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During meiosis, RNA polymerase II transcribes pachytene piRNA precursors with unusually long and unspliced transcripts from discrete autosomal loci in the mouse genome. Despite the importance of piRNA for male fertility and a well-defined maturation process, the transcriptional machinery remains poorly understood. Here, we document that D1PAS1, an ATP-dependent RNA helicase, is critical for pachytene piRNA expression from multiple genomic loci and subsequent translocation into the cytoplasm to ensure mature piRNA biogenesis. Depletion of D1PAS1 in gene-edited mice results in the accumulation of R-loops in pachytene spermatocytes, leading to DNA-damage-induced apoptosis, disruption of piRNA biogenesis, spermatogenic arrest, and male infertility. Transcriptome, genome-wide R-loop profiling, and proteomic analyses document that D1PAS1 regulates pachytene piRNA transcript elongation and termination. D1PAS1 subsequently forms a complex with nuclear export components to ensure pachytene piRNA precursor translocation from the nucleus to the cytoplasm for processing into small non-coding RNAs. Thus, our study defines D1PAS1 as a specific transcription activator that promotes R-loop unwinding and is a critical factor in pachytene piRNA biogenesis., (Published by Oxford University Press on behalf of Nucleic Acids Research 2024.)

Efficient homing of infused hematopoietic stem and progenitor cells (HSPCs) into the bone marrow (BM) is the prerequisite for successful hematopoietic stem cell transplantation. However, only a small part of infused HSPCs find their way to the BM niche. A better understanding of the mechanisms that facilitate HSPC homing will help to develop strategies to improve the initial HSPC engraftment and subsequent hematopoietic regeneration. Here, we show that irradiation upregulates the endomucin expression of endothelial cells in vivo and in vitro. Furthermore, depletion of endomucin in irradiated endothelial cells with short-interfering RNA (siRNA) increases the HSPC-endothelial cell adhesion in vitro. To abrogate the endomucin of BM sinusoidal endothelial cells (BM-SECs) in vivo, we develop a siRNA-loaded bovine serum albumin nanoparticle for targeted delivery. Nanoparticle-mediated siRNA delivery successfully silences endomucin expression in BM-SECs and improves HSPC homing during transplantation. These results reveal that endomucin plays a critical role in HSPC homing during transplantation and that gene-based manipulation of BM-SEC endomucin in vivo can be exploited to improve the efficacy of HSPC transplantation., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

PIWI-interacting RNAs (piRNAs), a class of small RNAs, are renowned for their roles in sequencing-dependent targeting and suppressing transposable elements (TEs). Nevertheless, a majority of mammalian piRNAs, expressing at pachytene stage of meiosis, known as pachytene piRNAs, are devoid of discernible targets, casting a veil of enigma over their functional significance. Overturning the notion that this unusual class of piRNAs functions beyond TE silencing, we recently demonstrated that pachytene piRNAs play an essential and conserved role in silencing young and actively transposing TEs across amniotes. However, only 1% of pachytene piRNAs target active TEs. The biological significance of the abundant non-TE piRNAs, coproduced from the same precursors as TE piRNAs, remains unclear. Here, we provide a comprehensive summary of the potential roles of non-TE piRNAs, and thus propose that these non-TE piRNAs either bolster the action of TE piRNAs or provide the host genome a preexisting mechanism to suppress the potential invasion of novel TEs in the future., Competing Interests: Conflict of interest The authors declare no competing financial interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The clinical adoption of small interfering RNAs (siRNAs) has prompted the development of various computational strategies for siRNA design, from traditional data analysis to advanced machine learning techniques. However, previous studies have inadequately considered the full complexity of the siRNA silencing mechanism, neglecting critical elements such as siRNA positioning on mRNA, RNA base-pairing probabilities, and RNA-AGO2 interactions, thereby limiting the insight and accuracy of existing models. Here, we introduce siRNADiscovery, a Graph Neural Network (GNN) framework that leverages both non-empirical and empirical rule-based features of siRNA and mRNA to effectively capture the complex dynamics of gene silencing. On multiple internal datasets, siRNADiscovery achieves state-of-the-art performance. Significantly, siRNADiscovery also outperforms existing methodologies in in vitro studies and on an externally validated dataset. Additionally, we develop a new data-splitting methodology that addresses the data leakage issue, a frequently overlooked problem in previous studies, ensuring the robustness and stability of our model under various experimental settings. Through rigorous testing, siRNADiscovery has demonstrated remarkable predictive accuracy and robustness, making significant contributions to the field of gene silencing. Furthermore, our approach to redefining data-splitting standards aims to set new benchmarks for future research in the domain of predictive biological modeling for siRNA., (© The Author(s) 2024. Published by Oxford University Press.)

Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides. The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript. 22nt siRNAs are under-represented due to competition between DCL2 and DCL4, while only a small number of 22nt miRNAs exist. Here we produce abundant 22nt siRNAs and other siRNA size classes using long hairpin RNA (hpRNA) transgenes. By introducing asymmetric bulges into the antisense strand of hpRNA, we shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs. The asymmetric hpRNAs effectively silenced a β-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes showed increased amounts of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes. This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. The 22nt asymmetric hpRNA constructs enhanced virus resistance in plants compared to the traditional hpRNA constructs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Coordination of gene regulatory networks is necessary for proper execution of cellular programs throughout development. RNA interference (RNAi) is an essential regulatory mechanism in all metazoans. Proper RNAi-mediated gene regulation requires coordination of several RNAi branches to ensure homeostasis. For example, in Caenorhabditis elegans, the Argonautes, ALG-3 and ALG-4, are expressed specifically during spermatogenesis (L4 stage) and bind small interfering RNAs (siRNAs) complementary to sperm-enriched genes. We find that alg-3 and alg-4 are regulated by siRNAs. Our work shows that gene switches are operated via these siRNAs to regulate the Argonautes' expression in a temporal manner. This RNAi-to-RNAi regulatory cascade is essential for coordinating ALG-3/4 pathway function, particularly during heat stress, to provide thermotolerant sperm-based fertility. This work provides insight into one regulatory motif used to maintain RNAi homeostasis, across developmental stages, despite environmental stressors. As RNAi pathways are evolutionarily conserved, other species likely use similar regulatory architectures to maintain RNAi homeostasis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Virus-induced drought tolerance presents a fascinating facet of biotic-abiotic interaction in plants, yet its molecular intricacies remain unclear. Our study shows that cowpea mild mottle virus (CPMMV) infection enhances drought tolerance in common bean (Phaseolus vulgaris) plants through a virus-derived small interfering RNA (vsiRNA)-activated autophagy pathway. Specifically, a 21 nt vsiRNA originating from the CPMMV Triple Gene Block1 (TGB1) gene targeted the 5' untranslated region (UTR) of the host Teosinte branched 1, Cycloidea, Proliferating Cell Factor (TCP) transcription factor gene PvTCP2, independent of the known role of TGB1 as an RNA silencing suppressor. This targeting attenuated the expression of PvTCP2, which encodes a transcriptional repressor, and in turn upregulated the core autophagy-related gene (ATG) PvATG8c, leading to activated autophagy activity surpassing the level induced by drought or CPMMV infection alone. The downstream EARLY RESPONSIVE TO DEHYDRATION (ERD) effector PvERD15 is a homologue of Arabidopsis thaliana AtERD15, which positively regulates stomatal aperture. PvERD15 was degraded in PvATG8c-mediated autophagy. Therefore, we establish a TGB1-PvTCP2-PvATG8c-PvERD15 module as a trans-kingdom fine-tuning mechanism that contributes to virus-induced drought tolerance in plant-drought-virus interactions., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The miR390-derived TAS3 trans-acting short-interfering RNAs (tasiRNAs) module represents a conserved RNA silencing pathway in the plant kingdom; however, its characterization in the bryophyte Marchantia polymorpha is limited. This study elucidated that MpDCL4 processes MpTAS3 double-stranded RNA (dsRNA) to generate tasiRNAs, primarily from the 5'- and 3'-ends of dsRNA. Notably, we discovered a novel tasiRNA, tasi78A, which can negatively regulate a cytochrome P450 gene, MpCYP78A101. Additionally, tasi78A was abundant in MpAGO1, and transient expression assays underscored the role of tasi78A in repressing MpCYP78A101. A microRNA, miR11700, also regulates MpCYP78A101 expression. This coordinate regulation suggests a role in modulating auxin signaling at apical notches of gemma, influencing the growth and sexual organ development of M. polymorpha and emphasizing the significance of RNA silencing in MpCYP78A101 regulation. However, phylogenetic analysis identified another paralog of the CYP78 family, Mp1g14150, which may have a redundant role with MpCYP78A101, explaining the absence of noticeable morphological changes in loss-of-function plants. Taken together, our findings provide new insights into the combined regulatory roles of miR390/MpTAS3/miR11700 in controlling MpCYP78A101 and expand our knowledge about the biogenesis and regulation of tasiRNAs in M. polymorpha., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Organisms with differentiated sex chromosomes must accommodate unequal gene dosage in males and females. Male fruit flies increase X-linked gene expression to compensate for hemizygosity of their single X chromosome. Full compensation requires localization of the Male-Specific Lethal (MSL) complex to active genes on the male X, where it modulates chromatin to elevate expression. The mechanisms that identify X chromatin are poorly understood. The euchromatic X is enriched for AT-rich, ∼359 bp satellites termed the 1.688X repeats. Autosomal insertions of 1.688X DNA enable MSL recruitment to nearby genes. Ectopic expression of dsRNA from one of these repeats produces siRNA and partially restores X-localization of MSLs in males with defective X recognition. Surprisingly, expression of double-stranded RNA from three other 1.688X repeats failed to rescue males. We reconstructed dsRNA-expressing transgenes with sequence from two of these repeats and identified phasing of repeat DNA, rather than sequence or orientation, as the factor that determines rescue of males with defective X recognition. Small RNA sequencing revealed that siRNA was produced in flies with a transgene that rescues, but not in those carrying a transgene with the same repeat but different phasing. We demonstrate that pericentromeric X heterochromatin promotes X recognition through a maternal effect, potentially mediated by small RNA from closely related heterochromatic repeats. This suggests that the sources of siRNAs promoting X recognition are highly redundant. We propose that enrichment of satellite repeats on Drosophilid X chromosomes facilitates the rapid evolution of differentiated sex chromosomes by marking the X for compensation., Competing Interests: Conflicts of interest The author(s) declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. The loss of TDRD5 and TDRKH interaction with MIWI results in attenuation of piRNA amplification. We find that piRNA amplification is necessary for transposon control and for sustaining piRNA levels including select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as self-serving genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Flowering plant genomes encode four or five DICER-LIKE (DCL) enzymes that produce small interfering RNAs (siRNAs) and microRNAs, which function in RNA interference (RNAi). Different RNAi pathways in plants effect transposon silencing, antiviral defense, and endogenous gene regulation. DCL2 acts genetically redundantly with DCL4 to confer basal antiviral defense. However, DCL2 may also counteract DCL4 since knockout of DCL4 causes growth defects that are suppressed by DCL2 inactivation. Current models maintain that RNAi via DCL2-dependent siRNAs is the biochemical basis of both effects. Here, we report that DCL2-mediated antiviral resistance and growth defects cannot be explained by the silencing effects of DCL2-dependent siRNAs. Both functions are defective in genetic backgrounds that maintain high levels of DCL2-dependent siRNAs, either with specific point mutations in DCL2 or with reduced DCL2 dosage because of heterozygosity for dcl2 knockout alleles. Intriguingly, all DCL2 functions require its catalytic activity, and the penetrance of DCL2-dependent growth phenotypes in dcl4 mutants correlates with DCL2 protein levels but not with levels of major DCL2-dependent siRNAs. We discuss this requirement and correlation with catalytic activity but not with resulting siRNAs, in light of other findings that reveal a DCL2 function in innate immunity activation triggered by cytoplasmic double-stranded RNA., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Competing Interests: Conflict of interest statement. None declared.

