RNA-mediated Interference Research Articles

A pipeline for validation of Serendipita indica effector-like sRNA suggests cross-kingdom communication in the symbiosis with Arabidopsis.

Bidirectional communication between pathogenic microbes and their plant hosts via small (s)RNA-mediated cross-kingdom RNA interference (ckRNAi) is a key element for successful host colonisation. Whether mutualistic fungi of the Serendipitaceae family, known for their extremely broad host range, use sRNAs to colonize plant roots is still under debate. To address this question, we developed a pipeline to validate the accumulation, translocation, and activity of fungal sRNAs in post-transcriptional silencing of Arabidopsis thaliana genes. Using stem-loop RT-qPCR, we detected the expression of a specific set of Serendipita indica (Si)sRNAs, targeting host genes involved in cell wall organization, hormonal signalling regulation, immunity, and gene regulation. To confirm the gene silencing activity of these sRNAs in plant cells, SisRNAs were transiently expressed in protoplasts. Stem-loop PCR confirmed sRNAs expression and accumulation, while qPCR validated post-transcriptional gene silencing of their predicted target genes. Furthermore, Arabidopsis ARGONAUTE 1 immunoprecipitation (AtAGO1-IP) revealed the loading of fungal SisRNAs into the plant RNAi machinery, suggesting the translocation of SisRNA from the fungus into root cells. In conclusion, this study provides a blueprint for rapid selection and analysis of sRNA effectors and further supports the model of cross-kingdom communication in the Sebacinoid symbiosis.

CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.

RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.

Gene-Silencing Therapeutic Approaches Targeting PI3K/Akt/mTOR Signaling in Degenerative Intervertebral Disk Cells: An In Vitro Comparative Study Between RNA Interference and CRISPR-Cas9.

The mammalian target of rapamycin (mTOR), a serine/threonine kinase, promotes cell growth and inhibits autophagy. The following two complexes contain mTOR: mTORC1 with the regulatory associated protein of mTOR (RAPTOR) and mTORC2 with the rapamycin-insensitive companion of mTOR (RICTOR). The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway is important in the intervertebral disk, which is the largest avascular, hypoxic, low-nutrient organ in the body. To examine gene-silencing therapeutic approaches targeting PI3K/Akt/mTOR signaling in degenerative disk cells, an in vitro comparative study was designed between small interfering RNA (siRNA)-mediated RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene editing. Surgically obtained human disk nucleus pulposus cells were transfected with a siRNA or CRISPR-Cas9 plasmid targeting mTOR, RAPTOR, or RICTOR. Both of the approaches specifically suppressed target protein expression; however, the 24-h transfection efficiency differed by 53.8-60.3% for RNAi and 88.1-89.3% for CRISPR-Cas9 (p < 0.0001). Targeting mTOR, RAPTOR, and RICTOR all induced autophagy and inhibited apoptosis, senescence, pyroptosis, and matrix catabolism, with the most prominent effects observed with RAPTOR CRISPR-Cas9. In the time-course analysis, the 168-h suppression ratio of RAPTOR protein expression was 83.2% by CRISPR-Cas9 but only 8.8% by RNAi. While RNAi facilitates transient gene knockdown, CRISPR-Cas9 provides extensive gene knockout. Our findings suggest that RAPTOR/mTORC1 is a potential therapeutic target for degenerative disk disease.

Corneal strain influences keratocyte proliferation and migration through upregulation of ALDH3A1 expression.

