Chemically modified short interfering RNAs (siRNAs) unequivocally represent a groundbreaking class of drugs. The deliberate chemical modification of the natural structure has been pivotal to their resounding success. Specific modifications at certain positions bolster their potency, safety, stability, and specificity. In clinical research, 2'-O-methyl and 2'-fluoro are the most used modifications. The effects of a wide range of chemical changes in fully modified siRNAs have not been thoroughly evaluated for tolerability. In this study, we utilized two sequences in a fully modified siRNA to systematically assess the tolerability of single nucleotide backbone and sugar modifications, including deoxyribonucleic acid, 2'-O-(2-methoxyethyl), locked nucleic acid, unlocked nucleic acid, mismatches, butane diol substitution, and butane diol insertion. We synthesized 522 siRNA variants and evaluated their efficacy in vitro. Our findings demonstrate that individual tolerability is significantly influenced by the modification's sequence, pattern, and position, with limited universal principles identifiable from this dataset. The efficacy results are probably driven by the thermodynamic balance defined by a combination of parameters. The framework presented here will serve as a reference dataset to facilitate the expansion of chemical diversity in therapeutic siRNAs.

Pterygium is a fibrovascular growth associated with chronic inflammation, tissue remodeling, and angiogenesis, which invades the cornea. Circular RNAs (circRNAs) are emerging as pivotal role in many diseases, but their role in pterygium remains unclear. We performed circRNA and miRNA expression profiling on pterygium and conjunctival tissues, then the circRNA-miRNA-mRNA regulatory network was constructed. Bioinformatics was used to predict downstream pathways. Pterygium fibroblasts were used for experiments assessing proliferation (CCK8, EdU), migration (wound healing, transwell), and apoptosis (AnnexinV-FITC/PI). We identified 162 differentially expressed circRNAs and 96 miRNAs. Key pathways involved in pterygium pathogenesis, including focal adhesion and PI3K-Akt signaling, were predicted. Hsa_circ_0081862 was downregulated in pterygium tissues and fibroblasts, inhibiting fibroblast proliferation and migration while promoting apoptosis. This research constructed a ceRNA network and identified hsa_circ_0081682 as the potential diagnostic marker for pterygium. This research contributes to the understanding of biochemical basis of pterygium, which may facilitate the development of targeted strategies for its management and prevention.

MicroRNAs (miRNAs) are conserved, short, non-coding RNAs that play a crucial role in regulating gene expression. Emerging evidence suggests that miRNAs are closely involved in the pathophysiology of various corneal diseases, particularly in regulating corneal wound healing, inflammation and neovascularization. In this review, we summarized the recent progress of miRNAs in corneal diseases, especially focused on their application as diagnostic biomarkers, regulators of cell biology, and therapeutic targets. Recent advances in miRNA detection technology have made it possible to analyze minimal miRNAs in samples such as tears or exosomes, further enhancing the ability to identify disease-specific miRNA profiles and providing potential objective indicators for the early diagnosis of disease. Meanwhile, we summarized the mechanisms and pathways of multiple miRNAs in regulating various biological processes of corneal cells, as well as the advantages of studying miRNA compared to proteins or genes. Furthermore, we explore the potential of miRNAs-based therapies, especially introduce various miRNA delivery systems and challenges associated with clinical translation. This review highlights the need for further research to harness the full potential of miRNAs in treating various corneal diseases.

The functional specificity of proteins is often attributed to their sequence and structural homology while frequently neglecting the underlying conformational dynamics occurring at different time scales that can profoundly impact biological consequences. Using 15N-CEST NMR and RDC-corrected metainference molecular dynamics simulations, here, we reveal differential substrate recognition mechanisms in two dsRNA-binding domain (dsRBD) paralogs, DRB2D1 and DRB3D1. Despite their nearly identical solution structures and conserved dsRNA interaction interfaces, DRB3D1 demonstrates structural plasticity that enables it to recognize conformationally flexible dsRNA, a feature notably absent in the more rigid DRB2D1. We present the pivotal role of intrinsic structural dynamics in driving functional divergence and provide insights into the mechanisms that govern specificity in dsRBD:dsRNA interactions. Importantly, our combined experimental and computational approach captures a cluster of intermediate conformations, complementing conventional methods to resolve the dominant ground state and sparsely populated excited states.

miRNA arm switching is a pivotal regulatory mechanism that allows organisms to fine-tune gene expression by selectively utilizing either the 5p or 3p strand of a miRNA duplex. This process, conserved across species, facilitates adaptive responses to developmental cues, environmental changes, and disease states. By dynamically altering strand selection, arm switching reshapes gene regulatory networks, contributing to phenotypic diversity and evolutionary innovation. Despite its growing recognition, the mechanisms driving arm switching-such as thermodynamic properties and enzyme-mediated processing-remain incompletely understood. This review synthesizes current findings, highlighting arm switching as a highly conserved mechanism with profound implications for the evolution of regulatory networks. We explore how this phenomenon expands miRNA functionality, drives phenotypic plasticity, and co-evolves with miRNA gene duplications to fuel the diversification of biological functions across taxa.

Small non-coding RNAs can be categorized into two main classes: structural RNAs and regulatory RNAs. Structural RNAs, which are abundant and ubiquitously expressed, have essential roles in the maturation of pre-mRNAs, modification of rRNAs and the translation of coding transcripts. By contrast, regulatory RNAs are often expressed in a developmental-specific, tissue-specific or cell-type-specific manner and exert precise control over gene expression. Reductions in cost and improvements in the accuracy of high-throughput RNA sequencing have led to the identification of many new small RNA species. In this Review, we provide a broad discussion of the genomic origins, biogenesis and functions of structural small RNAs, including tRNAs, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), vault RNAs (vtRNAs) and Y RNAs as well as their derived RNA fragments, and of regulatory small RNAs, such as microRNAs (miRNAs), endogenous small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs), in animals.

A phosphate or a phosphate mimic at the 5' terminus of the antisense strand of a small interfering RNA (siRNA) is required for efficient loading into the RISC complex through the MID domain binding pocket of Ago2. Introduction of 5'-E-vinylphosphonate improves this binding and siRNA potency, but the Z isomer does not. Here, we demonstrate that both the E and Z isomers of 6'-vinylphosphonate at the 5' ends of antisense strands of siRNAs have equivalent potencies.

