Artificial Small RNAs Research Articles

Retrotransposon addiction promotes centromere function via epigenetically activated small RNAs

Retrotransposons have invaded eukaryotic centromeres in cycles of repeat expansion and purging, but the function of centromeric retrotransposons has remained unclear. In Arabidopsis, centromeric ATHILA retrotransposons give rise to epigenetically activated short interfering RNAs in mutants in DECREASE IN DNA METHYLATION1 (DDM1). Here we show that mutants that lose both DDM1 and RNA-dependent RNA polymerase have pleiotropic developmental defects and mis-segregate chromosome 5 during mitosis. Fertility and segregation defects are epigenetically inherited with centromere 5, and can be rescued by directing artificial small RNAs to ATHILA5 retrotransposons that interrupt tandem satellite repeats. Epigenetically activated short interfering RNAs promote pericentromeric condensation, chromosome cohesion and chromosome segregation in mitosis. We propose that insertion of ATHILA silences centromeric transcription, while simultaneously making centromere function dependent on retrotransposon small RNAs in the absence of DDM1. Parallels are made with the fission yeast Schizosaccharomyces pombe, where chromosome cohesion depends on RNA interference, and with humans, where chromosome segregation depends on both RNA interference and HELLSDDM1.

Investigating the impact of silencing an RNA-binding protein gene SlRBP1 on tomato photosynthesis through RNA-sequencing analysis

Photosynthesis in plants directly affects the synthesis and accumulation of organic matter, which directly influences crop yield. RNA-binding proteins (RBPs) are involved in the regulation of a variety of physiological functions in plants, while the functions of RBPs in photosynthesis have not been clearly elucidated. To investigate the effect of a glycine-rich RNA-binding protein (SlRBP1) in tomato on plant photosynthesis, a stably inherited SlRBP1 silenced plant in Alisa Craig was obtained by plant tissue culture using artificial small RNA interference. It turns out that the size of the tomato fruit was reduced and leaves significantly turned yellow. Chlorophyll(Chl) content measurement, Chl fluorescence imaging and chloroplast transmission electron microscopy revealed that the chloroplast morphology and structure of the leaves of tomato amiR-SlRBP1 silenced plants were disrupted, and the chlorophyll content was significantly reduced. Measurement of photosynthesis rate of wild-type and amiR-SlRBP1 silenced plants in the same period demonstrated that the photosynthetic rate of these plants was significantly reduced, and analysis of RNA-seq data indicated that silencing of SlRBP1 significantly reduced the expression of photosynthesis-related genes, such as PsaE, PsaL, and PsbY, and affected the yield of tomato fruits through photosynthesis.

Exploring and validating key factors limiting cyanobacteria-based CO2 bioconversion: Case study to maximize myo-inositol biosynthesis

Cyanobacteria have been considered as an ideal photosynthetic chassis for CO2 recycle and bioconversion, which could make a significant contribution to carbon neutrality. However, several inherited issues associated with current cyanobacterial strains need to be addressed before their biotechnological application, such as slow growth, uneven of carbon distribution, biased production of reducing power and imbalance between growth and biosynthesis. To address the issues, myo-inositol (MI) was selected as an example target and a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 was utilized as chassis to improve cell growth. Second, several genes involved in MI synthesis were evaluated. Third, the MI biosynthesis was further increased by cofactors enhancement and down regulation of the competing pathways using multiplex artificial small RNAs (PTRNAs). Forth, a MI sensor was developed and optimized to trigger the automatic expression PTRNAs to achieve real-time intelligent regulation, avoiding the growth defect caused by blocking essential genes at early growth stage. Fifth, carbon re-direction was achieved using rhythmical cultivation, resulting in a final production of 262.6 mg/L MI in shaking flask, which represents the highest production from CO2 in cyanobacteria. Finally, the metabolomic analysis was applied to elucidate the shifted metabolic flux and predicted targets for future optimization. Together of all efforts, we can achieve the CO2-based MI biosynthesis to the same order of magnitude as heterogenous microorganisms with suitable scale-up in the future.

Targeting of genomic and negative-sense strands of viral RNA contributes to antiviral resistance mediated by artificial miRNAs and promotes the emergence of complex viral populations.