Glioblastoma multiforme is a universally lethal brain tumor that largely resists current surgical and drug interventions. Despite important advancements in understanding GBM biology, the invasiveness and heterogeneity of these tumors has made it challenging to develop effective therapies. Therapeutic oligonucleotides-antisense oligonucleotides and small-interfering RNAs-are chemically modified nucleic acids that can silence gene expression in the brain. However, activity of these oligonucleotides in brain tumors remains inadequately characterized. In this study, we developed a quantitative method to differentiate oligonucleotide-induced gene silencing in orthotopic GBM xenografts from gene silencing in normal brain tissue, and used this method to test the differential silencing activity of a chemically diverse panel of oligonucleotides. We show that oligonucleotides chemically optimized for pharmacological activity in normal brain tissue do not show consistent activity in GBM xenografts. We then survey multiple advanced oligonucleotide chemistries for their activity in GBM xenografts. Attaching lipid conjugates to oligonucleotides improves silencing in GBM cells across several different lipid classes. Highly hydrophobic lipid conjugates cholesterol and docosanoic acid enhance silencing but at the cost of higher neurotoxicity. Moderately hydrophobic, unsaturated fatty acid and amphiphilic lipid conjugates still improve activity without compromising safety. These oligonucleotide conjugates show promise for treating glioblastoma., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA interference (RNAi) is an endogenous process that can be harnessed using chemically modified small interfering RNAs (siRNAs) to potently modulate gene expression in many tissues. The route of administration and chemical architecture are the primary drivers of oligonucleotide tissue distribution, including siRNAs. Independently of the nature and type, oligonucleotides are eliminated from the body through clearance tissues, where their unintended accumulation may result in undesired gene modulation. Divalent siRNAs (di-siRNAs) administered into the CSF induce robust gene silencing throughout the central nervous system (CNS). Upon clearance from the CSF, they are mainly filtered by the kidneys and liver, with the most functionally significant accumulation occurring in the liver. siRNA- and miRNA-induced silencing can be blocked through substrate inhibition using single-stranded, stabilized oligonucleotides called antagomirs or anti-siRNAs. Using APOE as a model target, we show that undesired di-siRNA-induced silencing in the liver can be mitigated through administration of liver targeting GalNAc-conjugated anti-siRNAs, without impacting CNS activity. Blocking unwanted hepatic APOE silencing achieves fully CNS-selective silencing, essential for potential clinical translation. While we focus on CNS/liver selectivity, coadministration of differentially targeting siRNA and anti-siRNAs can be adapted as a strategy to achieve tissue selectivity in different organ combinations., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Much insight has been gained on how stem cells maintain genomic integrity, but less attention has been paid to how they maintain their transcriptome. Here, we report that the PIWI protein SMEDWI-1 plays a role in the filtering of dysfunctional transcripts from the transcriptome of planarian stem cells. SMEDWI-1 accomplishes this through association with the ribosomes during the pioneer round of translation, and processing of poorly translated transcripts into piRNAs. This results in the removal of such transcripts from the cytoplasmic pool and at the same time creates a dynamic pool of small RNAs for post-transcriptional surveillance through the piRNA pathway. Loss of SMEDWI-1 results in elevated levels of several non-coding transcripts, including rRNAs, snRNAs and pseudogene mRNAs, while reducing levels of several coding transcripts. In the absence of SMEDWI-1, stem cell colonies are delayed in their expansion and a higher fraction of descendants exit the stem cell state, indicating that this transcriptomic sanitation mediated by SMEDWI-1 is essential to maintain stem cell health. This study presents a new model for the function of PIWI proteins in stem cell maintenance, that complements their role in transposon repression, and proposes a new biogenesis pathway for piRNAs in stem cells., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Adaptation to external environmental challenges at the cellular level requires rapid responses and involves relay of information to the nucleus to drive key gene expression changes through downstream transcription factors. Here, we describe an alternative route of adaptation through a direct role for cellular signaling components in governing gene expression via RNA interference-mediated small RNA production. Calcium-calcineurin signaling is a highly conserved signaling cascade that plays central roles in stress adaptation and virulence of eukaryotic pathogens, including the human fungal pathogen Cryptococcus neoformans. Upon activation in C. neoformans, calcineurin localizes to P-bodies, membraneless organelles that are also the site for RNA processing. Here, we studied the role of calcineurin and its substrates in RNAi-mediated transgene silencing. Our results reveal that calcineurin regulates both the onset and the reversion of transgene silencing. We found that some calcineurin substrates that localize to P-bodies also regulate transgene silencing but in opposing directions. Small RNA sequencing in mutants lacking calcineurin or its targets revealed a role for calcineurin in small RNA production. Interestingly, the impact of calcineurin and its substrates was found to be different in genome-wide analysis, suggesting that calcineurin may