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Arabidopsis Apoplastic Fluids Contain sRNA- and Circular RNA-Protein Complexes That Are Located Outside of Extracellular Vesicles

ABSTRACT: The extracellular space within plant leaves is called the apoplast, and functions as a key battlefield between plants and pathogens. Previously, we have shown that apoplastic wash fluid purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these RNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside of EVs, but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside of EVs and are associated with proteins. Additional analyses of these extracellular RNAs (exRNAs) revealed that they are made up both sRNAs and long non-coding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the post-transcriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-binding protein in apoplastic wash fluids, GLYCINE-RICH RNA BINDING PROTEIN 7 (GRP7), as wells as the small RNA-binding protein ARGONAUTE2 (AGO2). These two proteins co-immunoprecipitated with each other, and with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of apoplastic wash fluid, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE183867 | GEO | 2022/04/18

REPOSITORIES: GEO

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Arabidopsis Apoplastic Fluid Contains sRNA- and Circular RNA-Protein Complexes that Are Located Outside Extracellular Vesicles

Project description:The extracellular space within plant leaves is called the apoplast, and functions as a key battlefield between plants and pathogens. Previously, we have shown that apoplastic wash fluid purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these RNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside of EVs, but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside of EVs and are associated with proteins. Additional analyses of these extracellular RNAs (exRNAs) revealed that they are made up both sRNAs and long non-coding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the post-transcriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-binding protein in apoplastic wash fluids, GLYCINE-RICH RNA BINDING PROTEIN 7 (GRP7), as wells as the small RNA-binding protein ARGONAUTE2 (AGO2). These two proteins co-immunoprecipitated with each other, and with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of apoplastic wash fluid, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs.

2022-04-18 | GSE185133 | GEO

Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses

Project description:Extracellular RNAs (exRNAs) can be released by numerous cell types in vitro, are often protected within vesicles, and can modify recipient cell function. To determine how the composition and cellular sources of exRNAs and the extracellular vesicles (EVs) that carry them change in vivo during tissue inflammation, we analyzed bronchoalveolar lavage fluid (BALF) from mice before and after lung allergen challenge. In the lung, extracellular microRNAs (ex-miRNAs) had a composition highly correlated with airway lining epithelium. Using cell-type specific membrane tagging and single vesicle flow, we also found that 80% of detected vesicles were of epithelial origin. After the induction of allergic airway inflammation, miRNAs selectively expressed by immune cells, including miR-223 and miR-142a, increased and hematopoietic cell derived EVs also increased >2-fold. Together these data demonstrate that infiltrating immune cells release ex-miRNAs and EVs in inflamed tissues to alter the local extracellular environment.

2018-10-30 | GSE121818 | GEO

Translocated Legionella pneumophila small RNAs mimic eukaryotic miRNAs to dampen the host immune response

Project description:Extracellular vesicles (EVs) produced by bacteria, archaea and eukaryotic cells, are increasingly recognized as important mediators of intercellular communication via transfer of a wide variety of molecular cargoes. To characterize and identify the RNA molecules enriched in Legionella pneumophila extracellular vesicles (Lp-EVs) we performed RNAseq analyses. Four independent RNAseq libraries were constructed from RNA isolated from purified Lp-EVs and as control from the bacterial pellets, from which the Lp-EVs had been shed and deep sequenced using an Illumina platform. The sequences obtained from the Lp-EVs RNAseq libraries were compared to those obtained from the RNA extracted from the bacterial pellets (Figure 1g). The Lp-EV-sRNA cargo was defined as RNAs for which after normalization at least 1000 reads were sequenced and that showed an enrichment of a log2FC > 5 as compared to the RNAs from the bacterial pellets were. Using these parameters the analysis identified 47 different sRNAs enriched in the Lp-EVs. The 20 highest enriched sRNA that were present in all four biological replicates comprised 8 tRNAs, 7 sRNAs and 5 fragments of mRNA located in the CDS or UTR.

2021-12-06 | GSE159109 | GEO

Plant Extracellular Vesicles Contain Diverse Small RNA Species and Are Enriched in 10 to 17 Nucleotide "Tiny" RNAs.

Project description:Small RNAs (sRNAs) are small non-coding RNAs of 20 to 24 nucleotides in size found across a wide range of eukaryote organisms, from plants to animals. sRNAs are master regulators of a wide range of biological functions, including development, reproduction and stress responses. They typically function via the control of the stability and translation of target mRNAs. sRNAs can be divided in two major classes, according to their biogenesis and mode of action: microRNAs (miRNAs) and small interfering RNAs (siRNAs). As a silencing signal, several different types of sRNAs have also the ability to move short distances, like cell-to-cell, or much longer distances like root to shoot. They are even trafficked between different species and organisms. However, the mechanisms by which sRNAs move remain unknown. Extracellular vesicles (EVs) are mobile lipid compartments that participate in intercellular communication; they are able to carry proteins, lipids, nucleic acids and metabolites. Despite the fact that very little is known about the origin, formation or function of EVs in plants, recent results suggest that they participate in both long distance movement and immune responses. In this study, we analysed the small RNA content of EVs, and we identified miRNAs and siRNAs that are preferentially loaded into the EVs. This EV enrichment of certain RNAs supports hypotheses for long-distance RNA movement and for RNA-based communication between species, including host-pathogen interactions.

