Fine-tune control of targeted RNAi efficacy by plant artificial small RNAs

Abstract Eukaryotic RNA interference (RNAi) results in gene silencing upon the sequence-specific degradation of target transcripts by complementary small RNAs (sRNAs). In plants, RNAi-based tools have been optimized for high efficacy and high specificity, and are extensively used in gene function studies and for crop improvement. However, efficient methods for finely adjusting the degree of induced silencing are missing. Here, we present two different strategies based on artificial sRNAs for fine-tuning targeted RNAi efficacy in plants. First, the degree of silencing induced by synthetic-trans-acting small interfering RNAs (syn-tasiRNAs) can be adjusted by modifying the precursor position from which the syn-tasiRNA is expressed. The accumulation and efficacy of Arabidopsis TAS1c-based syn-tasiRNAs progressively decrease as the syn-tasiRNA is expressed from positions more distal to the trigger miR173 target site. And second, syn-tasiRNA activity can also be tweaked by modifying the degree of base-pairing between the 3′ end of the syn-tasiRNA and the 5′ end of the target RNA. Both strategies were used to finely modulate the degree of silencing of endogenous and exogenous target genes in Arabidopsis thaliana and Nicotiana benthamiana. New high-throughput syn-tasiRNA vectors were developed and functionally analyzed, and should facilitate the precise control of gene expression in multiple plant species.

Download Full-text

Abiotic Stress Tolerance in Soybean : Regulated by ncRNA

Journal of AgriSearch ◽

10.21921/jas.v3i1.11399 ◽

2016 ◽

Vol 3 (1) ◽

Author(s):

YASIN JESHIMA KHAN ◽

HUSNARA Tyagi ◽

Anil kumar Singh ◽

Santosh kumar. Magadum

Keyword(s):

Abiotic Stresses ◽

Target Genes ◽

Abiotic Stress Tolerance ◽

Crop Improvement ◽

Vital Role ◽

Grain Legume ◽

Biological Processes ◽

Small Interfering Rnas ◽

Cellular Environment ◽

Non Coding Rnas

Plants respond through a cascade of reactions resulting in varied cellular environment leading to alterations in the patterns of protein expression resulting in phonotypic changes. Single cell genomics and global proteomics came out to be powerful tools and efficient techniques in studying stress tolerant plants. Non-coding RNAs are a distinct class of regulatory RNAs in plants and animals that control a variety of biological processes. Small ncRNAs play a vital role in post transcriptional gene regulation by either translational repression or by inducing mRNA cleavage. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs control the expression of cognate target genes by binding to complementary sequences, resulting in cleavage or translational inhibition of the target RNAs. siRNAs too have a similar structure, function, and biogenesis like miRNAs but are derived from long double-stranded RNAs and can often direct DNA methylation at target sequences.In this review, we focus on the involvement of ncRNAs in comabting abiotic stresses of soybean. This review emphasis on previously known miRNAs as they play important role in several abiotic stresses like drought, salinity, chilling and heat stress by their diverse roles in mediating biological processes like gene expression, chromatin formation, defense of genome against invading viruses. This review attempts to elucidate the various kinds of non-coding RNAs explored, their discovery, biogenesis, functions, and response for different type of abiotic stresses and future aspects for crop improvement in the context of soybean, a representative grain legume.

Download Full-text

Small RNAs in parasitic nematodes – forms and functions

Parasitology ◽

10.1017/s0031182019001689 ◽

2019 ◽

Vol 147 (8) ◽

pp. 855-864

Author(s):

Collette Britton ◽

Roz Laing ◽

Eileen Devaney

Keyword(s):

Gene Expression ◽

Small Rna ◽

Small Rnas ◽

Target Genes ◽

Parasitic Nematodes ◽

Small Rna Sequencing ◽

Small Interfering Rnas ◽

Rna Sequences ◽

C Elegans

AbstractSmall RNAs are important regulators of gene expression. They were first identified in Caenorhabditis elegans, but it is now apparent that the main small RNA silencing pathways are functionally conserved across diverse organisms. Availability of genome data for an increasing number of parasitic nematodes has enabled bioinformatic identification of small RNA sequences. Expression of these in different lifecycle stages is revealed by small RNA sequencing and microarray analysis. In this review we describe what is known of the three main small RNA classes in parasitic nematodes – microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) – and their proposed functions. miRNAs regulate development in C. elegans and the temporal expression of parasitic nematode miRNAs suggest modulation of target gene levels as parasites develop within the host. miRNAs are also present in extracellular vesicles released by nematodes in vitro, and in plasma from infected hosts, suggesting potential regulation of host gene expression. Roles of piRNAs and siRNAs in suppressing target genes, including transposable elements, are also reviewed. Recent successes in RNAi-mediated gene silencing, and application of small RNA inhibitors and mimics will continue to advance understanding of small RNA functions within the parasite and at the host–parasite interface.