regulate small RNA production in C. neoformans through additional pathways. Overall, these findings define a mechanism by which signaling machinery induced by external stimuli can directly alter gene expression to accelerate adaptative responses and contribute to genome defense., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Repeated implantation failure (RIF) leads to a waste of high-quality embryos and remains a challenge in assisted reproductive technology. During early human placentation, the invasion of trophoblast cells into the decidua is an essential step for the establishment of maternal-fetal interactions and subsequent successful pregnancy. Bone morphogenetic protein 2 (BMP2) has been reported to regulate endometrial receptivity and promote trophoblast invasion. However, whether there is dysregulation of endometrial BMP2 expression in patients with RIF remains unknown. Additionally, the molecular mechanisms underlying the effects of BMP2 on human trophoblast invasion and early placentation remain to be further elucidated., Methods: Midluteal phase endometrial samples were biopsied from patients with RIF and from routine control in vitro fertilization followed by quantitative polymerase chain reaction and immunoblotting analyses. Human trophoblast organoids, primary human trophoblast cells, and an immortalized trophoblast cell line (HTR8/SVneo) were used as study models., Results: We found that BMP2 was aberrantly low in midluteal phase endometrial tissues from patients with RIF. Recombinant human BMP2 treatment upregulated integrin β3 (ITGB3) in a SMAD2/3-SMAD4 signaling-dependent manner in both HTR8/SVneo cells and primary trophoblast cells. siRNA-mediated integrin β3 downregulation reduced both basal and BMP2-upregulated trophoblast invasion and vascular mimicry in HTR8/SVneo cells. Importantly, shRNA-mediated ITGB3 knockdown significantly decreased the formation ability of human trophoblast organoids., Conclusion: Our results demonstrate endometrial BMP2 deficiency in patients with RIF. ITGB3 mediates both basal and BMP2-promoted human trophoblast invasion and is essential for early placentation. These findings broaden our knowledge regarding the regulation of early placentation and provide candidate diagnostic and therapeutic targets for RIF clinical management., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, by both uncovering essential biological processes and creating new tools for developmental geneticists. However, the efficacy of exogenous RNA interference (RNAi) varies dramatically within the Caenorhabditis elegans natural population, raising questions about our understanding of RNAi in the lab relative to its activity and significance in nature. Here, we investigate why some wild strains fail to mount a robust RNAi response to germline targets. We observe diversity in mechanism: in some strains, the response is stochastic, either on or off among individuals, while in others, the response is consistent but delayed. Increased activity of the Argonaute PPW-1, which is required for germline RNAi in the laboratory strain N2, rescues the response in some strains but dampens it further in others. Among wild strains, genes known to mediate RNAi exhibited very high expression variation relative to other genes in the genome as well as allelic divergence and strain-specific instances of pseudogenization at the sequence level. Our results demonstrate functional diversification in the small RNA pathways in C. elegans and suggest that RNAi processes are evolving rapidly and dynamically in nature., Competing Interests: Conflicts of interest statement The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Wnt16 is expressed in bone and arteries, and maintains bone mass in mice and humans, but its role in cardiovascular physiology is unknown. We show that Wnt16 protein accumulates in murine and human vascular smooth muscle (VSM). WNT16 genotypes that convey risk for bone frailty also convey risk for cardiovascular events in the Dallas Heart Study. Murine Wnt16 deficiency, which causes postnatal bone loss, also reduced systolic blood pressure. Electron microscopy demonstrated abnormal VSM mitochondrial morphology in Wnt16-null mice, with reductions in mitochondrial respiration. Following angiotensin-II (AngII) infusion, thoracic ascending aorta (TAA) dilatation was greater in Wnt16-/- vs Wnt16+/+ mice (LDLR-/- background). Acta2 (vascular smooth muscle alpha actin) deficiency has been shown to impair contractile phenotype and worsen TAA aneurysm with concomitant reductions in blood pressure. Wnt16 deficiency reduced expression of Acta2, SM22 (transgelin), and other contractile genes, and reduced VSM contraction induced by TGFβ. Acta2 and SM22 proteins were reduced in Wnt16-/- VSM as was Ankrd1, a prototypic contractile target of Yap1 and Taz activation via TEA domain (TEAD)-directed transcription. Wnt16-/- VSM exhibited reduced nuclear Taz and Yap1 protein accumulation. SiRNA targeting Wnt16 or Taz, but not Yap1, phenocopied Wnt16 deficiency, and Taz siRNA inhibited contractile gene upregulation by Wnt16. Wnt16 incubation stimulated mitochondrial respiration and contraction (reversed by verteporfin, a Yap/Taz inhibitor). SiRNA targeting Taz inhibitors Ccm2 and Lats1/2 mimicked Wnt16 treatment. Wnt16 stimulated Taz binding to Acta2 chromatin and H3K4me3 methylation. TEAD cognates in the Acta2 promoter conveyed transcriptional responses to Wnt16 and Taz. Wnt16 regulates cardiovascular physiology and VSM contractile phenotype, mediated via Taz signaling., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aging is the consequence of intra- and extracellular events that promote cellular senescence. Dyskeratosis congenita (DC) is an example of a premature aging disorder caused by underlying telomere/telomerase-related mutations. Cells from these patients