scholarly journals Plant Extracellular Vesicles Contain Diverse Small RNA Species and Are Enriched in 10 to 17 Nucleotide “Tiny” RNAs

2018 ◽  
Author(s):  
Patricia Baldrich ◽  
Brian D. Rutter ◽  
Hana Zandkarimi ◽  
Ram Podicheti ◽  
Blake C. Meyers ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Stress Responses ◽  
Small Rnas ◽  
Degradation Products ◽  
Small Interfering Rnas ◽  
Diverse Species ◽  
Transposon Silencing ◽  
The Stability ◽  
Eukaryotic Organisms ◽  
Small Rna Species

ABSTRACTSmall RNAs (sRNAs) that are 21 to 24 nucleotides (nt) in length are found in most eukaryotic organisms and regulate numerous biological functions, including transposon silencing, development, reproduction, and stress responses, typically via control of the stability and/or translation of target mRNAs. Major classes of sRNAs in plants include microRNAs (miRNAs) and small interfering RNAs (siRNAs); sRNAs are known to travel as a silencing signal from cell to cell, root to shoot, and even between host and pathogen. In mammals, sRNAs are transported inside extracellular vesicles (EVs), which are mobile lipid compartments that participate in intercellular communication. In addition to sRNAs, EVs carry proteins, lipids, metabolites, and potentially other types of nucleic acids. Here we report that plant EVs also contain diverse species of sRNA. We found that specific miRNAs and siRNAs are preferentially loaded into plant EVs. We also report a previously overlooked class of “tiny RNAs” (10 to 17 nt) that are highly enriched in EVs. This new RNA category of unknown function has a broad and very diverse genome origin and might correspond to degradation products.

scholarly journals An atypical class of non-coding small RNAs produced in rice leaves upon bacterial infection

2021 ◽  
Author(s):  
Ganna Reshetnyak ◽  
Jonathan M. Jacobs ◽  
Florence Auguy ◽  
Coline Sciallano ◽  
Lisa Claude ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Stress Responses ◽  
Small Rnas ◽  
Early Stage ◽  
Kinase Domain ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Post Transcriptional Gene Silencing ◽  
Virulent Strains ◽  
Microbe Interactions

ABSTRACTNon-coding small RNAs (sRNA) act as mediators of gene silencing and regulate plant growth, development and stress responses. Early insights into plant sRNAs established a role in antiviral defense and they are now extensively studied across plant-microbe interactions. Here, sRNA sequencing discovered a class of sRNA in rice (Oryza sativa) specifically associated with foliar diseases caused by Xanthomonas oryzae bacteria. Xanthomonas-induced small RNAs (xisRNAs) loci were distinctively upregulated in response to diverse virulent strains at an early stage of infection producing a single duplex of 20-22nt sRNAs. xisRNAs production was dependent on the Type III secretion system, a major bacterial virulence factor for host colonization. xisRNA loci overlap with annotated transcripts sequences often encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and some xisRNA loci coincide with the coding sequence of a conserved kinase motif. xisRNAs exhibit features of small interfering RNAs and their biosynthesis depend on canonical components OsDCL1 and OsHEN1. xisRNA induction possibly mediates post-transcriptional gene silencing but they do not broadly suppress cis-genes expression on the basis of mRNA-seq data. Overall, our results identify a group of unusual sRNAs with a potential role in plant-microbe interactions.

scholarly journals RppH can faithfully replace TAP to allow cloning of 5’-triphosphate carrying small RNAs

2018 ◽  
Author(s):  
Miguel Vasconcelos Almeida ◽  
António Miguel de Jesus Domingues ◽  
Hanna Lukas ◽  
Maria Mendez-Lago ◽  
René F. Ketting
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Small Rna Sequencing ◽  
Cloning Efficiency ◽  
Library Preparation ◽  
Small Interfering Rnas ◽  
Rdrp Activity ◽  
C Elegans ◽  
Rna Pathways ◽  
Small Rna Species

AbstractRNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. Much like plants, but unlike Drosophila and mammals, worms have RNA-dependent RNA Polymerases (RdRPs) that directly synthesize small RNAs using other transcripts as a template. The very prominent secondary small interfering RNAs, also called 22G-RNAs, produced by the RdRPs RRF-1 and EGO-1 in C. elegans, maintain the 5’ triphosphate group, stemming from RdRP activity, also after loading into an Argonaute protein. This creates a technical issue, since 5’PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5’ pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison.

scholarly journals Cargo sorting to lysosome-related organelles regulates siRNA-mediated gene silencing

2011 ◽  
Vol 194 (1) ◽  
pp. 77-87 ◽  
Author(s):  
Dinari A. Harris ◽  
Kevin Kim ◽  
Kenji Nakahara ◽  
Constanza Vásquez-Doorman ◽  
Richard W. Carthew
Keyword(s):  
Small Rnas ◽  
Messenger Rna ◽  
Specific Class ◽  
Small Interfering Rnas ◽  
Ribonucleic Acids ◽  
Rna Transcripts ◽  
A Subunit ◽  
Lysosome Related Organelles ◽  
The Stability ◽  
Cargo Sorting

Mammals lacking BLOC-3 have impaired formation of melanosomes, a type of lysosome-related organelle (LRO), and, in earlier work, we found that a subunit of the BLOC-3 complex inhibits loading of Argonaute (Ago) proteins with small ribonucleic acids (RNAs) in Drosophila melanogaster cells. Small RNAs such as small interfering RNAs (siRNAs) direct Ago proteins to repress the stability of messenger RNA transcripts. In this paper, we show that BLOC-3 is required for biogenesis of Drosophila LROs called pigment granules. Other complexes that sort cargo to pigment LROs also negatively regulate siRNA activity. However, regulation is not obligately linked to biogenesis of LROs but instead to specific cargo-sorting processes. Negative regulation is also not linked to sorting into all LROs but only a specific class of pigment LRO. Thus, regulation of siRNA activity is tied to sorting of specific types of cargo to particular LROs.

scholarly journals Small RNAs: The Essential Regulators in Plant Thermotolerance

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhi-Fang Zuo ◽  
Wenbo He ◽  
Jing Li ◽  
Beixin Mo ◽  
Lin Liu
Keyword(s):  
Heat Stress ◽  
High Temperature ◽  
Stress Responses ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
High Temperature Stress ◽  
Temperature Acclimation ◽  
Regulatory Mechanisms ◽  
Small Interfering Rnas ◽  
Heat Stress Response

Small RNAs (sRNAs) are a class of non-coding RNAs that consist of 21–24 nucleotides. They have been extensively investigated as critical regulators in a variety of biological processes in plants. sRNAs include two major classes: microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis and functional pathways. Due to global warming, high-temperature stress has become one of the primary causes for crop loss worldwide. Recent studies have shown that sRNAs are involved in heat stress responses in plants and play essential roles in high-temperature acclimation. Genome-wide studies for heat-responsive sRNAs have been conducted in many plant species using high-throughput sequencing. The roles for these sRNAs in heat stress response were also unraveled subsequently in model plants and crops. Exploring how sRNAs regulate gene expression and their regulatory mechanisms will broaden our understanding of sRNAs in thermal stress responses of plant. Here, we highlight the roles of currently known miRNAs and siRNAs in heat stress responses and acclimation of plants. We also discuss the regulatory mechanisms of sRNAs and their targets that are responsive to heat stress, which will provide powerful molecular biological resources for engineering crops with improved thermotolerance.

scholarly journals An atypical class of non-coding small RNAs is produced in rice leaves upon bacterial infection

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ganna Reshetnyak ◽  
Jonathan M. Jacobs ◽  
Florence Auguy ◽  
Coline Sciallano ◽  
Lisa Claude ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Stress Responses ◽  
Small Rnas ◽  
Early Stage ◽  
Kinase Domain ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Post Transcriptional Gene Silencing ◽  
Virulent Strains ◽  
Microbe Interactions