Download Full-text

Artificial Small RNA-Based Silencing Tools for Antiviral Resistance in Plants

Plants ◽

10.3390/plants9060669 ◽

2020 ◽

Vol 9 (6) ◽

pp. 669 ◽

Cited By ~ 1

Author(s):

Adriana E. Cisneros ◽

Alberto Carbonell

Keyword(s):

Small Rnas ◽

Rational Design ◽

High Specificity ◽

Plant Viruses ◽

Antiviral Resistance ◽

Small Interfering Rnas ◽

Crop Species ◽

Dna Viruses ◽

Induce Resistance ◽

Rna And Dna

Artificial small RNAs (art-sRNAs), such as artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs), are highly specific 21-nucleotide small RNAs designed to recognize and silence complementary target RNAs. Art-sRNAs are extensively used in gene function studies or for improving crops, particularly to protect plants against viruses. Typically, antiviral art-sRNAs are computationally designed to target one or multiple sites in viral RNAs with high specificity, and art-sRNA constructs are generated and introduced into plants that are subsequently challenged with the target virus(es). Numerous studies have reported the successful application of art-sRNAs to induce resistance against a large number of RNA and DNA viruses in model and crop species. However, the application of art-sRNAs as an antiviral tool has limitations, such as the difficulty to predict the efficacy of a particular art-sRNA or the emergence of virus variants with mutated target sites escaping to art-sRNA-mediated degradation. Here, we review the different classes, features, and uses of art-sRNA-based tools to induce antiviral resistance in plants. We also provide strategies for the rational design of antiviral art-sRNAs and discuss the latest advances in developing art-sRNA-based methodologies for enhanced resistance to plant viruses.

Download Full-text

Removal of H3K27me3 by JMJ Proteins Controls Plant Development and Environmental Responses in Arabidopsis

Frontiers in Plant Science ◽

10.3389/fpls.2021.687416 ◽

2021 ◽

Vol 12 ◽

Author(s):

Nobutoshi Yamaguchi

Keyword(s):

Gene Expression ◽

Plant Development ◽

Transcriptional Repression ◽

Target Genes ◽

Regulation Of Gene Expression ◽

Histone H3 ◽

Control Of Gene Expression ◽

Precise Control ◽

Repressive Histone Modification ◽

Environmental Responses

Trimethylation of histone H3 lysine 27 (H3K27me3) is a highly conserved repressive histone modification that signifies transcriptional repression in plants and animals. In Arabidopsis thaliana, the demethylation of H3K27 is regulated by a group of JUMONJI DOMAIN-CONTANING PROTEIN (JMJ) genes. Transcription of JMJ genes is spatiotemporally regulated during plant development and in response to the environment. Once JMJ genes are transcribed, recruitment of JMJs to target genes, followed by demethylation of H3K27, is critically important for the precise control of gene expression. JMJs function synergistically and antagonistically with transcription factors and/or other epigenetic regulators on chromatin. This review summarizes the latest advances in our understanding of Arabidopsis H3K27me3 demethylases that provide robust and flexible epigenetic regulation of gene expression to direct appropriate development and environmental responses in plants.

Download Full-text

The interplay between small RNA pathways shapes chromatin landscapes in C. elegans

10.1101/320713 ◽

2018 ◽

Author(s):

Ekaterina Gushchanskaia ◽

Ruben Esse ◽

Qicheng Ma ◽

Nelson Lau ◽

Alla Grishok

Keyword(s):

