RNA-Mediated Silencing in Algae: Biological Roles and Tools for Analysis of Gene Function

ABSTRACTAlgae are a large group of aquatic, typically photosynthetic, eukaryotes that include species from very diverse phylogenetic lineages, from those similar to land plants to those related to protist parasites. The recent sequencing of several algal genomes has provided insights into the great complexity of these organisms. Genomic information has also emphasized our lack of knowledge of the functions of many predicted genes, as well as the gene regulatory mechanisms in algae. Core components of the machinery for RNA-mediated silencing show widespread distribution among algal lineages, but they also seem to have been lost entirely from several species with relatively small nuclear genomes. Complex sets of endogenous small RNAs, including candidate microRNAs and small interfering RNAs, have now been identified by high-throughput sequencing in green, red, and brown algae. However, the natural roles of RNA-mediated silencing in algal biology remain poorly understood. Limited evidence suggests that small RNAs may function, in different algae, in defense mechanisms against transposon mobilization, in responses to nutrient deprivation and, possibly, in the regulation of recently evolved developmental processes. From a practical perspective, RNA interference (RNAi) is becoming a promising tool for assessing gene function by sequence-specific knockdown. Transient gene silencing, triggered with exogenously synthesized nucleic acids, and/or stable gene repression, involving genome-integrated transgenes, have been achieved in green algae, diatoms, yellow-green algae, and euglenoids. The development of RNAi technology in conjunction with system level “omics” approaches may provide the tools needed to advance our understanding of algal physiological and metabolic processes.

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

Genome Biology ◽

10.1186/s13059-020-02242-6 ◽

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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RNA interference and the use of small interfering RNA to study gene function in mammalian systems

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01582 ◽

2004 ◽

Vol 33 (3) ◽

pp. 545-557 ◽

Cited By ~ 92

Author(s):

I Bantounas ◽

L A Phylactou ◽

J B Uney

Keyword(s):

Rna Interference ◽

Gene Function ◽

Viral Vector ◽

Sirna Delivery ◽

Interferon Response ◽

Rnai Pathway ◽

Small Interfering Rnas ◽

Rnai Technology

In the past 2 years, extraordinary developments in RNA interference (RNAi)-based methodologies have seen small interfering RNAs (siRNA) become the method of choice for researchers wishing to target specific genes for silencing. In this review, an historic overview of the biochemistry of the RNAi pathway is described together with the latest advances in the RNAi field. Particular emphasis is given to strategies by which siRNAs are used to study mammalian gene function. In this regard, the use of plasmid-based and viral vector-based systems to mediate long-term RNAi in vitro and in vivo are described. However, recent work has shown that non-specific silencing effects and activation of the interferon response may occur following the use of some siRNA and delivery vector combinations. Future goals must therefore be to understand the mechanisms by which siRNA delivery leads to unwanted gene silencing effects in cells and, in this way, RNAi technology can reach its tremendous potential as a scientific tool and ultimately be used for therapeutic purposes.

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Widespread roles for piRNAs and WAGO-class siRNAs in shaping the germline transcriptome of Caenorhabditis elegans

Nucleic Acids Research ◽

10.1093/nar/gkz1178 ◽

2019 ◽

Vol 48 (4) ◽

pp. 1811-1827 ◽

Cited By ~ 11

Author(s):

Kailee J Reed ◽

Joshua M Svendsen ◽

Kristen C Brown ◽

Brooke E Montgomery ◽

Taylor N Marks ◽

...

Keyword(s):

Gene Expression ◽

Caenorhabditis Elegans ◽

Small Rnas ◽

High Throughput Sequencing ◽

Critical Role ◽

Rnai Pathway ◽

Small Interfering Rnas ◽

Germline Development ◽

Histone Mrnas ◽

High Level

Abstract Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) are distinct classes of small RNAs required for proper germline development. To identify the roles of piRNAs and siRNAs in regulating gene expression in Caenorhabditis elegans, we subjected small RNAs and mRNAs from the gonads of piRNA and siRNA defective mutants to high-throughput sequencing. We show that piRNAs and an abundant class of siRNAs known as WAGO-class 22G-RNAs are required for proper expression of spermatogenic and oogenic genes. WAGO-class 22G-RNAs are also broadly required for transposon silencing, whereas piRNAs are largely dispensable. piRNAs, however, have a critical role in controlling histone gene expression. In the absence of piRNAs, histone mRNAs are misrouted into the nuclear RNAi pathway involving the Argonaute HRDE-1, concurrent with a reduction in the expression of many histone mRNAs. We also show that high-level gene expression in the germline is correlated with high level 22G-RNA production. However, most highly expressed genes produce 22G-RNAs through a distinct pathway that presumably involves the Argonaute CSR-1. In contrast, genes targeted by the WAGO branch of the 22G-RNA pathway are typically poorly expressed and respond unpredictably to loss of 22G-RNAs. Our results point to broad roles for piRNAs and siRNAs in controlling gene expression in the C. elegans germline.

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Small RNAs: The Essential Regulators in Plant Thermotolerance

Frontiers in Plant Science ◽

10.3389/fpls.2021.726762 ◽

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.

