  
Secondary Sirnas ◽  
And Function

In the last decade, many diverse RNAi (RNA interference) pathways have been discovered that mediate gene silencing at epigenetic, transcriptional and post-transcriptional levels. The diversity of RNAi pathways is inherently linked to the evolution of Ago (Argonaute) proteins, the central protein component of RISCs (RNA-induced silencing complexes). An increasing number of diverse Agos have been identified in different species. The functions of most of these proteins are not yet known, but they are generally assumed to play roles in development, genome stability and/or protection against viruses. Recent research in the nematode Caenorhabditis elegans has expanded the breadth of RNAi functions to include transgenerational epigenetic memory and, possibly, environmental sensing. These functions are inherently linked to the production of secondary siRNAs (small interfering RNAs) that bind to members of a clade of WAGOs (worm-specific Agos). In the present article, we review briefly what is known about the evolution and function of Ago proteins in eukaryotes, including the expansion of WAGOs in nematodes. We postulate that the rapid evolution of WAGOs enables the exceptional functional plasticity of nematodes, including their capacity for parasitism.

scholarly journals Highly efficacious antiviral protection of plants by small interfering RNAs identified in vitro

2019 ◽  
Vol 47 (17) ◽  
pp. 9343-9357 ◽  
Author(s):  
Selma Gago-Zachert ◽  
Jana Schuck ◽  
Claus Weinholdt ◽  
Marie Knoblich ◽  
Vitantonio Pantaleo ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Plant Protection ◽  
Small Interfering Rnas ◽  
Functional Identification ◽  
Protection Measures ◽  
Argonaute Proteins ◽  
Effective Sirnas ◽  
Target Rna ◽  
Model Virus

Abstract In response to a viral infection, the plant’s RNA silencing machinery processes viral RNAs into a huge number of small interfering RNAs (siRNAs). However, a very few of these siRNAs actually interfere with viral replication. A reliable approach to identify these immunologically effective siRNAs (esiRNAs) and to define the characteristics underlying their activity has not been available so far. Here, we develop a novel screening approach that enables a rapid functional identification of antiviral esiRNAs. Tests on the efficacy of such identified esiRNAs of a model virus achieved a virtual full protection of plants against a massive subsequent infection in transient applications. We find that the functionality of esiRNAs depends crucially on two properties: the binding affinity to Argonaute proteins and the ability to access the target RNA. The ability to rapidly identify functional esiRNAs could be of great benefit for all RNA silencing-based plant protection measures against viruses and other pathogens.

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 HOPF BIFURCATION FROM NEW-KEYNESIAN TAYLOR RULE TO RAMSEY OPTIMAL POLICY

2020 ◽  
pp. 1-33
Author(s):  
Jean-Bernard Chatelain ◽  
Kirsten Ralf
Keyword(s):  
Hopf Bifurcation ◽  
Optimal Policy ◽  
Ad Hoc ◽  
Taylor Rule ◽  
Dynamic Properties ◽  
Feedback Mechanism ◽  
New Keynesian ◽  
Negative Feedback Mechanism ◽  
Positive Feedback Mechanism ◽  
Forward Looking

This paper compares different implementations of monetary policy in a new-Keynesian setting. We can show that a shift from Ramsey optimal policy under short-term commitment (based on a negative feedback mechanism) to a Taylor rule (based on a positive feedback mechanism) corresponds to a Hopf bifurcation with opposite policy advice and a change of the dynamic properties. This bifurcation occurs because of the ad hoc assumption that interest rate is a forward-looking variable when policy targets (inflation and output gap) are forward-looking variables in the new-Keynesian theory.

scholarly journals A positive feedback mechanism ensures proper assembly of the functional inner centromere during mitosis in human cells

2018 ◽  
Vol 294 (5) ◽  
pp. 1437-1450 ◽  
Author(s):  
Cai Liang ◽  
Zhenlei Zhang ◽  
Qinfu Chen ◽  
Haiyan Yan ◽  
Miao Zhang ◽  
...  
Keyword(s):  
Positive Feedback ◽  
Histone H3 ◽  
Causal Link ◽  
Feedback Mechanism ◽  
Histone H2a ◽  
Defective Mutant ◽  
Chromosomal Passenger ◽  
Phosphatase 2A ◽  
Elevated Rate ◽  
Positive Feedback Mechanism

The inner centromere region of a mitotic chromosome critically regulates sister chromatid cohesion and kinetochore–microtubule attachments. However, the molecular mechanism underlying inner centromere assembly remains elusive. Here, using CRISPR/Cas9-based gene editing in HeLa cells, we disrupted the interaction of Shugoshin 1 (Sgo1) with histone H2A phosphorylated on Thr-120 (H2ApT120) to selectively release Sgo1 from mitotic centromeres. Interestingly, cells expressing the H2ApT120-binding defective mutant of Sgo1 have an elevated rate of chromosome missegregation accompanied by weakened centromeric cohesion and decreased centromere accumulation of the chromosomal passenger complex (CPC), an integral part of the inner centromere and a key player in the correction of erroneous kinetochore–microtubule attachments. When artificially tethered to centromeres, a Sgo1 mutant defective in binding protein phosphatase 2A (PP2A) is not able to support proper centromeric cohesion and CPC accumulation, indicating that the Sgo1–PP2A interaction is essential for the integrity of mitotic centromeres. We further provide evidence indicating that Sgo1 protects centromeric cohesin to create a binding site for the histone H3–associated protein kinase Haspin, which not only inhibits the cohesin release factor Wapl and thereby strengthens centromeric cohesion but also phosphorylates histone H3 at Thr-3 to position CPC at inner centromeres. Taken together, our findings reveal a positive feedback–based mechanism that ensures proper assembly of the functional inner centromere during mitosis. They further suggest a causal link between centromeric cohesion defects and chromosomal instability in cancer cells.

scholarly journals Hedgehog signaling activates a positive feedback mechanism involving insulin-like growth factors to induce osteoblast differentiation

2015 ◽  
Vol 112 (15) ◽  
pp. 4678-4683 ◽  
Author(s):  
Yu Shi ◽  
Jianquan Chen ◽  
Courtney M. Karner ◽  
Fanxin Long
Keyword(s):  
Transcriptional Activation ◽  
Positive Feedback ◽  
Hedgehog Signaling ◽  
Osteoblast Differentiation ◽  
Akt Signaling ◽  
Feedback Mechanism ◽  
Genetic Deletion ◽  
Positive Feedback Mechanism ◽  
Igf Signaling

Hedgehog (Hh) signaling is essential for osteoblast differentiation in the endochondral skeleton during embryogenesis. However, the molecular mechanism underlying the osteoblastogenic role of Hh is not completely understood. Here, we report that Hh markedly induces the expression of insulin-like growth factor 2 (Igf2) that activates the mTORC2-Akt signaling cascade during osteoblast differentiation. Igf2-Akt signaling, in turn, stabilizes full-length Gli2 through Serine 230, thus enhancing the output of transcriptional activation by Hh. Importantly, genetic deletion of the Igf signaling receptor Igf1r specifically in Hh-responding cells diminishes bone formation in the mouse embryo. Thus, Hh engages Igf signaling in a positive feedback mechanism to activate the osteogenic program.

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