Keratocytes are the primary resident cells in the corneal stroma. They play an essential role in maintaining corneal physiological function. Studying the factors that affect the phenotype and behavior of keratocytes offers meaningful perspectives for improving the understanding and treatment of corneal injuries. In this study, 3% strain was applied to human keratocytes using the Flexcell® Tension Systems. Real-time quantitative PCR (RT-qPCR) and western blot were used to investigate the influence of strain on the expression of intracellular aldehyde dehydrogenase 3A1 (ALDH3A1). ALDH3A1 knockdown was achieved using double-stranded RNA-mediated interference (RNAi). Immunofluorescence (IF) staining was employed to observe the impact of changes in ALDH3A1 expression on nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nuclear translocation. Keratocyte proliferation and migration were assessed by bromodeoxyuridine (BrdU) assay and scratch wound healing assay, respectively. Mouse injury models and single-cell RNA sequencing of keratocytes from keratoconus patients were used to assess how strain influenced ALDH3A1 invivo. Our results demonstrate that 3% strain suppresses keratocyte proliferation and increases ALDH3A1. Increased ALDH3A1 inhibits NF-κB nuclear translocation, a key step in the activation of the NF-κB signaling pathway. Conversely, ALDH3A1 knockdown promotes NF-κB nuclear translocation, ultimately enhancing keratocyte proliferation and migration. Elevated ALDH3A1 levels were also observed in mouse injury models with increased corneal strain and keratoconus patients. These findings provide valuable insights for further research into the role of corneal strain and its connection to corneal injury repair.

The biogenesis, regulation and functions of transitive siRNA in plants.

Small RNA (sRNA)-mediated RNA interference (RNAi) is a sequence-specific gene silencing mechanism that modulates gene expression in eukaryotes. As core molecules of RNAi, various sRNAs are encoded in the plant genome or derived from invading RNA molecules, and their biogenesis depends on distinct genetic pathways. Transitive small interfering RNAs (siRNAs), which are sRNAs produced from double-strand RNA (dsRNA) in a process that depends on RNA-dependent RNA polymerases (RDRs), can amplify and spread silencing signals to additional transcripts, thereby enabling a phenomenon termed "transitive RNAi". Members of this class of siRNAs function in various biological processes ranging from development to stress adaptation. In Arabidopsis thaliana, two RDRs participate in the generation of transitive siRNAs, acting cooperatively with various siRNA generation-related factors, such as the RNA-induced silencing complex (RISC) and aberrant RNAs. Transitive siRNAs are produced in diverse subcellular locations and structures under the control of various mechanisms, highlighting the intricacies of their biogenesis and functions. In this review, we discuss recent advances in understanding the molecular events of transitive siRNA biogenesis and its regulation, with a particular focus on factors involved in RDR recruitment. We aim to provide a comprehensive description of the generalized mechanism governing the biogenesis of transitive siRNAs. Additionally, we present an overview of the diverse biological functions of these siRNAs and raise some pressing questions in this area for further investigation.

Tau is required for glial lipid droplet formation and resistance to neuronal oxidative stress.

The accumulation of reactive oxygen species (ROS) is a common feature of tauopathies, defined by Tau accumulations in neurons and glia. High ROS in neurons causes lipid production and the export of toxic peroxidated lipids (LPOs). Glia uptake these LPOs and incorporate them into lipid droplets (LDs) for storage and catabolism. We found that overexpressing Tau in glia disrupts LDs in flies and rat neuron-astrocyte co-cultures, sensitizing the glia to toxic, neuronal LPOs. Using a new fly tau loss-of-function allele and RNA-mediated interference, we found that endogenous Tau is required for glial LD formation and protection against neuronal LPOs. Similarly, endogenous Tau is required in rat astrocytes and human oligodendrocyte-like cells for LD formation and the breakdown of LPOs. Behaviorally, flies lacking glial Tau have decreased lifespans and motor defects that are rescuable by administering the antioxidant N-acetylcysteine amide. Overall, this work provides insights into the important role that Tau has in glia to mitigate ROS in the brain.

Ectopic MYBL2-Mediated Regulation of Androglobin Gene Expression.