RNA interference (RNAi) is a tool for studying gene function and has emerged as a promising eco-friendly alternative to chemical pesticides. RNAi relies on delivering double-stranded RNA (dsRNA), which is processed into small interfering RNA (siRNA) to silence genes. However, so far, knowledge and tools for optimizing the dsRNA sequences for maximum efficacy are based on human data, which might not be optimal for insect pest control.

Here, we systematically tested different siRNA sequences in the red flour beetle Tribolium castaneum to identify sequence features that correlated with high efficacy using pest control as a study case. Thermodynamic asymmetry, the absence of secondary structures, and adenine at the 10th position in antisense siRNA were most predictive of insecticidal efficacy. Interestingly, we also found that, in contrast to results from human data, high, rather than low, GC content from the 9th to 14th nucleotides of antisense was associated with high efficacy. Consideration of these features for the design of insecticidal dsRNAs targeting essential genes in three insect species improved the efficacy of the treatment. The improvement was associated with a higher ratio of the antisense, rather than sense, siRNA strand bound to the RNA-induced silencing complex. Finally, we developed a web platform named dsRIP, which offers tools for optimizing dsRNA sequences, identifying effective target genes in pests, and minimizing risk to non-target species.

The identified sequence features and the dsRIP web platform allow optimizing dsRNA sequences for application of RNAi for pest control and research.

Osteoarthritis (OA) of the temporomandibular joint (TMJ) is a progressive disease characterized by the progressive destruction of the internal surfaces of the joint. Certain epigenetic biomarkers have been detected in TMJ-OA. We summarized the available evidence on the epigenetic biomarkers in TMJ-OA. There is an increase in the expression of non-coding RNAs related to the degradation of the extracellular matrix, chondrocyte apoptosis, and proinflammatory cytokines, while there is a decrease in the expression of those related to COL2A1, as well as the osteogenic and chondrogenic differentiation of mesenchymal stem cells. Certain methylated genes and histone modifications in TMJ-OA were also identified. In the early stage, DNA methylation was significantly decreased; that is, the expression of inflammation-related genes such as TNF and genes associated with extracellular matrix degradation, such as Adamts, were increased. While in the late stage, there was an increase in the expression of genes associated with the TGF-β and MAPK signaling pathway and angiogenesis-related genes. Although research on the role of epigenetic markers in TMJ-OA is still ongoing, the results here contribute to improving the basis for the identification of accurate diagnostic and prognostic markers and the development of new therapeutic molecules for the prevention and management of TMJ-OA. It also represents a significant advancement in elucidating its pathogenesis.

Ovarian cancer (OC) is one of the most common cancers in women. Biomarkers for OC are still being sought. The aim of this review was to evaluate microRNAs in the prognosis and diagnosis of OC. We conducted a literature review searching for articles published from January 2014 to September 2024. We included articles presenting the association of microRNAs with ovarian cancer prognosis, where patient survival was shown by the Kaplan-Meier curve, and articles presenting the association of microRNAs with ovarian cancer diagnosis, where the results were presented as an ROC curve. MicroRNAs are promising clinical markers in ovarian cancer patients. As is shown here, expression (high or low) of various miRNAs was differentially associated with survival in OC patients, with some miRNAs being associated with a longer survival and some with a shorter survival. In the absence of diagnostic markers for OC, the raised role of miRNAs in diagnosis seems all the more important. The diagnostic value of miRNAs has been shown, mostly as blood biomarkers, although they have also been evaluated as tissue or urine markers. MiRNAs have an important role as clinical biomarkers for ovarian cancer, not only as single molecules, but also as biomarker pairs or panels of miRNAs. It should be noted that most of the miRNAs reviewed here have been studied once, so despite the promising results, it seems necessary to conduct studies to confirm or negate the results obtained.

MicroRNAs (miRNAs) are small, yet profoundly influential, non-coding RNAs that base-pair with mRNAs to induce RNA silencing. Although the basic principles of miRNA biogenesis and function have been established, recent breakthroughs have yielded important new insights into the molecular mechanisms of miRNA biogenesis. In this Review, we discuss the metazoan miRNA biogenesis pathway step-by-step, focusing on the key biogenesis machinery, including the Drosha-DGCR8 complex (Microprocessor), exportin-5, Dicer and Argonaute. We also highlight newly identified cis-acting elements and their impact on miRNA maturation, informed by advanced high-throughput and structural studies, and discuss recently discovered mechanisms of clustered miRNA processing, target recognition and target-directed miRNA decay (TDMD). Lastly, we explore multiple regulatory layers of miRNA biogenesis, mediated by RNA-protein interactions, miRNA tailing (uridylation or adenylation) and RNA modifications.

Double-stranded RNA (dsRNA) biopesticides offer the potential for highly targeted pest control with minimal off-target impacts. Varroa mites (Varroa destructor) are an important pest of honey bees (Apis mellifera) that are primarily managed by synthetic pesticides. A next-generation treatment using a varroa-active dsRNA, vadescana, has been developed to target calmodulin expression in varroa. We evaluated the potential exposure of non-target species to vadescana. First, we assessed potential gene silencing effects on 39 arthropods with known genomes via bioinformatics. Three mite species, monarch butterflies (Danaus plexippus), fruit flies (Drosophila melanogaster), and European earwigs (Forficula auricularia) showed theoretical potential for off-target effects. These in silico results could be used to help inform risk assessments. Second, we conducted vadescana feeding trials on the greater wax moth (Galleria mellonella), a common beehive associate. There were no significant differences in wax moth reproduction, survival, or adult F2 wing length between vadescana-fed and control groups. Male F2 body weight was slightly but significantly lower in wax moths exposed to the highest vadescana dose, with no such effect observed in female moths. Calmodulin gene expression was unaffected in wax moths. Our hazard assessment of vadescana's lethal and sublethal effects on wax moths indicates minimal impact following continuous dietary exposure far greater than any exposure that might be expected in the field, in line with the bioinformatics findings. This biopesticide appears highly varroa-specific and likely has fewer non-target effects than many current varroa control methods.