Small Rnas ◽

Target Genes ◽

Noncoding Rnas ◽

Small Interfering Rnas ◽

Loss Of Function ◽

Partial Loss ◽

Rna Dependent Rna Polymerase ◽

C Elegans ◽

Highly Expressed Genes ◽

Rna Pathways

ABSTRACTThe nematode C. elegans contains several types of endogenous small interfering RNAs (endo-siRNAs) produced by RNA-dependent RNA polymerase (RdRP) complexes. Both “silencing” siRNAs bound by Worm-specific Argonautes (WAGO) and “activating” siRNAs bound by the CSR-1 Argonaute require the DRH-3 helicase, an RdRP component. Here we show that, in the drh-3(ne4253) mutant deficient in RdRP-produced secondary endo-siRNAs, the silencing histone mark H3K9me3 is largely depleted, whereas in the csr-1 partial loss-of-function mutant this mark is ectopically deposited on CSR-1 target genes. Moreover, we observe ectopic H3K9me3 at enhancer elements in both drh-3 and csr-1 partial loss-of-function mutants and describe small RNAs matching enhancers. Finally, we detect accumulation of H3K27me3 at highly expressed genes in the drh-3(ne4253) mutant, which correlates with their reduced transcription. Our study shows that when abundant RdRP-produced siRNAs are depleted, there is ectopic elevation of noncoding RNAs linked to increase in silencing chromatin marks. Moreover, our results suggest that enhancer small RNAs may guide local H3K9 methylation.

Download Full-text

Posttranscriptional gene silencing in nuclei

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1009805108 ◽

2010 ◽

Vol 108 (1) ◽

pp. 409-414 ◽

Cited By ~ 49

Author(s):

Paul Hoffer ◽

Sergey Ivashuta ◽

Olga Pontes ◽

Alexa Vitins ◽

Craig Pikaard ◽

...

Keyword(s):

Gene Silencing ◽

Target Genes ◽

Rna Degradation ◽

Small Interfering Rnas ◽

Double Stranded Rna ◽

Posttranscriptional Gene Silencing ◽

Subcellular Compartmentation ◽

Specific Degradation ◽

Precursor Mrna ◽

Specific Sequences

In plants, small interfering RNAs (siRNAs) with sequence homology to transcribed regions of genes can guide the sequence-specific degradation of corresponding mRNAs, leading to posttranscriptional gene silencing (PTGS). The current consensus is that siRNA-mediated PTGS occurs primarily in the cytoplasm where target mRNAs are localized and translated into proteins. However, expression of an inverted-repeat double-stranded RNA corresponding to the soybeanFAD2-1Adesaturase intron is sufficient to silenceFAD2-1, implicating nuclear precursor mRNA (pre-mRNA) rather than cytosolic mRNA as the target of PTGS. SilencingFAD2-1using intronic or 3′-UTR sequences does not affect transcription rates of the target genes but results in the strong reduction of target transcript levels in the nucleus. Moreover, siRNAs corresponding to pre-mRNA–specific sequences accumulate in the nucleus. In Arabidopsis, we find that two enzymes involved in PTGS, Dicer-like 4 and RNA-dependent RNA polymerase 6, are localized in the nucleus. Collectively, these results demonstrate that siRNA-directed RNA degradation can take place in the nucleus, suggesting the need for a more complex view of the subcellular compartmentation of PTGS in plants.

Download Full-text

GAPPromoter Library for Fine-Tuning of Gene Expression in Pichia pastoris

Applied and Environmental Microbiology ◽

10.1128/aem.02843-10 ◽

2011 ◽

Vol 77 (11) ◽

pp. 3600-3608 ◽

Cited By ~ 82

Author(s):

Xiulin Qin ◽

Jiangchao Qian ◽

Gaofeng Yao ◽

Yingping Zhuang ◽

Siliang Zhang ◽

...

Keyword(s):

Gene Expression ◽

Pichia Pastoris ◽

Fine Tuning ◽

Promoter Strength ◽

Control Of Gene Expression ◽

Content Type ◽

Expression Levels ◽

Precise Control ◽

Promoter Library

ABSTRACTA library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeastPichia pastorisis a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range inP. pastoris, we created a functional promoter library through mutagenesis of the constitutiveGAPpromoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ∼0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (β-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth andS-adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research inP. pastoris.

Download Full-text

Interplay between small RNA pathways shapes chromatin landscapes in C. elegans

Nucleic Acids Research ◽

10.1093/nar/gkz275 ◽

2019 ◽

Vol 47 (11) ◽

pp. 5603-5616 ◽

Cited By ~ 3

Author(s):

Ekaterina S Gushchanskaia ◽

Ruben Esse ◽

Qicheng Ma ◽

Nelson C Lau ◽

Alla Grishok

Keyword(s):