offer an opportunity to study telomere-related aging and senescence. Our previous work has found that telomere shortening stimulates DNA damage responses (DDRs) and increases reactive oxygen species (ROS), thereby promoting entry into senescence. This work also found that telomere elongation via TERT expression, the catalytic component of the telomere-elongating enzyme telomerase, or p53 shRNA could decrease ROS by disrupting this telomere-DDR-ROS pathway. To further characterize this pathway, we performed a CRISPR/Cas9 knockout screen to identify genes that extend life span in DC cells. Of the cellular clones isolated due to increased life span, 34% had a guide RNA (gRNA) targeting CEBPB, while gRNAs targeting WSB1, MED28, and p73 were observed multiple times. CEBPB is a transcription factor associated with activation of proinflammatory response genes suggesting that inflammation may be present in DC cells. The inflammatory response was investigated using RNA sequencing to compare DC and control cells. Expression of inflammatory genes was found to be significantly elevated (P < 0.0001) in addition to a key subset of these inflammation-related genes [IL1B, IL6, IL8, IL12A, CXCL1 (GROa), CXCL2 (GROb), and CXCL5]. which are regulated by CEBPB. Exogenous TERT expression led to downregulation of RNA/protein CEBPB expression and the inflammatory response genes suggesting a telomere length-dependent mechanism to regulate CEBPB. Furthermore, unlike exogenous TERT and p53 shRNA, CEBPB shRNA did not significantly decrease ROS suggesting that CEBPB's contribution in DC cells' senescence is ROS independent. Our findings demonstrate a key role for CEBPB in engaging senescence by mobilizing an inflammatory response within DC cells., Competing Interests: Conflicts of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Metazoans guard their germlines against transposons and other foreign transcripts with PIWI-interacting RNAs (piRNAs). Due to the robust heritability of the silencing initiated by piRNAs in Caenorhabditis elegans (C. elegans), previous screens using C. elegans were strongly biased to uncover members of this pathway in the maintenance process but not in the initiation process. To identify novel piRNA pathway members, we have utilized a sensitized reporter strain which detects defects in initiation, amplification, or regulation of piRNA silencing. Using our reporter, we have identified Integrator complex subunits, nuclear pore components, protein import components, and pre-mRNA splicing factors as essential for piRNA-mediated gene silencing. We found the small nuclear processing cellular machine termed the Integrator complex is required for both type I and type II piRNA production. Notably, we identified a role for nuclear pore and nucleolar components NPP-1/Nup54, NPP-6/Nup160, NPP-7/Nup153, and FIB-1 in promoting the perinuclear localization of anti-silencing CSR-1 Argonaute, as well as a role for Importin factor IMA-3 in nuclear localization of silencing Argonaute HRDE-1. Together, we have shown that piRNA silencing in C. elegans is dependent on evolutionarily ancient RNA processing machinery that has been co-opted to function in the piRNA-mediated genome surveillance pathway., Competing Interests: Conflicts of interest statement The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Our group previously found that the transmembrane protein 232 (TMEM232) gene was associated with atopic dermatitis (AD) by genome-wide association study and fine mapping study. However, its function is unclear so far., Objectives: To investigate the roles and mechanisms of TMEM232 in AD., Methods: The expression of TMEM232 was investigated in skin lesions of patients with AD, the MC903-induced AD mouse model, human primary keratinocytes and immortalized human keratinocyte cell line (HaCaT) cells stimulated with different inflammatory factors. The role of TMEM232 in AD was analysed in HaCaT cells and Tmem232 knockout (Tmem232-/-) mice. Tmem232-specific small interfering RNA (siRNA) was used to evaluate its therapeutic potential in the AD mouse model., Results: The expression of TMEM232 was significantly increased in skin lesions of patients with AD, the MC903-induced AD mouse model and human primary keratinocytes and HaCaT cells stimulated with different inflammatory factors compared with controls. In the presence of MC903, Tmem232-/- mice exhibited significantly reduced dermatitis severity, mast-cell infiltration in the back, and expression of T-helper (Th)1 and Th2-related inflammatory factors in skin tissue compared with wild-type mice. In vitro and in vivo experiments further showed that upregulation of TMEM232 in AD exacerbated the inflammation response through activating the pathway of nuclear factor-κB and signal transducer and activator of transcription (STAT) 3, and was regulated by the interleukin-4/STAT6 axis, which formed a self-amplifying loop. Finally, topical application of Tmem232 siRNA markedly ameliorated AD-like lesions in the AD model., Conclusions: This study is the first to outline the function of TMEM232. It is involved in regulating inflammation in AD and may be a potential target for AD treatment., Competing Interests: Conflicts of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Association of Dermatologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Reproductive tissues are a rich source of small RNAs, including several classes of short interfering (si)RNAs that are restricted to this stage of development. In addition to RNA polymerase IV-dependent 24-nt siRNAs that trigger canonical RNA-directed DNA methylation, abundant reproductive-specific siRNAs are produced from companion cells adjacent to the developing germ line or zygote and may move intercellularly before inducing methylation. In some cases, these siRNAs are produced via non-canonical biosynthesis