The ability of small nucleic acids to modulate gene expression via a range of processes has been widely explored. Compared with conventional treatments, small nucleic acid therapeutics have the potential to achieve long-lasting or even curative effects via gene editing. As a result of recent technological advances, efficient small nucleic acid delivery for therapeutic and biomedical applications has been achieved, accelerating their clinical translation. Here, we review the increasing number of small nucleic acid therapeutic classes and the most common chemical modifications and delivery platforms. We also discuss the key advances in the design, development and therapeutic application of each delivery platform. Furthermore, this review presents comprehensive profiles of currently approved small nucleic acid drugs, including 11 antisense oligonucleotides (ASOs), 2 aptamers and 6 siRNA drugs, summarizing their modifications, disease-specific mechanisms of action and delivery strategies. Other candidates whose clinical trial status has been recorded and updated are also discussed. We also consider strategic issues such as important safety considerations, novel vectors and hurdles for translating academic breakthroughs to the clinic. Small nucleic acid therapeutics have produced favorable results in clinical trials and have the potential to address previously "undruggable" targets, suggesting that they could be useful for guiding the development of additional clinical candidates.

RNA interference (RNAi)-based control technologies are gaining popularity as potential alternatives to synthetic fungicides in the ongoing effort to manage plant pathogenic fungi. Among these methods, spray-induced gene silencing (SIGS) emerges as particularly promising due to its convenience and feasibility for development. This approach is a new technology for plant disease management, in which double-stranded RNAs (dsRNAs) targeting essential or virulence genes are applied to plants or plant products and subsequently absorbed by plant pathogens, triggering a gene silencing effect and the inhibition of the infection process. Spray-induced gene silencing has demonstrated efficacy in laboratory settings against various fungal pathogens. However, as research progressed from the laboratory to the greenhouse and field environments, novel challenges arose, such as ensuring the stability of dsRNAs and their effective delivery to fungal targets. Here, we provide a practical guide to SIGS for the control of plant pathogenic fungi. This guide outlines the essential steps and considerations needed for designing and assessing dsRNA molecules. It also addresses key challenges inherent to SIGS, including delivery and stability of dsRNA molecules, and how nanoencapsulation of dsRNAs can aid in overcoming these obstacles. Additionally, the guide underscores existing knowledge gaps that warrant further research and aims to provide assistance to researchers, especially those new to the field, encouraging the advancement of SIGS for the control of a broad range of fungal pathogens.

Culex species mosquitoes are globally distributed and transmit several pathogens that impact animal and public health, including West Nile virus, Usutu virus, and Plasmodium relictum. Despite their relevance, Culex species are less widely studied than Aedes and Anopheles mosquitoes. To expand the genetic tools used to study Culex mosquitoes, we previously developed an optimized plasmid for transient Cas9 and single-guide RNA (sgRNA) expression in Culex quinquefasciatus cells to generate gene knockouts. Here, we established a monoclonal cell line that consistently expresses Cas9 and can be used for screens to determine gene function or antiviral activity.

We used this system to perform the successful gene editing of seven genes and subsequent testing for potential antiviral effects, using a simple single-guide RNA (sgRNA) transfection and subsequent virus infection.

We were able to show antiviral effects for the Cx. quinquefasciatus genes dicer-2, argonaute-2b, vago, piwi5, piwi6a, and cullin4a. In comparison to the RNAi-mediated gene silencing of dicer-2, argonaute-2b, and piwi5, our Cas9/sgRNA approach showed an enhanced ability to detect antiviral effects.

We propose that this cell line offers a new tool for studying gene function in Cx. quinquefasciatus mosquitoes that avoids the use of RNAi. This short study also serves as a proof-of-concept for future gene knock-ins in these cells. Our cell line expands the molecular resources available for vector competence research and will support the design of future research strategies to reduce the transmission of mosquito-borne diseases.

Mirtrons are a predominant class of noncanonical microRNAs derived from introns through a Drosha-independent, splicing-dependent pathway. Unregulated splicing of introns containing hairpins may adversely impact Dicer/Ago-mediated canonical microRNA biogenesis. However, the mechanism regulating mirtron biogenesis remains poorly understood. We found that the 5' arm of plant mirtrons and invertebrate mirtrons are enriched for uracils; in contrast, the 5' arm of vertebrate mirtrons are enriched for guanines. Further analysis revealed that most of the mammalian mirtrons contain an RNA G-quadruplex (rG4); this was not observed among plant/invertebrate mirtrons. Interestingly, almost all the rG4s in mammalian mirtrons were present in the 5' arm. Predicted rG4s in human mirtrons form a G-quadruplex structure in vitro and rG4 formation in the 5' arm of mirtrons facilitates splicing-mediated biogenesis of mirtrons. Notably, the disruption of rG4s in the 5' arm of mirtrons inhibits splicing and maturation; while mutations outside the rG4-motif do not impact mirtron biogenesis. Our findings support the notion that rG4s at the 5' arm are key regulatory elements in the evolutionary landscape of mammalian mirtrons. This work advances our current understanding of mirtron biogenesis and highlights additional roles for rG4s in small RNA biology.

Senile sarcopenia is a condition of age-associated muscular disorder and is a significant health issue around the world. In the current review, we curated the information from the NCBI, PubMed, and Google Scholar literature and explored the non-genetic and genetic causes of senile sarcopenia. Interestingly, the myomiRs such as miR-1, miR-206, miR-133a, miR-133b, miR-208b, and miR-499 are skeletal muscle's critical structural and functional regulators. However, very scattered information is available regarding the roles of myomiRs in different skeletal muscle phenotypes through a diverse list of known target genes. Therefore, these pieces of information must be organized to focus on the conserved target genes and comparable effects of the myomiRs in regulating senile sarcopenia. Hence, in the present review, the roles of pathogenetic factors in regulating senile sarcopenia were highlighted. The literature was further curated for the roles of myomiRs such as hsa-miR-1-3p/206, hsa-miR-27-3p, hsa-miR-146-5p, and hsa-miR-499-5p and their target genes. Additionally, we used different bioinformatics tools and predicted target genes of the myomiRs and found the most critical target genes, shared pathways, and their standard functions in regulating muscle structure and functions. The information gathered in the current review will help the researchers to explore their possible therapeutic potential, especially the use of the myomiRs for the treatment of senile sarcopenia.