Technology based on artificial small RNAs, including artificial microRNAs (amiRNAs), exploits natural RNA silencing mechanisms to achieve silencing of endogenous genes or pathogens. This technology has been successfully employed to generate resistance against different eukaryotic viruses. However, information about viral RNA molecules effectively targeted by these small RNAs is rather conflicting, and factors contributing to the selection of virus mutants escaping the antiviral activity of virus‐specific small RNAs have not been studied in detail. In this work, we transformed Nicotiana benthamiana plants with amiRNA constructs designed against the potyvirus plum pox virus (PPV), a positive‐sense RNA virus, and obtained lines highly resistant to PPV infection and others showing partial resistance. These lines have allowed us to verify that amiRNA directed against genomic RNA is more efficient than amiRNA targeting its complementary strand. However, we also provide evidence that the negative‐sense RNA strand is cleaved by the amiRNA‐guided RNA silencing machinery. Our results show that the selection pressure posed by the amiRNA action on both viral RNA strands causes an evolutionary explosion that results in the emergence of a broad range of virus variants, which can further expand in the presence, and even in the absence, of antiviral challenges.

An Artificial Small RNA Editor by Chimeric dsRNase with RNA Binding Protein

RNA plays a vital role in cell functions, but tools to manipulate it is limited. RNA interference (RNAi) is an important approach for biological and clinical applications, but the prone of non-target knockdown effects limited the usage. CRISPR-Cas13 systems recently have been identified for RNA-guided RNA-interfering activity, and can be used in therapeutics, but the large size of Cas13 proteins and the off-targets effect also limit their further usage. Here we report that the chimeric protein containing a double strand nuclease/domain and a structure RNA binding domain (dsRNase-stRBD) with structure guided RNA (sgRNA) can be engineered for mammalian RNA silencing effectively. The RNA knockdown mediated by this method was durable, efficient and stringent without off-target interfering by the sense strand of shRNA base method. Moreover, at size of only 307 aa, allowing dsRNase-stRBD fitting for the versatile scAAV, while the most recent report displays that the smallest Cas13 protein is 775 aa. These results establish sgRNA-dsRBD-RNase as an excellent method for studying RNA function of cells and further clinical application.

Systemic silencing of an endogenous plant gene by two classes of mobile 21‐nucleotide artificial small RNAs

SUMMARYArtificial small RNAs (art‐sRNAs) are 21‐nucleotide small RNAs (sRNAs) computationally designed to silence plant genes or pathogenic RNAs with high efficacy and specificity. They are typically produced in transgenic plants to induce silencing at the whole‐organism level, although their expression in selected tissues for inactivating genes in distal tissues has not been reported. Here, art‐sRNAs designed against the magnesium chelatase subunit CHLI‐encoding SULFUR gene (NbSu) were agroinfiltrated in Nicotiana benthamiana leaves, and the induction of local and systemic silencing was analyzed phenotypically by monitoring the appearance of the characteristic bleached phenotype, as well as molecularly by analyzing art‐sRNA processing, accumulation and targeting activity and efficacy. We found that the two classes of art‐sRNAs, artificial microRNAs (amiRNAs) and synthetic trans‐acting small interfering RNAs (syn‐tasiRNAs), are able to induce systemic silencing of NbSu, which requires high art‐sRNA expression in the vicinity of the leaf petiole but is independent on the production of secondary sRNAs from NbSu mRNAs. Moreover, we revealed that 21‐nucleotide amiRNA and syn‐tasiRNA duplexes, and not their precursors, are the molecules moving between cells and through the phloem to systemically silence NbSu in upper leaves. In sum, our results indicate that 21‐nucleotide art‐sRNAs can move throughout the plant to silence plant genes in tissues different from where they are produced. This highlights the biotechnological potential of art‐sRNAs, which might be applied locally for triggering whole‐plant and highly specific silencing to regulate gene expression or induce resistance against pathogenic RNAs in next‐generation crops. The present study demonstrates that artificial small RNAs, such as artificial microRNAs and synthetic trans‐acting small interfering RNAs, can move long distances in plants as 21‐nucleotide duplexes, specifically silencing endogenous genes in tissues different from where they are applied. This highlights the biotechnological potential of artificial small RNAs, which might be applied locally for triggering whole‐plant, highly specific silencing to regulate gene expression or induce resistance against pathogenic RNAs in next‐generation crops.