mechanisms or from sequences with little similarity to transposons. While the precise role of these siRNAs and the methylation they trigger is unclear, they have been implicated in specifying a single megaspore mother cell, silencing transposons in the male germ line, mediating parental dosage conflict to ensure proper endosperm development, hypermethylation of mature embryos, and trans-chromosomal methylation in hybrids. In this review, we summarize the current knowledge of reproductive siRNAs, including their biosynthesis, transport, and function., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glucose 6-phosphate isomerase (G6PI) is an indicator to assist in diagnosis of rheumatoid arthritis (RA) and monitor the disease. It also plays a key role in proliferating RA synovial tissues, pannus formation, and invasion and destruction of articular cartilage. In this study, we synthesized nanoparticles targeting G6PI (siG6PI-MSN) using mesoporous silica nanocarriers (MSN) and small interfering RNA (siRNA), followed by identifying the characteristics and functions, and preliminarily exploring their application in the treatment of RA in vivo with a type II collagen-induced arthritis (CIA) rat model. It showed that the synthetic functionalized carrier had a regular pore structure and a specific volume and surface area. No obvious hemolysis or toxicity of the carrier was found when its concentration was below 100 µg/ml. Cytological results in vitro suggested that siG6PI-MSN significantly inhibited G6PI expression and reduced the ability of proliferation, migration, and invasion of FLSs, compared with the siNC-MSN group. In vivo results in the CIA rat model showed that the arthritis index and degree of joint swelling among rats in the siG6PI-MSN-treatment group were significantly lower than those in the control group. Moreover, the number of FLSs in Synovium and the levels of TNF α and IL-1 β were also significantly decreased in the siG6PI-MSN group. Histopathology of the synovial tissue and cartilage revealed siG6PI-MSN treatment significantly reduced the pathological manifestations of arthritis. In conclusion, siG6PI-MSN effectively suppresses the proliferation and invasive growth of synovial tissue and improve joint swelling and inflammatory infiltration, thereby preventing joint damage in RA. This carrier may be a new therapeutic measure for RA, with potential social and economic benefits., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

piRNA and PIWI proteins have been confirmed for disease diagnosis and treatment as novel biomarkers due to its abnormal expression in various cancers. However, the current research is not strong enough to further clarify the functions of piRNA in cancer and its underlying mechanism. Therefore, how to provide large-scale and serious piRNA candidates for biological research has grown up to be a pressing issue. In this study, a novel computational model based on the structural perturbation method is proposed to predict potential disease-associated piRNAs, called SPRDA. Notably, SPRDA belongs to positive-unlabeled learning, which is unaffected by negative examples in contrast to previous approaches. In the 5-fold cross-validation, SPRDA shows high performance on the benchmark dataset piRDisease, with an AUC of 0.9529. Furthermore, the predictive performance of SPRDA for 10 diseases shows the robustness of the proposed method. Overall, the proposed approach can provide unique insights into the pathogenesis of the disease and will advance the field of oncology diagnosis and treatment., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Targeting the type I insulin-like growth factor receptor (IGF-IR) has not been successful in breast cancer. Data suggest the highly homologous insulin receptor (IR) may be an alternate growth stimulatory pathway used by cancer cells. Since both receptors phosphorylate the insulin receptor substrate 1 (IRS-1) protein as an immediate consequence of ligand binding, disruption of both receptors could be accomplished by suppression of IRS-1. IRS-1 gene deletion by CRISPR/Cas9 editing resulted in suppression of IGF-I, insulin, and estrogen-stimulated growth in hormone-dependent MCF-7L breast cancer cells. A doxycycline-inducible IRS-1 shRNA lentiviral construct was also used to infect MCF-7L breast cancer cells. IRS-1 shRNA downregulation resulted in decreased responses to IGF-I, insulin, and estradiol in monolayer and anchorage-independent growth assays. Decreased IRS-1 levels also suppressed estradiol-stimulated gene expression and estrogen receptor binding to DNA. Xenograft growth was also inhibited by induction of IRS-1 shRNA. These data show that IRS-1 is a critical regulator of endocrine responsive breast cancer. Efforts to target this adaptor protein could have broader growth inhibitory effects and receptor targeting., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Ribonucleic acid (RNA)-targeted therapeutics, including antisense oligonucleotide technologies as well as small interfering RNAs (siRNAs), represent a new class of medications that may overcome several of the disadvantages of small molecule drugs or monoclonal antibodies. Specifically, upstream targeting at the messenger RNA (mRNA) level renders any disease-related protein a potential target, even those pathways previously deemed 'undruggable'. Additional advantages include the comparably simple and cost-effective way of manufacturing and the long dosing intervals. A few agents are already approved and a wide array of cardiovascular drugs is in development, aimed at hypercholesterolaemia, hypertension, myocardial storage diseases, and the coagulation system. Here, we provide an update on the current status of RNA-targeted therapeutics in the cardiovascular arena., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Society of Cardiology.)