Endogenous small interfering RNAs (endo-siRNAs or esiRNAs) originate from either elongated endogenous transcripts capable of forming complex fold-back structures or from double-stranded regions generated through intermolecular base pairing of convergently transcribed mRNAs. The mechanism of maturation and functionality of esiRNAs exhibit significant variation across diverse species. In Drosophila melanogaster, esiRNAs reside in both somatic and germline cells, where they serve as post-transcriptional modulators for specific target RNAs. Their maturation process critically relies on Dicer-2 (Dcr-2), with the assistance of its cofactor Loqs-PD. In this study, we have successfully elucidated the cryo-EM structures of Dcr-2/Loqs-PD complex bound to esiRNA precursors (pre-esiRNAs) in various states. Our structural and biochemical results reveal that ATP is essential for the cleavage of esiRNAs by the Dcr-2/Loqs-PD complex, a process analogous to the cleavage of double-stranded RNA (dsRNA). When Loqs-PD is present, pre-esiRNAs are preferentially loaded onto the Helicase domain of Dcr-2. Moreover, as the Helicase domain exhibits a preference for binding to the rigid end of double-stranded RNA, Dcr-2 tends to cleave pre-esiRNA from the small closed loop end, rather than the loose and flexible open end.

Diabetic nephropathy (DN) is a common complication of diabetes and a leading cause of end-stage renal disease, characterized by progressive kidney fibrosis and inflammation. The transforming growth factor-beta (TGF-β) signaling pathway plays a crucial role in the pathogenesis of diabetes nephropathy, and SMAD7 is a key negative regulator of this pathway. Recent studies have highlighted the involvement of miRNA in the progression of DN. Computational analysis identified 11 potential miRNAs such as miR-424, miR-195, miR-216a, miR-503, miR-15a-5p, miR-15b-5p, miR-665, miR-520h, miR16-5p, miR-21 and miR-32-5p which are predicted to target 3'UTR of SMAD7 mRNA. This review aims to explore the role of these miRNAs in the progression of DN. Notably, these miRNAs have shown therapeutic potential in mitigating fibrosis and inflammation by modulating SMAD7 expression in DN. Future directions can be to investigate the mechanistic pathways through which these miRNAs exert their effects, as well as optimizing delivery systems for effective clinical application. Targeting miRNAs that modulate SMAD7 expression represents a promising strategy for developing specific and effective therapies for diabetic nephropathy.

MicroRNAs (miRNAs) are small regulatory RNAs that are expressed in a tissue-specific manner during the development of plants and animals. The genes of miRNAs have been found to produce the following two products: (i) primary transcripts of these genes (pri-miRNA) are processed to give rise to mature miRNA, and (ii) in some cases, the pri-miRNA molecules can be translated to form small peptides, named as miPEPs. Gene silencing by artificial microRNAs (amiRNAs) is one of the potential crucial methods for the regulation of desired genes to improve horticultural plants. Likewise, external application of chemically synthesized miPEPs may help plants to resist biotic/abiotic stresses and grow faster. These potent and reliable derivatives of miRNA genes can be applied for improving useful traits in crop plants. This review summarizes the progress in research on the artificial gene derivatives involved in regulating plant development, virus and pest diseases, and abiotic stress resistance pathways. We also briefly discuss the molecular mechanisms of relevant target genes for future research on breeding in plants. In general, this review may be useful to researchers who are implementing amiRNA and miPEP for accelerating breeding programs and developmental studies in crop plants.

microRNAs (miRNAs) are short single-stranded non-coding RNAs between 21 and 25 nt in length in eukaryotic organisms, which control post-transcriptional gene expression. Through complementary base pairing, miRNAs generally bind to their target messenger RNAs and repress protein production by destabilizing the messenger RNA and translational silencing. They regulate almost all life activities, such as cell proliferation, differentiation, apoptosis, tumorigenesis, and host-pathogen interactions. Methylation modification is the most common RNA modification in eukaryotes. miRNA methylation exists in different types, mainly N6-methyladenosine, 5-methylcytosine, and 7-methylguanine, which can change the expression level and biological mode of action of miRNAs and improve the activity of regulating gene expression in a very fine-tuned way with flexibility. In this review, we will summarize the recent findings concerning methylation modifications of miRNA, focusing on their biogenesis and the potential role of miRNA fate and functions.

MicroRNAs (miRNAs) are endogenous, small molecules that negatively regulate gene expression to control the normal development and stress response in plants. They mediate epigenetic changes and regulate gene expression at both transcriptional and post-transcriptional levels. Synthetic biology approaches have been utilized to design efficient artificial miRNAs (amiRNAs) or target-mimics to regulate specific gene expression for understanding the biological function of genes and crop improvement. The amiRNA based gene silencing is an effective technique to "turn off" gene expression, while miRNA target-mimics or decoys are used for efficiently down regulating miRNAs and "turn on" gene expression. In this context, the development of endogenous target-mimics (eTMs) and short tandem target mimics (STTMs) represent promising biotechnological tools for enhancing crop traits like stress tolerance and disease resistance. Through this review, we present the recent developments in understanding plant miRNA biogenesis, which is utilized for the efficient design and development of amiRNAs. This is important to incorporate the artificially synthesized miRNAs as internal components and utilizing miRNA biogenesis pathways for the programming of synthetic circuits to improve crop tolerance to various abiotic and biotic stress factors. The review also examines the recent developments in the use of miRNA target-mimics or decoys for efficiently down regulating miRNAs for trait improvement. A perspective analysis and challenges on the use of amiRNAs and STTM as potent tools to engineer useful traits in plants have also been presented.

Mesial temporal lobe epilepsy (mTLE) is the most prevalent type of epilepsy in adults. First and subsequent generations of anti-epileptic therapy regimens fail to decrease seizures in a large number of patients suffering from mTLE, leaving surgical ablation of part of the hippocampus as the only therapeutic option to potentially reach seizure freedom. GluK2 has recently been identified as a promising target for the treatment of mTLE using gene therapy. Here, we engineered an adeno-associated virus serotype 9 vector expressing a cluster of two synthetic microRNAs (miRNAs), expressed from the human synapsin promoter, that target GRIK2 mRNA. Intra-hippocampal delivery of this vector in a mouse model of mTLE significantly reduced GRIK2 expression and daily seizure frequency. This treatment also improved the animals' health, reduced their anxiety, and restored working memory. Focal administration of the vector to the hippocampus of cynomolgus monkeys in GLP toxicology studies led to the selective transduction of hippocampal neurons with little exposure elsewhere in the brain and no transduction outside the central nervous system. Expression of miRNAs in hippocampal neurons resulted in substantially decreased GRIK2 mRNA expression. These data suggest that the intra-hippocampal delivery of a GMP-grade AAV9 encoding a synthetic miRNAs targeting GRIK2 is a promising treatment strategy for mTLE.