Androglobin (ADGB) is a highly conserved and recently identified member of the globin superfamily. Although previous studies revealed a link to ciliogenesis and an involvement in murine spermatogenesis, its physiological function remains mostly unknown. Apart from FOXJ1-dependent regulation, the transcriptional landscape of the ADGB gene remains unexplored. We, therefore, aimed to obtain further insights into regulatory mechanisms governing ADGB expression. To this end, changes in ADGB promoter activity were examined using luciferase reporter gene assays in the presence of a set of more than 475 different exogenous transcription factors. MYBL2 and PITX2 resulted in the most pronounced increase in ADGB promoter-dependent luciferase activity. Subsequent truncation strategies of the ADGB promoter fragment narrowed down the potential MYBL2 and PITX2 binding sites within the proximal ADGB promoter. Furthermore, MYBL2 binding sites on the ADGB promoter were further validated via a guide RNA-mediated interference strategy using reporter assays. Chromatin immunoprecipitation (ChIP)-qPCR experiments illustrated enrichment of the endogenous ADGB promoter region upon MYBL2 and PITX2 overexpression. Consistently, ectopic MYBL2 expression induced endogenous ADGB mRNA levels. Collectively, our data indicate that ADGB is strongly regulated at the transcriptional level and might have functions beyond ciliogenesis.

Structural insights into double-stranded RNA recognition and transport by SID-1.

RNA uptake by cells is critical for RNA-mediated gene interference (RNAi) and RNA-based therapeutics. In Caenorhabditis elegans, RNAi is systemic as a result of SID-1-mediated double-stranded RNA (dsRNA) across cells. Despite the functional importance, the underlying mechanisms of dsRNA internalization by SID-1 remain elusive. Here we describe cryogenic electron microscopy structures of SID-1, SID-1-dsRNA complex and human SID-1 homologs SIDT1 and SIDT2, elucidating the structural basis of dsRNA recognition and import by SID-1. The homodimeric SID-1 homologs share conserved architecture, but only SID-1 possesses the molecular determinants within its extracellular domains for distinguishing dsRNA from single-stranded RNA and DNA. We show that the removal of the long intracellular loop between transmembrane helix 1 and 2 attenuates dsRNA uptake and systemic RNAi in vivo, suggesting a possible endocytic mechanism of SID-1-mediated dsRNA internalization. Our study provides mechanistic insights into dsRNA internalization by SID-1, which may facilitate the development of dsRNA applications based on SID-1.

Host Cell-dependent Modulatory Role of Ras Homolog Enriched in Brain-Like-1 (RhebL1) Protein in Influenza A/NWS/33 Virus-infected Mammalian Cells.

The Mammalian Target of Rapamycin (mTOR) signaling pathway regulates protein phosphorylation and exerts control over major cellular processes. mTOR is activated by the small G-protein Ras Homolog Enriched in Brain (Rheb), which is encoded by the Rheb1 and Rheb-like-1 (RhebL1) genes. There is currently a paucity of information on the role of RhebL1, and specifically its involvement in viral infection. In the present study we investigated the role of RhebL1 during human influenza A/NWS/33 (NWS/33) (H1N1) virus infection of rhesus monkey-kidney (LLC-MK2) cells and human type II alveolar epithelial (A549) cells. To assess the efficiency of NWS/33 virus replication, the expression of viral nucleoprotein was examined by indirect immunofluorescence (IIF) and the viral yield by fifty percent tissue culture infectious dose assay. An RNA-mediated RNA interference approach was used to investigate the role of RhebL1 during NWS/33 infection. RhebL1 expression was evaluated by IIF, Western blotting, and enzyme-linked immunosorbent assays. A two-tailed Student's t-test was applied to evaluate differences between groups. RhebL1 was differentially expressed in the cell models used in this study. Silencing of the RhebL1 gene led to increased NWS/33 virus infection in A549 cells, but not in LLC-MK2 cells. Moreover, the expression of hyperphosphorylated cytokeratin 8, a marker of NWS/33 virus infection efficiency, increased in A549 cells depleted of RhebL1 but remained almost unchanged in LLC-MK2 cells. These are the first results showing involvement of the endogenous RhebL1 protein during viral infection. Our data suggests that RhebL1 exerts a host cell-dependent modulatory role during influenza virus infection. RhebL1 appears to be a restrictive factor against NWS/33 virus replication in A549 cells, but not in LLC-MK2.

Collagen and actin network mediate antiviral immunity against Orsay virus in C. elegans intestinal cells.