RNAi tools for controlling viroid diseases

Viroids are small (250–400 nucleotides), single-stranded, circular RNAs without protein-coding capacity that infect a large number of ornamental and crop plant species, causing high economic losses worldwide. Strategies to control viroid diseases have included the use of naturally resistant cultivars in breeding programs, the superinfection exclusion with mild strains, the expression of ribonucleases, sense or antisense (catalytic) RNAs and, more recently, RNA interference (RNAi)-based tools. Here, I review the different RNAi strategies used to control viroid infections in plants, with particular focus on highly specific artificial small RNA (art-sRNA)-based tools such as artificial microRNAs and synthetic trans-acting small interfering RNAs. The advantages and future perspectives of art-sRNA-based RNAi for controlling viroid diseases are discussed.

Rational design of self-assembled RNA nanostructures for HIV-1 virus assembly blockade.

Many pathological processes are driven by RNA-protein interactions, making such interactions promising targets for molecular interventions. HIV-1 assembly is one such process, in which the viral genomic RNA interacts with the viral Gag protein and serves as a scaffold to drive Gag multimerization that ultimately leads to formation of a virus particle. Here, we develop self-assembled RNA nanostructures that can inhibit HIV-1 virus assembly, achieved through hybridization of multiple artificial small RNAs with a stem–loop structure (STL) that we identify as a prominent ligand of Gag that can inhibit virus particle production via STL-Gag interactions. The resulting STL-decorated nanostructures (double and triple stem–loop structures denoted as Dumbbell and Tribell, respectively) can elicit more pronounced viral blockade than their building blocks, with the inhibition arising as a result of nanostructures interfering with Gag multimerization. These findings could open up new avenues for RNA-based therapy.

An Effective Method for Specific Gene Silencing in Escherichia coli Using Artificial Small RNA.

Knockdown or silencing of a specific gene presents a powerful strategy for elucidating gene function in a variety of organisms. To date, efficient silencing methods have been established in eukaryotes, but not bacteria. In this chapter, an efficient and versatile gene silencing method using artificial small RNA (afsRNA) is described. For this purpose, target-recognizing sequences were introduced in specially designed RNA scaffolds to exist as single-stranded stretches in afsRNA. The translation initiation region of target genes was used as the sequence for afsRNA recognition, based on the theory that this site is usually highly accessible to ribosomes, and therefore, possibly, afsRNA. Two genes transcribed as monocistrons were tested with our protocol. Both genes were effectively silenced by their cognate afsRNAs.

Designing and Constructing Artificial Small RNAs for Gene Regulation and Carbon Flux Redirection in Photosynthetic Cyanobacteria.

Photosynthetic cyanobacteria are not only model organisms for studying photosynthesis and biological cycling of carbon in biosphere but also potential "green microbial factories" to produce renewable fuels and chemicals, due to their capability to utilizing solar energy and CO2. Therefore, strategies for gene regulation and carbon flux redirection are important for both fundamental research and metabolic engineering of cyanobacteria. To address the challenges, regulatory tools based on artificial small RNAs have been developed with satisfactory effects for single or multiple gene(s) regulation in various cyanobacterial species. When combined with the promoters of varying gradient strength and the inducible switches developed in recent years, it is now feasible to realize precise gene regulation in photosynthetic cyanobacteria for producing fuels and chemicals. Here in this chapter, we provide a detailed introduction of the design principles and constructing methods of the artificial sRNA tools to achieve accurate inducible regulation of cyanobacterial gene(s).

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

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.

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

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.

Design and High-Throughput Generation of Artificial Small RNA Constructs for Plants.

Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are two classes of 21-nucleotide artificial small RNAs (sRNAs) designed to selectively silence transcripts in plants with high efficacy and specificity. Despite their extensive use during the last decade, methods for designing and generating artificial sRNA constructs have not been optimized for time- and cost-effectiveness and high-throughput applicability since recently. In this chapter, I detail the protocols for both the rationale design and high-throughput generation of plant artificial sRNA constructs using the P-SAMS ("Plant Small RNA Maker Suite") web tool and a new generation of BsaI/ccdB (B/c) vectors optimized for one-step cloning of artificial sRNA inserts. These protocols allow for the efficient generation of large number of amiRNA and syn-tasiRNA constructs for potent, selective, and specific gene silencing in plants.