Since the introduction of the central dogma of molecular biology in 1958, various RNA species have been discovered. Messenger RNAs transmit genetic instructions from DNA to make proteins, a process facilitated by housekeeping non-coding RNAs (ncRNAs) such as small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs), and transfer RNAs (tRNAs). Over the past four decades, a wide array of regulatory ncRNAs have emerged as crucial players in gene regulation. In celebration of Cell's 50th anniversary, this Review explores our current understanding of the most extensively studied regulatory ncRNAs-small RNAs and long non-coding RNAs (lncRNAs)-which have profoundly shaped the field of RNA biology and beyond. While small RNA pathways have been well documented with clearly defined mechanisms, lncRNAs exhibit a greater diversity of mechanisms, many of which remain unknown. This Review covers pivotal events in their discovery, biogenesis pathways, evolutionary traits, action mechanisms, functions, and crosstalks among ncRNAs. We also highlight their roles in pathophysiological contexts and propose future research directions to decipher the unknowns of lncRNAs by leveraging lessons from small RNAs.

MicroRNAs (miRNAs) are small, endogenous, single-stranded RNAs that act on gene silencing at the post-transcriptional level by binding to a target messenger RNA (mRNA), leading to its degradation or inhibiting translation into functional proteins. The key role of miRNAs in development, proliferation, differentiation andapoptosis has been deeply investigated, revealing that deregulation in their expression is critical in various diseases, such as metabolic disorders and cancer. Since these small molecules initially evolved as a mechanism of protection against viruses and transposable elements, the fascinating hypothesis that they can move between organisms both of the same or different species has been postulated. Trans-kingdom is the term used to define the migration that occurs between species. This mechanism has been well analyzed between plants and their pests, in order to boost defense and increase pathogenicity, respectively. Intriguingly, in the last decades, the plant-human trans-kingdom migration via food intake hypothesis arose. In particular, various studies highlighted the ability of exogenous miRNAs, abundant in the mainly consumed plant-derived food, to enter the human body affecting gene expression. Notably, plant miRNAs can resist the strict conditions of the gastrointestinal tract through a methylation step that occurs during miRNA maturation, conferring high stability to these small molecules. Recent studies observed the anti-tumoral, immune modulator and anti-inflammatory abilities of trans-kingdom interaction between plant and human. Here, we depict the existing knowledge and discuss the fascinating plant-human trans-kingdom interaction, highlighting first the eventual role of plant miRNAs from foods on our somatic gene identity card and then the potential impact of using plant miRNAs as novel therapeutic avenues.

In this study, we report the synthesis of 2'-formamidonucleoside phosphoramidite derivatives and their incorporation into siRNA strands to reduce seed-based off-target effects of small interfering RNAs (siRNAs). Formamido derivatives of all four nucleosides (A, G, C and U) were synthesized in 5-11 steps from commercial compounds. Introducing these derivatives into double-stranded RNA slightly reduced its thermodynamic stability, but X-ray crystallography and CD spectrum analysis confirmed that the RNA maintained its natural A-form structure. Although the introduction of the 2'-formamidonucleoside derivative at the 2nd position in the guide strand of the siRNA led to a slight decrease in the on-target RNAi activity, the siRNAs with different sequences incorporating 2'-formamidonucleoside with four kinds of nucleobases into any position other than 2nd position in the seed region revealed a significant suppression of off-target activity while maintaining on-target RNAi activity. This indicates that 2'-formamidonucleosides represent a promising approach for mitigating off-target effects in siRNA therapeutics.

Temporomandibular joint osteoarthritis (TMJOA) is the most common and severe subtype of temporomandibular disease characterized by inflammation and cartilage matrix degradation. Compared with traditional conservative treatment, small interfering RNAs (siRNAs) have emerged as a more efficient gene-targeted therapeutic tool for TMJOA treatment. Nuclear factor kappaB (NF-κB) is a transcription factor orchestrating the inflammatory processes in the pathogenesis of TMJOA. Employing siRNA-NF-κB could theoretically control the development of TMJOA. However, the clinical applications of siRNA-NF-κB are limited by its structural instability, poor cellular uptake, and short TMJ retention. To overcome these shortcomings, we developed a tetrahedral framework nucleic acid (tFNA) system carrying siRNA-NF-κB, named Tsi. The results indicated that Tsi exhibited excellent structural stability and excellent cellular uptake efficiency. It also demonstrated a superior NF-κB silencing effect over siRNA alone, attenuating the activation of NF-κB and upregulating the NRF2/HO-1 pathway. This system effectively reduced the release of inflammatory factors and reactive oxygen species (ROS), inhibiting cellular oxidative stress and apoptosis. In vivo, Tsi displayed enhanced TMJ retention capacity in comparison to siRNA alone and offered significant protective effects on both the cartilage matrix and subchondral bone, presenting a promising approach for TMJOA treatment.

Air pollution is one of the leading causes of early deaths worldwide, with particulate matter (PM) as an emerging factor contributing to this trend. PM is classified based on its physical size, which ranges from PM10 (diameter ≤10 μm) to PM2.5 (≤2.5 μm) and PM0.5 (≤0.5 μm). Smaller-sized PM can move freely through the air and readily infiltrate deep into the lungs, intensifying existing health issues and exacerbating complications. Lung complications are the most common issues arising from PM exposure due to the primary site of deposition in the respiratory system. Conditions such as asthma, COPD, idiopathic pulmonary fibrosis, lung cancer and various lung infections are all susceptible to worsening due to PM exposure. PM can epigenetically modify specific target sites, further complicating its impact on these conditions. Understanding these epigenetic mechanisms holds promise for addressing these complications in cases of PM exposure. This involves studying the effect of PM on different gene expressions and regulation through epigenetic modifications, including DNA methylation, histone modifications and microRNAs. Targeting and manipulating these epigenetic modifications and their mechanisms could be promising strategies for future treatments of lung complications. This review mainly focuses on different epigenetic modifications due to PM2.5 exposure in the various lung complications mentioned above.

Sustained siRNA release from nanocarriers is difficult to achieve inside the cell after entry: typically, all nanocarriers exhibit burst release of the cargo into the cytoplasm.

Layer-by-layer (LbL) nanoparticles (NPs) can be constructed so that they escape endosomes intact, and subsequently exhibit sustained release of the cargo. Our work quantifies intra-cellular siRNA release from multilayered NPs, evaluates mechanism behind the sustained release, and optimizes the duration of release.