C. elegans is a free-living nematode that is widely used as a small animal model for studying fundamental biological processes and disease mechanisms. Since the discovery of the Orsay virus in 2011, C. elegans also holds the promise of dissecting virus-host interaction networks and innate antiviral immunity pathways in an intact animal. Orsay virus primarily targets the worm intestine, causing enlarged intestinal lumen as well as visible changes to infected cells such as liquefaction of cytoplasm and convoluted apical border. Previous studies of Orsay virus identified that C. elegans is able to mount antiviral responses by DRH-1/RIG-I mediated RNA interference and Intracellular Pathogen Response, a uridylyltransferase that destabilizes viral RNAs by 3' end uridylation, and ubiquitin protein modifications and turnover. To comprehensively search for novel antiviral pathways in C. elegans, we performed genome-wide RNAi screens by bacterial feeding using existing bacterial RNAi libraries covering 94% of the entire genome. Out of the 106 potential antiviral gene hits identified, we investigated those in three new pathways: collagens, actin remodelers, and epigenetic regulators. By characterizing Orsay virus infection in RNAi and mutant worms, our results indicate that collagens likely form a physical barrier in intestine cells to inhibit viral infection by preventing Orsay virus entry. Furthermore, evidence suggests that actin remodeling proteins (unc-34, wve-1 and wsp-1) and chromatin remodelers (nurf-1 and isw-1) exert their antiviral activities by regulating the intestinal actin (act-5), a critical component of the terminal web which likely function as another physical barrier to prevent Orsay infection.

A genetic screen identifies C. elegans eif-3.H and hrpr-1 as pro-apoptotic genes and potential activators of egl-1 expression.

During C. elegans development, 1090 somatic cells are generated of which 131 reproducibly die, many through apoptosis. The C. elegans BH3-only gene egl-1 is the key activator of apoptosis in somatic tissues, and it is predominantly expressed in 'cell death' lineages i.e. lineages in which apoptotic cell death occurs. egl-1 expression is regulated at the transcriptional and post-transcriptional level. For example, we previously showed that the miR-35 and miR-58 families of miRNAs repress egl-1 expression in mothers of 'unwanted' cells by binding to the 3' UTR of egl-1 mRNA, thereby increasing egl-1 mRNA turnover. In a screen for RNA-binding proteins with a role in the post-transcriptional control of egl-1 expression, we identified EIF-3.H (ortholog of human eIF3H) and HRPR-1 (ortholog human hnRNP R/Q) as potential activators of egl-1 expression. In addition, we demonstrate that the knockdown of the eif-3.H or hrpr-1 gene by RNA-mediated interference (RNAi) results in the inappropriate survival of unwanted cells during C. elegans development. Our study provides novel insight into how egl-1 expression is controlled to cause the reproducible pattern of cell death observed during C. elegans development.

Evolutionary dynamics of whole-body regeneration across planarian flatworms

Regenerative abilities vary dramatically across animals. Even amongst planarian flatworms, well-known for complete regeneration from tiny body fragments, some species have restricted regeneration abilities while others are almost entirely regeneration incompetent. Here, we assemble a diverse live collection of 40 planarian species to probe the evolution of head regeneration in the group. Combining quantification of species-specific head-regeneration abilities with a comprehensive transcriptome-based phylogeny reconstruction, we show multiple independent transitions between robust whole-body regeneration and restricted regeneration in freshwater species. RNA-mediated genetic interference inhibition of canonical Wnt signalling in RNA-mediated genetic interference-sensitive species bypassed all head-regeneration defects, suggesting that the Wnt pathway is linked to the emergence of planarian regeneration defects. Our finding that Wnt signalling has multiple roles in the reproductive system of the model species Schmidtea mediterranea raises the possibility that a trade-off between egg-laying, asexual reproduction by fission/regeneration and Wnt signalling drives regenerative trait evolution. Although quantitative comparisons of Wnt signalling levels, yolk content and reproductive strategy across our species collection remained inconclusive, they revealed divergent Wnt signalling roles in the reproductive system of planarians. Altogether, our study establishes planarians as a model taxon for comparative regeneration research and presents a framework for the mechanistic evolution of regenerative abilities.