2018-11-19 | GSE114696 | GEO

Profiling of Argonaute (AGO) 2-associated sRNAs after infection of bacterial pathogen Pseudomonas syringae

Project description:Transgenic Arabidopsis plants (AGO2::HA:AGO2) were treated with either mock (10 mM MgCl2) or Pseudomonas syringae pv. tomato (Pst) expressing avrRpt2 (R2) at a concentration of 2 x 107 cfu/ml for 14 hours. sRNAs associated with AGO1 and AGO2 were co-immunoprecipitated using antibodies against either AGO1 (AGO1-IP), or HA (hemagglutinin) (AGO2-IP). As controls, we also gel-purified the 18-28 nt fraction of the total RNAs from an AGO2 mutant (ago2-1). The co-immunoprecipitated or gel-purified RNAs were cloned and sequenced by Illumina deep sequencing. Examination of AGO-associated sRNAs in pathogen-treated or control plants

2011-05-10 | E-GEOD-26161 | biostudies-arrayexpress

Profiling of Argonaute (AGO) 2-associated sRNAs after infection of bacterial pathogen Pseudomonas syringae

2011-05-10 | GSE26161 | GEO

Arabidopsis AGO3 predominantly recruits 24-nucleotide small RNAs to regulate epigenetic silencing [miRNA-seq]

Project description:AGO3 predominantly bound 24-nt sRNAs with 5â?? terminal adenine. The spectrum of AGO3-associated sRNAs was different from those bound to AGO2. By contrast, approximately 30% of AGO3-bound 24-nt sRNAs overlapped with those bound to AGO4 and over 60% of AGO3-associated 24-nt sRNA enriched loci were identical to those of AGO4. In addition, expression of AGO3 driven by AGO4 native promoter partially complemented AGO4 function and rescued DNA methylation defect in ago4-1 background. Examination of AGO3 and AGO2 bound small RNAs with/without salt stress.

2016-05-31 | E-GEOD-78088 | biostudies-arrayexpress

Unique extracellular RNA profiles in different fractions of plasma

Project description:Circulating extracellular RNAs (exRNAs) have great promise as novel clinically plasma-based biomarkers for cancer diagnosis and prognosis. knowledge of the difference or association between these different sources of exRNAs is very limited. We performed a sequential physical and biochemical precipitation to receive 4 plasma fractions (platelets and cell debris, Thrombin-treated fraction, extracellular vesicles and supernatant) from each plasma sample. After total RNA extraction, we made ligation-free libraries and performed RNA-seq to evaluate full spectrum of RNA abundance in each fraction. All RNAs were included without size selection during the library preparation. We utilized a successive stepwise alignment strategy to map the RNA sequences to different RNA categories. We found that each plasma fraction had its own unique distribution of RNA species. Hierarchical cluster analysis showed similarity in samples with same fraction and significant differences between different fractions. In addition, we also observed abundance difference of unique transcript. Furthermore, a considerable proportion of exogenous RNAs and novel predicted miRNAs in all plasma fractions were detected. These results demonstrate that thorough inspection of all plasma fractions is necessary for exRNA-based biomarker study and appropriate sampling processes is needed for illustrating the complexity of plasma-based EVs study.

2021-06-07 | GSE145767 | GEO

Comparative transcriptomic analysis of human and Drosophila extracellular vesicles reveals extensive conservation

Project description:Extracellular vesicles (EVs) are membrane-enclosed nanoparticles containing specific repertoires of genetic material. In mammals, EVs can mediate the horizontal transfer of various cargos and signaling molecules, notably miRNA and mRNA species. Whether this form of intercellular communication prevails in other metazoans remains unclear. Here, we report the first parallel comparative morphologic and transcriptomic characterization of EVs from Drosophila and human cellular models. Electronic microscopy revealed that Drosophila, like human cells release exosome-like EVs with diameter ranging from 30 to 200 nm, which contain complex populations of transcripts. RNA-seq identified abundant ribosomal RNA pseudogenes and retrotransposons in human and Drosophila EVs. Vault RNAs and Y RNAs abounded in human samples, whereas small nucleolar RNAs involved in pseudouridylation were most prevalent in Drosophila EVs. Numerous mRNAs were identified, largely consisting of exonic sequences displaying full-length read coverage and enriched for translation and electronic transport chain functions. By analogy with human systems, these extensive similarities suggest that EVs could also enable RNA-mediated intercellular communication in Drosophila. We performed RNA-seq on extracellular vesicles purified from of human and Drosophila cell line cultures. S2R+ and D17 Drosophila EVs were analyzed, along with human A431 and HepG2 EVs. No ribosomal RNA depletion or polyA selection was performed on EV samples. For comparative analyses, we also analyzed total cellular RNA from Drosophila D17 and human HepG2. Ribodepletion was performed on cellular samples.

2016-06-15 | E-GEOD-76173 | biostudies-arrayexpress

exRNA reference profiling of 13 individuals addressing the effect of biofluid (serum vs. EDTA plasma), individual, gender, fasting and female hormonal cycle

Project description:Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using novel combinations of denaturants, reducing agents, proteolysis and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and extracellular DNA (exDNA) from the same biofluid sample. We applied the method to characterize exRNAs from 312 plasma and serum samples collected from thirteen healthy volunteers at twelve time points over a two-month period.

2018-05-04 | GSE113994 | GEO

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