Intra-cellular studies showed that NPs developed with four layers of poly-L-arginine, alternated with three layers of siRNA layers, were able to elicit effective and prolonged SPARC knockdown activity over 21 days with a single-dose treatment. For the first time, we have quantified the amounts of released siRNA in the cytoplasm and the amount of siRNA remaining inside the NPs at each timepoint. Furthermore, we have correlated the amount of released siRNA within cells by LbL NPs to the cellular knockdown efficiency of multilayered delivery system.

This methodology may provide an excellent screening tool for assessing the duration of gene silencing by various nanocarrier formulations.

Adipose tissue remodeling plays a critical role in obesity-induced metabolic dysfunction, but the underlying molecular mechanisms remain incompletely understood. This study investigates the role of miR-10a-5p in adipose tissue inflammation and metabolic dysfunction induced by a high-fat diet (HFD).

Male miR-10a knockout (KO) mice were fed a HFD to induce obesity for up to 16 weeks. RNA sequencing (RNA-seq) analysis was performed to profile mRNA expression and assess the effects of miR-10a-5p KO in gonadal white adipose tissue (gWAT). Additional analyses included immunoblotting, qPCR, histological examination, and validation of the miR-10a-5p target sequence using a dual-luciferase reporter assay.

miR-10a-5p was highly expressed in gWAT but decreased after 8 weeks of HFD feeding. Over the 16-week HFD period, miR-10a KO mice exhibited greater weight gain and reduced energy expenditure compared to wild-type (WT) controls. gWAT of miR-10a KO mice on a HFD showed an increased population of proinflammatory macrophages, elevated inflammation, and increased cell death, characterized by upregulated apoptosis and necrosis markers. This was also associated with increased triglyceride accumulation in liver. Mechanistically, the proapoptotic gene Bcl2l11 was identified as a direct target of miR-10a-5p. Loss of miR-10a-5p led to BIM-mediated adipocyte death and inflammation, contributing to mitochondrial metabolic dysregulation, increased fibrosis marker expression, and the onset of inflammation in adipose tissue.

This study demonstrates the significant role of miR-10a-5p and its downstream target BIM in regulating adipocyte death during diet-induced obesity. This signaling pathway presents a potential therapeutic target for modulating obesity-induced inflammation and cell death in adipose tissue.

Ischaemic stroke, a leading cause of global morbidity and mortality, necessitates effective biomarkers for enhanced diagnostic and prognostic stratification. MicroRNAs (miRNAs), particularly miR-210, have emerged as promising candidates due to their intricate regulatory roles in cellular responses to hypoxia and neuroprotective effects. This study explores the potential of miR-210 as a biomarker for ischaemic stroke, considering its expression patterns, regulatory functions and diagnostic/prognostic implications. A literature search was conducted on PubMed, Scopus, Google Scholar and Web of Science to identify studies focusing on miR-210 in ischaemic stroke. Inclusion criteria comprised reports on miR-210 expression in ischaemic stroke patients, excluding non-English studies, reviews, commentaries and conference abstracts lacking primary data. Studies investigating miR-210 levels in ischaemic stroke patients revealed significant alterations in expression patterns compared to healthy controls. Diagnostic potential was explored, indicating miR-210's sensitivity and specificity in distinguishing ischaemic stroke from other neurological conditions. Prognostic value was evident through associations with infarct size, functional outcomes and long-term survival. Challenges included variability in miR-210 levels, limited diagnostic specificity, absence of standardised assays and concerns regarding cost-effectiveness and accessibility. While miR-210 holds promise as an ischaemic stroke biomarker, challenges must be addressed for its successful integration into clinical practice. Standardised reference ranges, validation studies in diverse populations and collaborative efforts for assay standardisation are crucial. Despite challenges, miR-210's diagnostic and prognostic potential, particularly in predicting therapeutic responses, suggests a significant role in advancing ischaemic stroke management.

Virus-encoded microRNAs (miRNAs) exert diverse regulatory roles in the biological processes of both viruses and hosts. This study delves into the functions of AcMNPV-miR-2, an early miRNA encoded by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). AcMNPV-miR-2 targets viral early genes ac28 (lef-6), ac37 (lef-11), ac49, and ac63. Overexpression of AcMNPV-miR-2 leads to reduced production of infectious budded virions (BVs) and diminished viral DNA replication. Delayed polyhedron formation was observed through light and transmission electron microscopy, and the larval lifespan extended in oral infection assays. Moreover, the mRNA expression levels of two Lepidoptera-specific immune-related proteins, Gloverin and Spod-11-tox, significantly decreased. These findings indicate that AcMNPV-miR-2 restrains viral load, reducing host immune sensitivity. This beneficial effect enables the virus to combat host defense mechanisms and reside within the host for an extended duration.

Virus-encoded miRNAs have been extensively studied for their pivotal roles in finetuning viral infections. Baculoviruses, highly pathogenic in insects, remain underexplored concerning their encoded miRNAs. Previous reports outlined three AcMNPV-encoded miRNAs, AcMNPV-miR-1, -miR-3, and -miR-4. This study delves into the functions of another AcMNPV-encoded miRNA, AcMNPV-miR-2 (Ac-miR-2). Through a comprehensive analysis of target gene expression, the impact on larvae, and variations in host immune-related gene expression, we elucidate a functional pathway for Ac-miR-2. This miRNA suppresses viral load and infectivity and prolongs lifespans of infected larva by downregulating specific viral early genes and host immune-related genes. These mechanisms ultimately serve the virus's primary goal of enhanced propagation. Our study significantly contributes to understanding of the intricate regulatory mechanisms of virus-encoded miRNAs in baculovirus infections.

To date, the US Food and Drug Administration (FDA) has approved six small interfering RNA (siRNA) drugs: patisiran, givosiran, lumasiran, inclisiran, vutrisiran, and nedosiran, serving as compelling evidence of the promising potential of RNA interference (RNAi) therapeutics. The successful implementation of siRNA therapeutics is improved through a combination of various chemical modifications and diverse delivery approaches. The utilization of chemically modified siRNA at specific sites on either the sense strand (SS) or antisense strand (AS) has the potential to enhance resistance to ribozyme degradation, improve stability and specificity, and prolong the efficacy of drugs. Herein, we provide comprehensive analyses concerning the correlation between chemical modifications and structure-guided siRNA design. Various modifications, such as 2'-modifications, 2',4'-dual modifications, non-canonical sugar modifications, and phosphonate mimics, are crucial for the activity of siRNA. We also emphasize the essential strategies for enhancing overhang stability, improving RISC loading efficacy and strand selection, reducing off-target effects, and discussing the future of targeted delivery.