Mitochondrially mediated RNA interference, a retrograde signaling system affecting nuclear gene expression.

Several functional classes of short noncoding RNAs are involved in manifold regulatory processes in eukaryotes, including, among the best characterized, miRNAs. One of the most intriguing regulatory networks in the eukaryotic cell is the mito-nuclear crosstalk: recently, miRNA-like elements of mitochondrial origin, called smithRNAs, were detected in a bivalve species, Ruditapes philippinarum. These RNA molecules originate in the organelle but were shown in vivo to regulate nuclear genes. Since miRNA genes evolve easily de novo with respect to protein-coding genes, in the present work we estimate the probability with which a newly arisen smithRNA finds a suitable target in the nuclear transcriptome. Simulations with transcriptomes of 12 bivalve species suggest that this probability is high and not species specific: one in a hundred million (1 × 10-8) if five mismatches between the smithRNA and the 3' mRNA are allowed, yet many more are allowed in animals. We propose that novel smithRNAs may easily evolve as exaptation of the pre-existing mitochondrial RNAs. In turn, the ability of evolving novel smithRNAs may have played a pivotal role in mito-nuclear interactions during animal evolution, including the intriguing possibility of acting as speciation trigger.

Sprayable double-stranded RNA mediated RNA interference reduced enzymatic browning of fresh-cut potatoes

Postharvest quality deterioration is the major reason for food loss and waste, which hinder global food security, environmental sustainability and human health. Spray-induced gene silencing (SIGS) is a technique involving spraying double-stranded RNA (dsRNA) onto the plant surface for targeted RNA interference, providing a new solution for pests and disease control in agriculture products. Here, fresh-cut browning, which is a common and visible quality deterioration in potatoes, was taken as the indicator for the exploratory SIGS technology in postharvest quality deterioration control. The polyphenol oxidase (PPO) encoding genes StPPO and StPPOD, and the phenylalanine ammonia-lyase (PAL) encoding gene StPAL2, were identified as important transcripts associated with the browning process in freshly-cut potatoes of the Yunshu 505 cultivar. Inhibition of StPPO and StPAL2 using dsRNA-PPO and dsRNA-PALX resulted in a decrease in the expression levels of the two genes, consequently leading to reduced activities of polyphenol oxidase and phenylalanine ammonia-lyase, as well as a decrease in the brown index. Spraying a mixture of these two dsRNAs demonstrated browning control capabilities similar to those of ascorbic acid. The distribution of dsRNA-Cy5 in cells confirmed its uptake and internalization by fresh-cut potatoes. In addition, the application of dsRNA-MYB12 resulted in a reduction of up to 78 % in tuber StMYB12 levels and decreased StPPO and StPAL1 expression. Spraying dsRNAs effectively reduced fresh-cut browning, highlighting the potential application of SIGS in reducing the browning of fresh-cut potatoes.

Synthesis and characterization of novel (S)-5′-C-aminopropyl-2′-fluorouridine modified oligonucleotides as therapeutic siRNAs

The lack of stability of natural nucleosides limits their application in small interfering RNA (siRNA)-mediated RNA interference (RNAi). Various chemical modifications have been reported to improve their pharmacokinetic behavior; however, the development of potential candidates is still underway. In this study, we designed and synthesized (S)-5′-C-aminopropyl-2′-fluorouridine (5′-AP-2′-FU) and evaluated the properties of siRNAs containing this analog. A comparative thermodynamic study revealed the enhanced thermal stability of double-stranded RNAs (dsRNAs) containing 5′-AP-2′-FU in a position-specific manner, whereas (S)-5′-C-aminopropyl-2′-O-methyluridine (5′-AP-2′-MoU)-modified dsRNAs exhibited lower melting temperatures. This improved thermal stability of RNA duplexes is attributed to favorable entropy loss, which induces the duplex into an N-type (C3′-endo) conformation and enhances duplex binding in this case. The 5′-AP-2′-FU analog was also suitable for incorporation into the passenger strand to induce gene-silencing activity. Gene knockdown efficacy was comparable to that of unmodified siRNAs, and the best response was observed by introducing 5′-AP-2′-FU near the 3′-terminal end of the passenger strand. In addition, the single-stranded RNAs (ssRNAs) modified with 5′-AP-2′-FU showed strong resistance against decomposition by nucleases when treated with buffer containing bovine serum, which was similar to 5′-AP-2′-MoU.