Design, Synthesis, and Functional Analysis of Highly Specific Artificial Small RNAs with Antiviral Activity in Plants.

Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are two classes of artificial small RNAs (sRNAs) that have been broadly used to confer antiviral resistance in plants. However, methods for designing, synthesizing and functionally analyzing antiviral artificial sRNAs have not been optimized for time and cost-effectiveness and high-throughput applicability since recently. Here we present a systematic methodology for the simple and fast-forward design, generation, and functional analysis of large numbers of artificial sRNA constructs engineered to induce high levels of antiviral resistance in plants. Artificial sRNA constructs are transiently expressed in Nicotiana benthamiana plants, which are subsequently inoculated with the virus of interest. The antiviral activity of each artificial sRNA construct is assessed by monitoring viral symptom appearance, and through molecular analysis of virus accumulation in plant tissues. This approach is aimed to easily identify artificial sRNAs with high antiviral activity that could be expressed in transgenic plants for highly durable antiviral resistance.

Fast-Forward Identification of Highly Effective Artificial Small RNAs Against Different Tomato spotted wilt virus Isolates.

Artificial small RNAs (sRNAs), including artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs), are used to silence viral RNAs and confer antiviral resistance in plants. Here, the combined use of recent high-throughput methods for generating artificial sRNA constructs and the Tomato spotted wilt virus (TSWV)-Nicotiana benthamiana pathosystem allowed for the simple and rapid identification of amiRNAs with high anti-TSWV activity. A comparative analysis between the most effective amiRNA construct and a syn-tasiRNA construct including the four most effective amiRNA sequences showed that both were highly effective against two different TSWV isolates. These results highlight the usefulness of this high-throughput methodology for the fast-forward identification of artificial sRNAs with high antiviral activity prior to time-consuming generation of stably transformed plants.

Re-direction of carbon flux to key precursor malonyl-CoA via artificial small RNAs in photosynthetic Synechocystis sp. PCC 6803

BackgroundPhotosynthetic cyanobacteria have attracted a significant attention as promising chassis to produce renewable fuels and chemicals due to their capability to utilizing solar energy and CO2. Notably, the enhancing supply of key precursors like malonyl-CoA would benefit the production of many bio-compounds. Nevertheless, the lacking of genetic tools in cyanobacteria, especially the knockdown strategies for essential pathways, has seriously restricted the attempts to re-direct carbon flux from the central carbohydrate metabolism to the synthesis of bioproducts.ResultsAiming at developing new genetic tools, two small RNA regulatory tools are reported for the model cyanobacterium Synechocystis sp. PCC6803, based on paired termini RNAs as well as the exogenous Hfq chaperone and MicC scaffold (Hfq-MicC) previously developed in Escherichia coli. Both regulatory tools functioned well in regulating exogenous reporter gene lacZ and endogenous glgC gene in Synechocystis sp. PCC6803, achieving a downregulation of gene expression up to 90% compared with wildtype. In addition, the Hfq-MicC tool was developed to simultaneously regulate multiple genes related to essential fatty acids biosynthesis, which led to decreased fatty acids content by 11%. Furthermore, aiming to re-direct the carbon flux, the Hfq-MicC tool was utilized to interfere the competing pathway of malonyl-CoA, achieving an increased intracellular malonyl-CoA abundance up to 41% (~ 698.3 pg/mL/OD730 nm) compared to the wildtype. Finally, the Hfq-MicC system was further modified into an inducible system based on the theophylline-inducible riboswitch.ConclusionsIn this study, two small RNA regulatory tools for manipulating essential metabolic pathways and re-directing carbon flux are reported for Synechocystis sp. PCC6803. The work introduces efficient and valuable metabolic regulatory strategies for photosynthetic cyanobacteria.

Artificial microRNAs and synthetic trans-acting small interfering RNAs interfere with viroid infection.