In recent years, therapeutic siRNA projects are booming in the biotech and pharmaceutical industries. As these drugs act by silencing the target gene expression, a critical step is the binding of antisense strands of siRNA to RNA-induced silencing complex (RISC) and then degrading their target mRNA. However, data that we recently obtained suggest that double-stranded siRNA can also load to RISC. This brings a new understanding of the mechanism of RISC loading which may have a potential impact on how quantification of RISC loaded siRNA should be performed. By combining RNA immune precipitation and probe-based hybridization LC-fluorescence approach, we have developed a novel assay that can accurately quantify the RISC-bound antisense strand, irrespective of which form (double-stranded or single-stranded) is loaded on RISC. In addition, this novel assay can discriminate between the 5'-phosphorylated antisense (5'p-AS) and the nonphosphorylated forms, therefore specifically quantifying the RISC bound 5'p-AS. In comparison, stem-loop qPCR assay does not provide discrimination and accurate quantification when the oligonucleotide analyte exists as a mixture of double and single-stranded forms. Taking together, RISC loading assay with probe-hybridization LC-fluorescence technique would be a more accurate and specific quantitative approach for RISC-associated pharmacokinetic assessment.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression by inhibiting the translation of target transcripts. The expression profiles of miRNAs vary in different tissues and change with the development of diseases, including cancer. This feature has begun to be used for the modification of oncolytic viruses (OVs) in order to increase their selectivity and efficacy. OVs represent a relatively new class of anticancer drugs; they are designed to replicate in cancer tumors and destroy them. These can be natural viruses that can replicate within cancer tumor cells, or recombinant viruses created in laboratories. There are some concerns regarding OVs' toxicity, due to their ability to partially replicate in healthy tissues. In addition, lytic and immunological responses upon OV therapy are not always sufficient, so various OV editing methods are used. This review discusses the latest results of preclinical and clinical studies of OVs, modifications of which are associated with the miRNA-mediated mechanism of gene silencing.

Drug development costs can be significantly reduced if proven "platform" technologies are allowed to be used without having to validate their use. The most recent US Food and Drug Administration (FDA) guideline brings more clarity, as well as a greater focus on the most complex technologies that can now be used for faster drug development. The FDA has highlights the use of lipid nanoparticles (LNPs) to package and deliver mRNA vaccines, gene therapy, and short (2-20 length) synthetic nucleotides (siRNA). Additionally, monoclonal antibody cell development is targeted. The FDA provides a systematic process of requesting platform status to benefit from its advantages. It brings advanced science and rationality into regulatory steps for the FDA's approval of drugs and biologicals.

Drosophila is a well-established insect model system for studying various physiological phenomena and developmental processes, with a focus on gene regulation. Drosophila development is controlled by programmed regulatory mechanisms specific to individual tissues. When key developmental processes are shared among various insects, the associated regulatory networks are believed to be conserved across insects. Thus, studies of developmental regulation in Drosophila have substantially contributed to our understanding of insect development. Over the past two decades, studies on microRNAs (miRNAs) in Drosophila have revealed their crucial regulatory roles in various developmental processes. This review focuses on the biological roles of miRNAs in specific tissues and processes associated with Drosophila development. Additionally, as a future direction, we discuss sequencing technologies that can analyze the interactions between miRNAs and their target genes, with the aim of enhancing miRNA studies in Drosophila development.

Chimeric antigen receptor (CAR) T-cell therapy has proven a breakthrough in cancer treatment in the last decade, giving unprecedented results against hematological malignancies. All approved CAR T-cell products, as well as many being assessed in clinical trials, are generated using viral vectors to deploy the exogenous genetic material into T-cells. Viral vectors have a long-standing clinical history in gene delivery, and thus underwent iterations of optimization to improve their efficiency and safety. Nonetheless, their capacity to integrate semi-randomly into the host genome makes them potentially oncogenic via insertional mutagenesis and dysregulation of key cellular genes. Secondary cancers following CAR T-cell administration appear to be a rare adverse event. However several cases documented in the last few years put the spotlight on this issue, which might have been underestimated so far, given the relatively recent deployment of CAR T-cell therapies. Furthermore, the initial successes obtained in hematological malignancies have not yet been replicated in solid tumors. It is now clear that further enhancements are needed to allow CAR T-cells to increase long-term persistence, overcome exhaustion and cope with the immunosuppressive tumor microenvironment. To this aim, a variety of genomic engineering strategies are under evaluation, most relying on CRISPR/Cas9 or other gene editing technologies. These approaches are liable to introduce unintended, irreversible genomic alterations in the product cells. In the first part of this review, we will discuss the viral and non-viral approaches used for the generation of CAR T-cells, whereas in the second part we will focus on gene editing and non-gene editing T-cell engineering, with particular regard to advantages, limitations, and safety. Finally, we will critically analyze the different gene deployment and genomic engineering combinations, delineating strategies with a superior safety profile for the production of next-generation CAR T-cell.

Many microRNA (miRNA)-guided Argonaute proteins can cleave RNA ('slicing'), even though miRNA-mediated target repression is generally cleavage-independent. Here we use Caenorhabditis elegans to examine the role of catalytic residues of miRNA Argonautes in organismal development. In contrast to previous work, mutations in presumed catalytic residues did not interfere with development when introduced by CRISPR. We find that unwinding and decay of miRNA star strands is weakly defective in the catalytic residue mutants, with the largest effect observed in embryos. Argonaute-Like Gene 2 (ALG-2) is more dependent on catalytic residues for unwinding than ALG-1. The miRNAs that displayed the greatest (albeit minor) dependence on catalytic residues for unwinding tend to form stable duplexes with their star strand, and in some cases, lowering duplex stability alleviates dependence on catalytic residues. While a few miRNA guide strands are reduced in the mutant background, the basis of this is unclear since changes were not dependent on EBAX-1, an effector of Target-Directed miRNA Degradation (TDMD). Overall, this work defines a role for the catalytic residues of miRNA Argonautes in star strand decay; future work should examine whether this role contributes to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny.