AbstractNon-coding small RNAs (sRNA) act as mediators of gene silencing and regulate plant growth, development and stress responses. Early insights into plant sRNAs established a role in antiviral defense and they are now extensively studied across plant–microbe interactions. Here, sRNA sequencing discovered a class of sRNA in rice (Oryza sativa) specifically associated with foliar diseases caused by Xanthomonas oryzae bacteria. Xanthomonas-induced small RNAs (xisRNAs) loci were distinctively upregulated in response to diverse virulent strains at an early stage of infection producing a single duplex of 20–22 nt sRNAs. xisRNAs production was dependent on the Type III secretion system, a major bacterial virulence factor for host colonization. xisRNA loci overlap with annotated transcripts sequences, with about half of them encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and xisRNA loci predominantly coincide with the coding sequence of a conserved kinase motif. xisRNAs exhibit features of small interfering RNAs and their biosynthesis depend on canonical components OsDCL1 and OsHEN1. xisRNA induction possibly mediates post-transcriptional gene silencing but they do not broadly suppress cis-genes expression on the basis of mRNA-seq data. Overall, our results identify a group of unusual sRNAs with a potential role in plant–microbe interactions.

scholarly journals InsP7is a small-molecule regulator of NUDT3-mediated mRNA decapping and processing-body dynamics

2020 ◽  
Vol 117 (32) ◽  
pp. 19245-19253 ◽  
Author(s):  
Soumyadip Sahu ◽  
Zhenzhen Wang ◽  
Xinfu Jiao ◽  
Chunfang Gu ◽  
Nikolaus Jork ◽  
...  
Keyword(s):  
Stress Responses ◽  
Messenger Rna ◽  
Control Process ◽  
Stem Cell Differentiation ◽  
Wild Type ◽  
Inositol Pyrophosphate ◽  
Body Dynamics ◽  
Processing Body ◽  
The Stability

Regulation of enzymatic 5′ decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5′ decapping promotes accumulation of mRNAs into processing (P) bodies—membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP7(5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP7inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout ofPPIP5Ks(diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e.,PPIP5KKO), which elevates cellular 5-InsP7levels by two- to threefold (i.e., within the physiological rheostatic range). ThePPIP5KKO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP7synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP7analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP7levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

scholarly journals Different miRNA Profiles in Plasma Derived Small and Large Extracellular Vesicles from Patients with Neurodegenerative Diseases

2021 ◽  
Vol 22 (5) ◽  
pp. 2737
Author(s):  
Daisy Sproviero ◽  
Stella Gagliardi ◽  
Susanna Zucca ◽  
Maddalena Arigoni ◽  
Marta Giannini ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Extracellular Vesicles ◽  
Small Rnas ◽  
Biomarker Discovery ◽  
Toll Like Receptor ◽  
Neurotrophin Signaling ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Lateral Sclerosis ◽  
Glycosphingolipid Biosynthesis

Identifying biomarkers is essential for early diagnosis of neurodegenerative diseases (NDs). Large (LEVs) and small extracellular vesicles (SEVs) are extracellular vesicles (EVs) of different sizes and biological functions transported in blood and they may be valid biomarkers for NDs. The aim of our study was to investigate common and different miRNA signatures in plasma derived LEVs and SEVs of Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Dementia (FTD) patients. LEVs and SEVs were isolated from plasma of patients and healthy volunteers (CTR) by filtration and differential centrifugation and RNA was extracted. Small RNAs libraries were carried out by Next Generation Sequencing (NGS). MiRNAs discriminate all NDs diseases from CTRs and they can provide a signature for each NDs. Common enriched pathways for SEVs were instead linked to ubiquitin mediated proteolysis and Toll-like receptor signaling pathways and for LEVs to neurotrophin signaling and Glycosphingolipid biosynthesis pathway. LEVs and SEVs are involved in different pathways and this might give a specificity to their role in the spreading of the disease. The study of common and different miRNAs transported by LEVs and SEVs can be of great interest for biomarker discovery and for pathogenesis studies in neurodegeneration.