SummaryArtificial microRNAs (amiRNAs) and synthetic trans‐acting small interfering RNAs (syn‐tasiRNAs) are two classes of artificial small RNAs (sRNAs) engineered to silence endogenous transcripts as well as viral RNAs in plants. Here, we explore the possibility of using amiRNAs and syn‐tasiRNAs to specifically interfere with infections by viroids, small (250–400‐nucleotide) non‐coding circular RNAs with compact secondary structure infecting a wide range of plant species. The combined use of recent high‐throughput methods for artificial sRNA construct generation and the Potato spindle tuber viroid (PSTVd)–Nicotiana benthamiana pathosystem allowed for the simple and time‐effective screening of multiple artificial sRNAs targeting sites distributed along PSTVd RNAs of (+) or (–) polarity. The majority of amiRNAs were highly active in agroinfiltrated leaves when co‐expressed with an infectious PSTVd transcript, as were syn‐tasiRNAs derived from a construct including the five most effective amiRNA sequences. A comparative analysis showed that the effects of the most effective amiRNA and of the syn‐tasiRNAs were similar in agroinfiltrated leaves, as well as in upper non‐agroinfiltrated leaves in which PSTVd accumulation was significantly delayed. These results suggest that amiRNAs and syn‐tasiRNAs can be used effectively to control viroid infections in plants.

Systematic characterization of artificial small RNA-mediated inhibition of Escherichia coli growth

ABSTRACTA new screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli was constructed. In this system, we used a plasmid library to express RNAs of ∼120 nucleotides, each with a random 30-nucleotide sequence that can recognize its target mRNA(s). After approximately 60,000 independent colonies were screened, several plasmids that inhibited bacterial growth were isolated. To understand the inhibitory mechanism, we focused on one sRNA, S-20, that exerted a strong inhibitory effect. A time-course analysis of the proteome of S-20-expressing E. coli and a bioinformatic analysis were used to identify potential S-20 target mRNAs, and suggested that S-20 binds the translation initiation sites of several mRNAs encoding enzymes such as peroxiredoxin (osmC), glycyl-tRNA synthetase α subunit (glyQ), uncharacterized protein ygiM, and tryptophan synthase β chain (trpB). An in vitro translation analysis of chimeric luciferase-encoding mRNAs, each containing a potential S-20 target sequence, indicated that the translation of these mRNAs was inhibited in the presence of S-20. A gel shift analysis combined with the analysis of a series of S-20 mutants suggested that S-20 targets multiple mRNAs that are responsible for inhibiting E. coli growth. These data also suggest that S-20 acts like an endogenous sRNA and that E. coli can utilize artificial sRNAs.

Natural and artificial small RNAs: a promising avenue of nucleic acid therapeutics for cancer.

Since the failure of traditional therapy, gene therapy using functional DNA sequence and small RNA/DNA molecules (oligonucleotide) has become a promising avenue for cancer treatment. The discovery of RNA molecules has impelled researchers to investigate small regulatory RNA from various natural and artificial sources and determine a cogent target for controlling tumor progression. Small regulatory RNAs are used for therapeutic silencing of oncogenes and aberrant DNA repair response genes. Despite their advantages, therapies based on small RNAs exhibit limitations in terms of stability of therapeutic drugs, precision-based delivery in tissues, precision-based intercellular and intracellular targeting, and tumor heterogeneity-based responses. In this study, we summarize the potential and drawbacks of small RNAs in nucleic acid therapeutics for cancer.

Visualized and precise design of artificial small RNAs for regulating T7 RNA polymerase and enhancing recombinant protein folding in Escherichia coli

Small non-coding RNAs (sRNAs) have received much attention in recent years due to their unique biological properties, which can efficiently and specifically tune target gene expressions in bacteria. Inspired by natural sRNAs, recent works have proposed the use of artificial sRNAs (asRNAs) as genetic tools to regulate desired gene that has been applied in several fields, such as metabolic engineering and bacterial physiology studies. However, the rational design of asRNAs is still a challenge. In this study, we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs. T7 expression system was one of the most useful recombinant protein expression systems. However, it was deeply limited by the formation of inclusion body. To settle this problem, we designed a series of asRNAs to inhibit the T7 RNA polymerase (Gene1) expression to balance the rate between transcription and folding of recombinant protein. Based on the heterologous expression of Aspergillus oryzae Li-3 glucuronidase in E. coli, the asRNA-antigene1-17bp can effectively decrease the inclusion body and increase the enzyme activity by 169.9%.