More than 25 years after its discovery, the post-transcriptional gene regulation mechanism termed RNAi is now transforming pharmaceutical development, proved by the recent FDA approval of multiple small interfering RNA (siRNA) drugs that target the liver. Synthetic siRNAs that trigger RNAi have the potential to specifically silence virtually any therapeutic target with unprecedented potency and durability. Bringing this innovative class of medicines to patients, however, has been riddled with substantial challenges, with delivery issues at the forefront. Several classes of siRNA drug are under clinical evaluation, but their utility in treating extrahepatic diseases remains limited, demanding continued innovation. In this Review, we discuss principal considerations and future directions in the design of therapeutic siRNAs, with a particular emphasis on chemistry, the application of informatics, delivery strategies and the importance of careful target selection, which together influence therapeutic success.

Argonaute (Ago) proteins act as key elements in RNA interference (RNAi) pathway, orchestrating the intricate machinery of gene regulation within eukaryotic cells. Within the RNAi pathway, small RNA molecules, including microRNA (miRNA), small interfering RNA (siRNA), and PIWI-interacting RNA (piRNA), collaborate with Ago family member proteins such as Ago1, Ago2, and Ago3 to form the RNA-induced silencing complex (RISC). This RISC complex, in turn, either cleaves the target mRNA or inhibits the process of protein translation. The precise contributions of Ago proteins have been well-established in numerous animals and plants, although they still remain unclear in some insect species. This review aims to shed light on the specific roles played by Ago proteins within the RNAi mechanism in a destructive lepidopteran pest, the diamondback moth (Plutella xylostella). Furthermore, we explore the potential of double-stranded RNA (dsRNA)-mediated RNAi as a robust genetic tool in pest management strategies. Through an in-depth examination of Ago proteins and dsRNA-mediated RNAi, this review seeks to contribute to our understanding of innovative approaches for controlling this pest and potentially other insect species of agricultural significance.

Pancreatic cancer (PC) is characterized by its extremely aggressive nature and ranks 14th in the number of new cancer cases worldwide. However, due to its complexity, it ranks 7th in the list of the most lethal cancers worldwide. The pathogenesis of PC involves several complex processes, including familial genetic factors associated with risk factors such as obesity, diabetes mellitus, chronic pancreatitis, and smoking. Mutations in genes such as KRAS, TP53, and SMAD4 are linked to the appearance of malignant cells that generate pancreatic lesions and, consequently, cancer. In this context, some therapies are used for PC, one of which is immunotherapy, which is extremely promising in various other types of cancer but has shown little response in the treatment of PC due to various resistance mechanisms that contribute to a drop in immunotherapy efficiency. It is therefore clear that the tumor microenvironment (TME) has a huge impact on the resistance process, since cellular and non-cellular elements create an immunosuppressive environment, characterized by a dense desmoplastic stroma with cancer-associated fibroblasts, pancreatic stellate cells, extracellular matrix, and immunosuppressive cells. Linked to this are genetic mutations in TP53 and immunosuppressive factors that act on T cells, resulting in a shortage of CD8+ T cells and limited expression of activation markers such as interferon-gamma. In this way, finding new strategies that make it possible to manipulate resistance mechanisms is necessary. Thus, techniques such as the use of TME modulators that block receptors and stromal molecules that generate resistance, the use of genetic manipulation in specific regions, such as microRNAs, the modulation of extrinsic and intrinsic factors associated with T cells, and, above all, therapeutic models that combine these modulation techniques constitute the promising future of PC therapy. Thus, this study aims to elucidate the main mechanisms of resistance to immunotherapy in PC and new ways of manipulating this process, resulting in a more efficient therapy for cancer patients and, consequently, a reduction in the lethality of this aggressive cancer.

The use of non-coding RNAs (ncRNAs) as drug targets is being researched due to their discovery and their role in disease. Targeting ncRNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), is an attractive approach for treating various diseases, such as cardiovascular disease and cancer. This seminar discusses the current status of ncRNAs as therapeutic targets in different pathological conditions. Regarding miRNA-based drugs, this approach has made significant progress in preclinical and clinical testing for cardiovascular diseases, where the limitations of conventional pharmacotherapy are evident. The challenges of miRNA-based drugs, including specificity, delivery, and tolerability, will be discussed. New approaches to improve their success will be explored. Furthermore, it extensively discusses the potential development of targeted therapies for cardiovascular disease. Finally, this document reports on the recent advances in identifying and characterizing microRNAs, manipulating them, and translating them into clinical applications. It also addresses the challenges and perspectives towards clinical application.

MicroRNA (miRNA) maturation is critically dependent on structural features of primary transcripts (pri-miRNAs). However, the scarcity of determined pri-miRNA structures has limited our understanding of miRNA maturation. Here, we employed selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), a high-throughput RNA structure probing method, to unravel the secondary structures of 476 high-confidence human pri-miRNAs. Our SHAPE-based structures diverge substantially from those inferred solely from computation, particularly in the apical loop and basal segments, underlining the need for experimental data in RNA structure prediction. By comparing the structures with high-throughput processing data, we determined the optimal structural features of pri-miRNAs. The sequence determinants are influenced substantially by their structural contexts. Moreover, we identified an element termed the bulged GWG motif (bGWG) with a 3' bulge in the lower stem, which promotes processing. Our structure-function mapping better annotates the determinants of pri-miRNA processing and offers practical implications for designing small hairpin RNAs and predicting the impacts of miRNA mutations.

MicroRNAs (miRNAs) are small non-coding RNAs that play a fundamental role in enabling miRNA-mediated target repression, a post-transcriptional gene regulatory mechanism preserved across metazoans. Loss of certain animal miRNA genes can lead to developmental abnormalities, disease, and various degrees of embryonic lethality. These short RNAs normally guide Argonaute (AGO) proteins to target RNAs, which are in turn translationally repressed and destabilized, silencing the target to fine-tune gene expression and maintain cellular homeostasis. Delineating miRNA-mediated target decay has been thoroughly examined in thousands of studies, yet despite these exhaustive studies, comparatively less is known about how and why miRNAs are directed for decay. Several key observations over the years have noted instances of rapid miRNA turnover, suggesting endogenous means for animals to induce miRNA degradation. Recently, it was revealed that certain targets, so-called target-directed miRNA degradation (TDMD) triggers, can "trigger" miRNA decay through inducing proteolysis of AGO and thereby the bound miRNA. This process is mediated in animals via the ZSWIM8 ubiquitin ligase complex, which is recruited to AGO during engagement with triggers. Since its discovery, several studies have identified that ZSWIM8 and TDMD are indispensable for proper animal development. Given the rapid expansion of this field of study, here, we summarize the key findings that have led to and followed the discovery of ZSWIM8-dependent TDMD. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA in Disease and Development > RNA in Development.