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High-pressure-sprayed double stranded RNA does not induce RNA interference of a reporter gene

ABSTRACT: High-pressure-sprayed double stranded RNA does not induce RNA interference of a reporter gene

PROVIDER: PRJNA671866 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12898887.fastq.gz	Fastqsanger.gz
	SRR12898888.fastq.gz	Fastqsanger.gz
	SRR12898889.fastq.gz	Fastqsanger.gz
	SRR12898890.fastq.gz	Fastqsanger.gz
	SRR12898891.fastq.gz	Fastqsanger.gz

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High-Pressure-Sprayed Double Stranded RNA Does Not Induce RNA Interference of a Reporter Gene.

Project description:In plants, RNA interference (RNAi) is an effective defense mechanism against pathogens and pests. RNAi mainly involves the micro RNA and the small interfering RNA (siRNA) pathways. The latter pathway is generally based on the processing of long double stranded RNAs (dsRNA) into siRNAs by DICER-LIKE endonucleases (DCLs). SiRNAs are loaded onto ARGONAUTE proteins to constitute the RNA-induced silencing complex (RISC). Natural dsRNAs derive from transcription of inverted repeats or of specific RNA molecules that are transcribed by RNA-directed RNA polymerase 6 (RDR6). Moreover, replication of infecting viruses/viroids results in the production of dsRNA intermediates that can serve as substrates for DCLs. The high effectiveness of RNAi both locally and systemically implicated that plants could become resistant to pathogens, including viruses, through artificial activation of RNAi by topical exogenous application of dsRNA. The most preferable procedure to exploit RNAi would be to simply spray naked dsRNAs onto mature plants that are specific for the attacking pathogens serving as a substitute for pesticides applications. However, the plant cell wall is a difficult barrier to overcome and only few reports claim that topical application of naked dsRNA triggers RNAi in plants. Using a transgenic Nicotiana benthamiana line, we found that high-pressure-sprayed naked dsRNA did not induce silencing of a green fluorescence protein (GFP) reporter gene. Small RNA sequencing (sRNA-seq) of the samples from dsRNA sprayed leaves revealed that the dsRNA was, if at all, not efficiently processed into siRNAs indicating that the dsRNA was insufficiently taken up by plant cells.

| S-EPMC7773025 | biostudies-literature

High-pressure-sprayed double stranded RNA does not induce RNA interference of a reporter gene

Project description:We report the sRNA profile upon dsRNA spraying on 16C (Nicotiana benthamiana) plants. In this experiment we could detect only the degradation products of the dsRNA, which indicates absence of further processing of the dsRNA by RNAi machinery

2020-12-01 | GSE160110 | GEO

The RIG-I like receptor LGP2 inhibits Dicer-dependent processing of long double-stranded RNA and blocks RNA interference in mammalian cells.

Project description:In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG-I like receptor (RLR) family , which signal to induce production of type I interferons (IFN). These key anti-viral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon-stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG-I, MDA5 and the least-studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus-derived double stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We identify Dicer as an LGP2-associated protein and show that LGP2 inhibits Dicer cleavage of dsRNA into siRNAs both in vitro and in vivo. Further, we show that in cells lacking an IFN response, ectopic expression of LGP2 interferes with RNAi-dependent suppression of gene expression. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs.

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Project description:Study on the MS1 interference problem for reporter ion quantification using orthogonal proteomes: human and pyrococcus furiosus.

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Project description:Reporter ion interference remains a limitation of isobaric tag-based sample multiplexing. Advances in instrumentation and data acquisition modes, such as the recently developed real-time database search (RTS), can reduce interference. However, interference persists as does the need to benchmark upstream sample preparation and data acquisition strategies. Here, we present an updated Triple yeast KnockOut (TKO) standard as well as corresponding upgrades to the TKO Viewing Tool (TVT2.5, http://tko.hms.harvard.edu/). Specifically, we expand the TKO standard to incorporate the TMTpro18-plex reagents (TKO18). We also construct a variant thereof which has been digested only with LysC (TKO18L). We compare proteome coverage and interference levels of TKO18 and TKO18L data that were acquired under different data acquisition modes and analyzed using TVT2.5. Our data illustrate that RTS reduces interference while improving proteome coverage and suggest that digesting with LysC alone only modestly reduces interference, albeit at the expense of proteome depth. Collectively, the two new TKO standards coupled with the updated TVT represent a convenient and versatile platform for assessing and developing methods to reduce interference in isobaric tag-based experiments.

2022-01-06 | PXD029458 | Pride

A double-stranded-RNA response program important for RNA interference efficiency.

Project description:When recognized by the RNA interference (RNAi) pathway, double-stranded RNA (dsRNA) produced in eukaryotic cells results in posttranscriptional gene silencing. In addition, dsRNA can trigger the interferon response as part of the immune response in vertebrates. In this study, we show that dsRNA, but not short interfering RNA (siRNA), induces the expression of qde-2 (an Argonaute gene) and dcl-2 (a Dicer gene), two central components of the RNAi pathway in the filamentous fungus Neurospora crassa. The induction of QDE-2 by dsRNA is required for normal gene silencing, indicating that this is a regulatory mechanism that allows the optimal function of the RNAi pathway. In addition, we demonstrate that Dicer proteins (DCLs) regulate QDE-2 posttranscriptionally, suggesting a role for DCLs or siRNA in QDE-2 accumulation. Finally, a genome-wide search revealed that additional RNAi components and homologs of antiviral and interferon-stimulated genes are also dsRNA-activated genes in Neurospora. Together, our results suggest that the activation of the RNAi components is part of a broad ancient host defense response against viral and transposon infections.

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A causal model of ion interference enables assessment and correction of ratio compression in multiplex proteomics

Project description:We thoroughly characterize isobaric labeling (TMT)-associated reporter ion interference at the MS2 level using a defined two-proteome experimental system with known ground truth. We discover remarkably poor agreement between the apparent precursor purity in the isolation window and the actual level of observed reporter ion interference in MS2-scans - a discrepancy that we find resolved by considering co-fragmentation of peptide ions hidden within the spectral “noise” of the MS1 isolation window. On this basis, we establish a comprehensive regression modeling strategy for predicting accurate, feature-wise estimates of reporter ion interference. Finally, we demonstrate the utility of accurate ion purity estimates for accurate fold change estimation and unbiased PTM site-to-protein normalization.

2023-12-19 | PXD040449 | Pride

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