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Transcriptome Profile Alterations with Carbon Nanotubes, Quantum Dots, and Silver Nanoparticles: A Review

Genes ◽

10.3390/genes12060794 ◽

2021 ◽

Vol 12 (6) ◽

pp. 794

Author(s):

Cullen Horstmann ◽

Victoria Davenport ◽

Min Zhang ◽

Alyse Peters ◽

Kyoungtae Kim

Keyword(s):

Carbon Nanotubes ◽

Quantum Dots ◽

High Throughput Sequencing ◽

The Body ◽

Rna Seq ◽

Mechanisms Of Toxicity ◽

Promising Tool ◽

Engineered Nanomaterial ◽

Next Generation Sequencing Ngs ◽

Transcriptomic Changes

Next-generation sequencing (NGS) technology has revolutionized sequence-based research. In recent years, high-throughput sequencing has become the method of choice in studying the toxicity of chemical agents through observing and measuring changes in transcript levels. Engineered nanomaterial (ENM)-toxicity has become a major field of research and has adopted microarray and newer RNA-Seq methods. Recently, nanotechnology has become a promising tool in the diagnosis and treatment of several diseases in humans. However, due to their high stability, they are likely capable of remaining in the body and environment for long periods of time. Their mechanisms of toxicity and long-lasting effects on our health is still poorly understood. This review explores the effects of three ENMs including carbon nanotubes (CNTs), quantum dots (QDs), and Ag nanoparticles (AgNPs) by cross examining publications on transcriptomic changes induced by these nanomaterials.

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

Microorganisms ◽

10.3390/microorganisms9020249 ◽

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.

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The intestinal microbiota, gastrointestinal environment and colorectal cancer: a putative role for probiotics in prevention of colorectal cancer?

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00110.2011 ◽

2011 ◽

Vol 301 (3) ◽

pp. G401-G424 ◽

Cited By ~ 125

Author(s):

M. Andrea Azcárate-Peril ◽

Michael Sikes ◽

José M. Bruno-Bárcena

Keyword(s):

Colorectal Cancer ◽

Intestinal Microbiota ◽

Defense Mechanisms ◽

High Throughput Sequencing ◽

Safety Evaluation ◽

Health Benefit ◽

The United States ◽

Dietary Factors ◽

Colorectal Adenomas ◽

Food Allergies

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the United States, and, even though 5–15% of the total CRC cases can be attributed to individual genetic predisposition, environmental factors could be considered major factors in susceptibility to CRC. Lifestyle factors increasing the risks of CRC include elevated body mass index, obesity, and reduced physical activity. Additionally, a number of dietary elements have been associated with higher or lower incidence of CRC. In this context, it has been suggested that diets high in fruit and low in meat might have a protective effect, reducing the incidence of colorectal adenomas by modulating the composition of the normal nonpathogenic commensal microbiota. In addition, it has been demonstrated that changes in abundance of taxonomic groups have a profound impact on the gastrointestinal physiology, and an increasing number of studies are proposing that the microbiota mediates the generation of dietary factors triggering colon cancer. High-throughput sequencing and molecular taxonomic technologies are rapidly filling the knowledge gaps left by conventional microbiology techniques to obtain a comprehensive catalog of the human intestinal microbiota and their associated metabolic repertoire. The information provided by these studies will be essential to identify agents capable of modulating the massive amount of gut bacteria in safe noninvasive manners to prevent CRC. Probiotics, defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” ( 219 ), are capable of transient modulation of the microbiota, and their beneficial effects include reinforcement of the natural defense mechanisms and protection against gastrointestinal disorders. Probiotics have been successfully used to manage infant diarrhea, food allergies, and inflammatory bowel disease; hence, the purpose of this review was to examine probiotic metabolic activities that may have an effect on the prevention of CRC by scavenging toxic compounds or preventing their generation in situ. Additionally, a brief consideration is given to safety evaluation and production methods in the context of probiotics efficacy.

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Graph-based sequence annotation using a data integration approach

Journal of Integrative Bioinformatics ◽

10.1515/jib-2008-94 ◽

2008 ◽

Vol 5 (2) ◽

Cited By ~ 1

Author(s):

Robert Pesch ◽

Artem Lysenko ◽

Matthew Hindle ◽

Keywan Hassani-Pak ◽

Ralf Thiele ◽

...

Keyword(s):

Data Integration ◽

Gene Function ◽

High Throughput Sequencing ◽

Prediction Method ◽

Function Prediction ◽

Function Annotation ◽

Automated Annotation ◽

Data Integration System ◽

Reference Databases ◽

Function Assignment

SummaryThe automated annotation of data from high throughput sequencing and genomics experiments is a significant challenge for bioinformatics. Most current approaches rely on sequential pipelines of gene finding and gene function prediction methods that annotate a gene with information from different reference data sources. Each function prediction method contributes evidence supporting a functional assignment. Such approaches generally ignore the links between the information in the reference datasets. These links, however, are valuable for assessing the plausibility of a function assignment and can be used to evaluate the confidence in a prediction. We are working towards a novel annotation system that uses the network of information supporting the function assignment to enrich the annotation process for use by expert curators and predicting the function of previously unannotated genes. In this paper we describe our success in the first stages of this development. We present the data integration steps that are needed to create the core database of integrated reference databases (UniProt, PFAM, PDB, GO and the pathway database Ara- Cyc) which has been established in the ONDEX data integration system. We also present a comparison between different methods for integration of GO terms as part of the function assignment pipeline and discuss the consequences of this analysis for improving the accuracy of gene function annotation.The methods and algorithms presented in this publication are an integral part of the ONDEX system which is freely available from http://ondex.sf.net/.

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