The model plant Arabidopsis thaliana encodes as many as ten Argonaute proteins (AGO1-10) with different functions. Each AGO selectively loads a set of small RNAs by recognizing their length and 5' nucleotide identity to properly regulate target genes. Previous studies showed that AGO4 and AGO6, key factors in DNA methylation, incorporate 24-nt small-interfering RNAs with 5' adenine (24A siRNAs). However, it has been unclear how these AGOs specifically load 24A siRNAs. Here, we biochemically investigated the siRNA preference of AGO4, AGO6 and their chimeric mutants. We found that AGO4 and AGO6 use distinct mechanisms to preferentially load 24A siRNAs. Moreover, we showed that the 5' A specificity of AGO4 and AGO6 is not determined by the previously known nucleotide specificity loop in the MID domain but rather by the coordination of the MID and PIWI domains. These findings advance our mechanistic understanding of how small RNAs are accurately sorted into different AGO proteins in plants., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Study Question: Does basigin (BSG) regulate human endometrial stromal cell (HESC) decidualization in vitro?, Summary Answer: BSG regulates HESCs proliferation and decidualization., What Is Known Already: Studies have shown that in the human endometrium, BSG expression is menstrual-cycle dependent and its expression was significantly lower in uterine endometrium during the luteal phase of women experiencing multiple implantation failures after IVF than in women with normal fertility., Study Design, Size, Duration: We utilized a telomerase-immortalized HESCs in an in vitro cell culture model system to investigate whether BSG regulates decidualization of stromal cells. Further, we used microarray analysis to identify changes in the gene expression profile of HESCs treated with BSG small interfering RNA (siRNA). All experiments were repeated at least three times., Participants/materials, Setting, Methods: The effect of BSG knockdown (using siRNA) on HESC proliferation was determined by counting cell number and by tritiated thymidine incorporation assays. The effect of BSG on decidualization of HESCs was determined by RT-qPCR for the decidualization markers insulin-like growth factor-binding protein 1 (IGFBP1) and prolactin (PRL). Immunoblotting was used to determine the effect of BSG siRNA on the expression of MMP-2,3. Microarray analysis was used to identify BSG-regulated genes in HESCs at Day 6 of decidualization. Functional and pathway enrichment analyses were then carried out on the differentially expressed genes (DEGs). The STRING online database was used to analyze protein-protein interaction (PPI) between DEG-encoded proteins, and CytoScape software was used to visualize the interaction. MCODE and CytoHubba were used to construct functional modules and screen hub genes separately. Several BSG-regulated genes identified in the microarray analysis were confirmed by qPCR., Main Results and the Role of Chance: Knockdown of BSG expression in cultured stromal cells by siRNA significantly (P < 0.05) inhibited HESC proliferation, disrupted cell decidualization and down-regulated MMP-2 and MMP-3 expression. Microarray analysis identified 721 genes that were down-regulated, and 484 genes up-regulated with P < 0.05 in BSG siRNA treated HESCs. GO term enrichment analysis showed that the DEGs were significantly enriched in cell communication, signaling transduction and regulation, response to stimulus, cell adhesion, anatomical structure morphogenesis, extracellular matrix organization, as well as other functional pathways. KEGG pathway analysis identified upregulated gene enriched in pathways such as the MAPK signaling pathway, colorectal cancer, melanoma and axon guidance. In contrast, downregulated genes were mainly enriched in pathways including ECM-receptor interaction, PI3K-Akt signaling pathway, pathways in cancer, antigen processing, type I diabetes mellitus and focal adhesion. The top 10 hub nodes were identified using 12 methods analyses. The hub genes that showed up in two methods were screened out. Among these genes, upregulated genes included EGFR, HSP90AA1, CCND1, PXN, PRKACB, MGAT4A, EVA1A, LGALS1, STC2, HSPA4; downregulated genes included WNT4/5, FOXO1, CDK1, PIK3R1, IGF1, JAK2, LAMB1, ITGAV, HGF, MXRA8, TMEM132A, UBE2C, QSOX1, ERBB2, GNB4, HSP90B1, LAMB2, LAMC1 and ITGA1. Hub genes and module genes involved in the top three modules of PPI analysis were analyzed through the string database. Analysis showed that hub and module genes were related mainly to the WNT signaling pathway, PI3K-AKT signaling pathway and pathways in cancer., Large Scale Data: The microarray data set generated in this study has been published online at databank.illinois.edu., Limitations, Reasons for Caution: Most of the findings were obtained using an in vitro cell culture system that may not necessarily reflect in vivo functions., Wider Implications of the Findings: Our results demonstrate that BSG plays a vital role in decidualization and that downregulation of BSG in the uterine endometrium may be associated with infertility in women. The identified hub genes and pathways increase our understanding of the genetic etiology and molecular mechanisms underlying the regulation of decidualization by BSG., Study Funding/competing Interest(s): This work was supported by the NIH U54 HD40093 (R.A.N.). The authors have no competing interests to declare., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