Retraction notice to Retrovirally mediated RNA interference targeting the M2 subunit of ribonucleotide reductase (RRM2): a novel therapeutic strategy in pancreatic cancer [Surgery 136 (2004) 261-269

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editors-in-Chief, because an investigation by Harvard Medical School and the Brigham and Women’s Hospital has concluded that Figure 1A is invalid and no underlying research data are available to resolve the discrepancies or validate the reported results. RETRACTED: Retrovirally mediated RNA interference targeting the M2 subunit of ribonucleotide reductase: A novel therapeutic strategy in pancreatic cancerSurgeryVol. 136Issue 2PreviewThis article has been retracted: please see Elsevier Policy on Article Withdrawal ( http://www.elsevier.com/locate/withdrawalpolicy ). Full-Text PDF

In Silico Identification of Cassava Genome-Encoded MicroRNAs with Predicted Potential for Targeting the ICMV-Kerala Begomoviral Pathogen of Cassava.

Cassava mosaic disease (CMD) is caused by several divergent species belonging to the genus Begomovirus (Geminiviridae) transmitted by the whitefly Bemisia tabaci cryptic species group. In India and other parts of Asia, the Indian cassava mosaic virus-Kerala (ICMV-Ker) is an emergent begomovirus of cassava causing damage that results in reduced yield loss and tuber quality. Double-stranded RNA-mediated interference (RNAi) is an evolutionary conserved mechanism in eukaryotes and highly effective, innate defense system to inhibit plant viral replication and/or translation. The objective of this study was to identify and characterize cassava genome-encoded microRNAs (mes-miRNA) that are predicted to target ICMV-Ker ssDNA-encoded mRNAs, based on four in silico algorithms: miRanda, RNA22, Tapirhybrid, and psRNA. The goal is to deploy the predicted miRNAs to trigger RNAi and develop cassava plants with resistance to ICMV-Ker. Experimentally validated mature cassava miRNA sequences (n = 175) were downloaded from the miRBase biological database and aligned with the ICMV-Ker genome. The miRNAs were evaluated for base-pairing with the cassava miRNA seed regions and to complementary binding sites within target viral mRNAs. Among the 175 locus-derived mes-miRNAs evaluated, one cassava miRNA homolog, mes-miR1446a, was identified to have a predicted miRNA target binding site, at position 2053 of the ICMV-Ker genome. To predict whether the cassava miRNA might bind predicted ICMV-Ker mRNA target(s) that could disrupt viral infection of cassava plants, a cassava locus-derived miRNA-mRNA regulatory network was constructed using Circos software. The in silico-predicted cassava locus-derived mes-miRNA-mRNA network corroborated interactions between cassava mature miRNAs and the ICMV-Ker genome that warrant in vivo analysis, which could lead to the development of ICMV-Ker resistant cassava plants.

Blocking Translation of Oncogenic mRNA

Double-stranded RNA-mediated interference (RNAi), antisense oligonucleotides (ASO), and ribozymes have excellent specificity to their target oncogenic mRNA. They also seem to show great promise when it comes to treating cancer. The problem is that RNAi, ASO, and ribozymes have poor stability and are constantly being degraded by nucleases. Researchers have made some efforts to increase antisense oligonucleotides’ stability by creating phospharimidate and Phosphorothioate. Currently, ribozymes, antisense oligonucleotides, and (RNAi) are the three main methods used to target RNA. These methods are currently undergoing clinical trials for the purpose of focusing on specific RNAs involved in disorders like cancer and neurodegeneration. In fact, ASOs that target amyotrophic lateral sclerosis and spinal muscular atrophy have produced promising results in clinical trials. The formation of chemical alterations that boost affinity and selectivity while reducing noxiousness owing to off-target impacts are two benefits of ASOs. Another benefit is increased affinity. With a focus on RNAi and ASOs, this review illustrated the main therapeutic strategies of RNA therapy now in use.