scholarly journals Increasing Solvent Tolerance to Improve Microbial Production of Alcohols, Terpenoids and Aromatics

2021 ◽  
Vol 9 (2) ◽  
pp. 249
Author(s):  
Thomas Schalck ◽  
Bram Van den Bergh ◽  
Jan Michiels
Keyword(s):  
Stress Responses ◽  
Small Rnas ◽  
Defense Mechanisms ◽  
Industrial Processes ◽  
Solvent Tolerance ◽  
Adaptive Responses ◽  
Environment Friendly ◽  
Pathway Optimization ◽  
Polymer Precursors ◽  
Product Accumulation

Fuels and polymer precursors are widely used in daily life and in many industrial processes. Although these compounds are mainly derived from petrol, bacteria and yeast can produce them in an environment-friendly way. However, these molecules exhibit toxic solvent properties and reduce cell viability of the microbial producer which inevitably impedes high product titers. Hence, studying how product accumulation affects microbes and understanding how microbial adaptive responses counteract these harmful defects helps to maximize yields. Here, we specifically focus on the mode of toxicity of industry-relevant alcohols, terpenoids and aromatics and the associated stress-response mechanisms, encountered in several relevant bacterial and yeast producers. In practice, integrating heterologous defense mechanisms, overexpressing native stress responses or triggering multiple protection pathways by modifying the transcription machinery or small RNAs (sRNAs) are suitable strategies to improve solvent tolerance. Therefore, tolerance engineering, in combination with metabolic pathway optimization, shows high potential in developing superior microbial producers.

scholarly journals Widespread occurrence of microRNA-mediated target cleavage on membrane-bound polysomes

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xiaoyu Yang ◽  
Chenjiang You ◽  
Xufeng Wang ◽  
Lei Gao ◽  
Beixin Mo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Gene Silencing ◽  
High Frequency ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Noncoding Rnas ◽  
Small Interfering Rnas ◽  
Widespread Occurrence ◽  
Membrane Bound ◽  
Different Tissues

Abstract Background Small RNAs (sRNAs) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) serve as core players in gene silencing at transcriptional and post-transcriptional levels in plants, but their subcellular localization has not yet been well studied, thus limiting our mechanistic understanding of sRNA action. Results We investigate the cytoplasmic partitioning of sRNAs and their targets globally in maize (Zea mays, inbred line “B73”) and rice (Oryza sativa, cv. “Nipponbare”) by high-throughput sequencing of polysome-associated sRNAs and 3′ cleavage fragments, and find that both miRNAs and a subset of 21-nucleotide (nt)/22-nt siRNAs are enriched on membrane-bound polysomes (MBPs) relative to total polysomes (TPs) across different tissues. Most of the siRNAs are generated from transposable elements (TEs), and retrotransposons positively contributed to MBP overaccumulation of 22-nt TE-derived siRNAs (TE-siRNAs) as opposed to DNA transposons. Widespread occurrence of miRNA-mediated target cleavage is observed on MBPs, and a large proportion of these cleavage events are MBP-unique. Reproductive 21PHAS (21-nt phasiRNA-generating) and 24PHAS (24-nt phasiRNA-generating) precursors, which were commonly considered as noncoding RNAs, are bound by polysomes, and high-frequency cleavage of 21PHAS precursors by miR2118 and 24PHAS precursors by miR2275 is further detected on MBPs. Reproductive 21-nt phasiRNAs are enriched on MBPs as opposed to TPs, whereas 24-nt phasiRNAs are nearly completely devoid of polysome occupancy. Conclusions MBP overaccumulation is a conserved pattern for cytoplasmic partitioning of sRNAs, and endoplasmic reticulum (ER)-bound ribosomes function as an independent regulatory layer for miRNA-induced gene silencing and reproductive phasiRNA biosynthesis in maize and rice.

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