Small Rnas ◽

Target Genes ◽

Noncoding Rnas ◽

Null Mutant ◽

Small Interfering Rnas ◽

Nematode Caenorhabditis Elegans ◽

Rna Dependent Rna Polymerase ◽

C Elegans ◽

Highly Expressed Genes ◽

Rna Pathways

Abstract The nematode Caenorhabditis elegans contains several types of endogenous small interfering RNAs (endo-siRNAs) produced by RNA-dependent RNA polymerase (RdRP) complexes. Both ‘silencing’ siRNAs bound by Worm-specific Argonautes (WAGO) and ‘activating’ siRNAs bound by the CSR-1 Argonaute require the DRH-3 helicase, an RdRP component. Here, we show that, in the drh-3(ne4253) mutant deficient in RdRP-produced secondary endo-siRNAs, the silencing histone mark H3K9me3 is largely depleted, whereas in the csr-1 partially rescued null mutant strain (WM193), this mark is ectopically deposited on CSR-1 target genes. Moreover, we observe ectopic H3K9me3 at enhancer elements and an increased number of small RNAs that match enhancers in both drh-3 and csr-1 mutants. Finally, we detect accumulation of H3K27me3 at highly expressed genes in the drh-3(ne4253) mutant, which correlates with their reduced transcription. Our study shows that when abundant RdRP-produced siRNAs are depleted, there is ectopic elevation of noncoding RNAs linked to sites with increased silencing chromatin marks. Moreover, our results suggest that enhancer small RNAs may guide local H3K9 methylation.

Download Full-text

Gene Regulation Mediated by microRNA-Triggered Secondary Small RNAs in Plants

Plants ◽

10.3390/plants8050112 ◽

2019 ◽

Vol 8 (5) ◽

pp. 112 ◽

Cited By ~ 5

Author(s):

Felipe Fenselau de Felippes

Keyword(s):

Gene Regulation ◽

Small Rnas ◽

Target Genes ◽

Regulation Of Gene Expression ◽

Small Interfering Rnas ◽

Expression Of Genes ◽

Secondary Sirnas ◽

In Trans ◽

Production Pattern ◽

In Cis

In plants, proper development and response to abiotic and biotic stimuli requires an orchestrated regulation of gene expression. Small RNAs (sRNAs) are key molecules involved in this process, leading to downregulation of their target genes. Two main classes of sRNAs exist, the small interfering RNAs (siRNAs) and microRNAs (miRNAs). The role of the latter class in plant development and physiology is well known, with many examples of how miRNAs directly impact the expression of genes in cells where they are produced, with dramatic consequences to the life of the plant. However, there is an aspect of miRNA biology that is still poorly understood. In some cases, miRNA targeting can lead to the production of secondary siRNAs from its target. These siRNAs, which display a characteristic phased production pattern, can act in cis, reinforcing the initial silencing signal set by the triggering miRNA, or in trans, affecting genes that are unrelated to the initial target. In this review, the mechanisms and implications of this process in the gene regulation mediated by miRNAs will be discussed. This work will also explore techniques for gene silencing in plants that are based on this unique pathway.

Download Full-text

Fish-Ing for Enhancers in the Heart

International Journal of Molecular Sciences ◽

10.3390/ijms22083914 ◽

2021 ◽

Vol 22 (8) ◽

pp. 3914

Author(s):

Costantino Parisi ◽

Shikha Vashisht ◽

Cecilia Lanny Winata

Keyword(s):

Gene Expression ◽

Heart Development ◽

Target Genes ◽

Regulatory Elements ◽

Model Organisms ◽

Sequence Motifs ◽

Specific Sequence ◽

Control Of Gene Expression ◽

Precise Control

Precise control of gene expression is crucial to ensure proper development and biological functioning of an organism. Enhancers are non-coding DNA elements which play an essential role in regulating gene expression. They contain specific sequence motifs serving as binding sites for transcription factors which interact with the basal transcription machinery at their target genes. Heart development is regulated by intricate gene regulatory network ensuring precise spatiotemporal gene expression program. Mutations affecting enhancers have been shown to result in devastating forms of congenital heart defect. Therefore, identifying enhancers implicated in heart biology and understanding their mechanism is key to improve diagnosis and therapeutic options. Despite their crucial role, enhancers are poorly studied, mainly due to a lack of reliable way to identify them and determine their function. Nevertheless, recent technological advances have allowed rapid progress in enhancer discovery. Model organisms such as the zebrafish have contributed significant insights into the genetics of heart development through enabling functional analyses of genes and their regulatory elements in vivo. Here, we summarize the current state of knowledge on heart enhancers gained through studies in model organisms, discuss various approaches to discover and study their function, and finally suggest methods that could further advance research in this field.

Download Full-text