Genome-Wide Identification and Characterization of Argonaute, Dicer-like and RNA-Dependent RNA Polymerase Gene Families and Their Expression Analyses in Fragaria spp.

In the growth and development of plants, some non-coding small RNAs (sRNAs) not only mediate RNA interference at the post-transcriptional level, but also play an important regulatory role in chromatin modification at the transcriptional level. In these processes, the protein factors Argonaute (AGO), Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) play very important roles in the synthesis of sRNAs respectively. Though they have been identified in many plants, the information about these gene families in strawberry was poorly understood. In this study, using a genome-wide analysis and a phylogenetic approach, 13 AGO, six DCL, and nine RDR genes were identified in diploid strawberry Fragaria vesca. We also identified 33 AGO, 18 DCL, and 28 RDR genes in octoploid strawberry Fragaria × ananassa, studied the expression patterns of these genes in various tissues and developmental stages of strawberry, and researched the response of these genes to some hormones, finding that almost all genes respond to the five hormone stresses. This study is the first report of a genome-wide analysis of AGO, DCL, and RDR gene families in Fragaria spp., in which we provide basic genomic information and expression patterns for these genes. Additionally, this study provides a basis for further research on the functions of these genes and some evidence for the evolution between diploid and octoploid strawberries.

Metabolic functional redundancy of the CYP9A subfamily members leads to P450-mediated lambda-cyhalothrin resistance in Cydia pomonella.

The evolution of insect resistance to pesticides poses a continuing threat to sustainable pest management. While much is known about the molecular mechanisms that confer resistance in model insects and few agricultural pests, far less is known about fruit pests. Field-evolved resistance to synthetic insecticides such as lambda-cyhalothrin has been widely documented in Cydia pomonella, a major invasive pest of pome fruit worldwide, and the increased production of cytochrome P450 monooxygenases (P450s) has been linked to resistance in field-evolved resistant populations. However, the underlying molecular mechanisms of P450-mediated insecticide resistance remain largely unknown. Here we found that functional redundancy and preference of metabolism by P450s genes in the CYP9A subfamily confer resistance to lambda-cyhalothrin in Cydia pomonella. A total of four CYP9A genes, including CYP9A61, CYP9A120, CYP9A121, and CYP9A122, were identified from Cydia pomonella. Among these, CYP9A120, CYP9A121, and CYP9A122 were predominantly expressed in the midgut of larvae. The expression levels of these P450 genes were significantly induced by a lethal dose that would kill 10% (LD10 ) of lambda-cyhalothrin and were overexpressed in a field-evolved lambda-cyhalothrin resistant population. Knockdown of CYP9A120 and CYP9A121 by RNA-mediated interference (RNAi) increased the susceptibility of larvae to lambda-cyhalothrin. In vitro assays demonstrated that recombinant P450s expressed in Sf9 cells can metabolize lambda-cyhalothrin, but with functional redundancy and divergence through regioselectivity of metabolism. CYP9A121 preferred to convert lambda-cyhalothrin to 2'-hydroxy-lambda-cyhalothrin, whereas CYP9A122 only generated 4'-hydroxy metabolite of lambda-cyhalothrin. Although possesses a relatively low metabolic capability, CYP9A120 balanced catalytic competence to generate both 2'- and 4'-metabolites. Collectively, these results reveal that metabolic functional redundancy of three members of the CYP9A subfamily leads to P450-mediated lambda-cyhalothrin resistance in Cydia pomonella, thus representing a potential adaptive evolutionary strategy during its worldwide expansion. © 2022 Society of Chemical Industry.