PIWI proteins and Piwi-Interacting RNAs (piRNAs) are commonly detected in human cancers, especially in germline and somatic tissues, and correlate with poorer clinical outcomes, suggesting that they play a functional role in cancer. As the problem of combinatorial explosions between ncRNA and disease exposes gradually, new bioinformatics methods for large-scale identification and prioritization of potential associations are therefore of interest. However, in the real world, the network of interactions between molecules is enormously intricate and noisy, which poses a problem for efficient graph mining. Line graphs can extend many heterogeneous networks to replace dichotomous networks. In this study, we present a new graph neural network framework, line graph attention networks (LGAT). And we apply it to predict PiRNA disease association (GAPDA). In the experiment, GAPDA performs excellently in 5-fold cross-validation with an AUC of 0.9038. Not only that, it still has superior performance compared with methods based on collaborative filtering and attribute features. The experimental results show that GAPDA ensures the prospect of the graph neural network on such problems and can be an excellent supplement for future biomedical research., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Our previous study indicates that Silent information regulator 1 (Sirt1) is involved in macroautophagy by upregulating light chain 3 (LC3) expression in astrocyte to exert a neuroprotective effect. Chaperon-mediated autophagy (CMA), another form of autophagy, is also upregulated after brain injury. However, little is known about the role of Sirt1 in regulation of the CMA. In the present study, an in vivo model of closed head injury (CHI) and an in vitro model of primary cortical astrocyte stimulated with interleukin-1β were employed to mimic the astrocyte activation induced by traumatic brain injury. Lentivirus carrying target complementary DNA (cDNA) or short hairpin RNA (shRNA) sequence was used to overexpress Sirt1 or knockdown DnaJ heat shock protein family member B1 (Dnajb1) (a molecular chaperone). We found that Sirt1 overexpression ameliorated neurological deficits, reduced tissue loss, and attenuated astrocyte activation after CHI, which was reversed by Dnajb1-shRNA administration. The upregulation of CMA activity induced by CHI in vivo and in vitro was inhibited after Dnajb1 knockdown. Sirt1 potently promoted CMA activity via upregulating Dnajb1 expression. Mechanically, Sirt1 could interact with Dnajb1 and modulate the deacetylation and ubiquitination of Dnajb1. These findings collectively suggest that Sirt1 plays a protective role against astrocyte activation, which may be associated with the regulation of the CMA activity via modulating the deacetylation and ubiquitination of Dnajb1 after CHI., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

STIP1-homologous U-Box containing protein 1 (STUB1) is involved in the development of immune pathologies and the regulation of T cell. However, the potential role of STUB1 in the pathogenesis of rheumatoid arthritis (RA), especially in the regulation of T cells, remains elusive. Here we show that STUB1 promotes the imbalance of Th17/Treg cells through non-degradative ubiquitination of aryl hydrocarbon receptor (AHR). Using Western blot and flow cytometry analysis, we observe that the level of STUB1 was increased in RA patients compared with healthy controls. In particular, the expression of STUB1 protein was different in Th17 cells and Treg cells of RA patients. We also demonstrated that STUB1 facilitates Th17/Treg imbalance by up- or downregulating the expression of STUB1. In a subsequent series of in vitro experiments, we revealed that STUB1 promoted the imbalance of Th17 and Treg cells through non-degradative ubiquitination of AHR. Both knockdown of the AHR expression by siRNA and assays of CYP1A1 enzymatic activity by ethoxyresorufin-O-deethylase (EROD) supported this conclusion. Furthermore, we explored the ubiquitination sites of AHR responsible for STUB1-mediated ubiquitination and revealed that STUB1 promotes ubiquitination of AHR via K63 chains. Together, STUB1 may induce the imbalance of Th17/Treg cells via ubiquitination of AHR and serve as a potential therapeutic target for RA., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Immunology.)

Twenty-four-nucleotide (nt) small interfering RNAs (siRNAs) maintain asymmetric DNA methylation at thousands of euchromatic transposable elements in plant genomes in a process called RNA-directed DNA methylation (RdDM). RdDM is dispensable for growth and development in Arabidopsis thaliana, but is required for reproduction in other plants, such as Brassica rapa. The 24-nt siRNAs are abundant in maternal reproductive tissue, due largely to overwhelming expression from a few loci in the ovule and developing seed coat, termed siren loci. A recent study showed that 24-nt siRNAs produced in the anther tapetal tissue can methylate male meiocyte genes in trans. Here we show that in B. rapa, a similar process takes place in female tissue. siRNAs are produced from gene fragments embedded in some siren loci, and these siRNAs can trigger methylation in trans at related protein-coding genes. This trans-methylation is associated with silencing of some target genes and may be responsible for seed abortion in RdDM mutants. Furthermore, we demonstrate that a consensus sequence in at least two families of DNA transposons is associated with abundant siren expression, most likely through recruitment of CLASSY3, a putative chromatin remodeler. This research describes a mechanism whereby RdDM influences gene expression and sheds light on the role of RdDM during plant reproduction., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

SLN360 is a liver-targeted N-acetyl galactosamine (GalNAc)-conjugated small interfering RNA (siRNA) with a promising profile for addressing lipoprotein (a)-related cardiovascular risk. Here, we describe the findings from key preclinical safety studies. In vitro, SLN360 specifically reduced LPA expression in primary human hepatocytes with no relevant off-target effects. In rats, 10 mg/kg subcutaneous SLN360 was distributed specifically to the liver and kidney (peak 126 or 246 mg/g tissue at 6 h, respectively), with &lt;1% of peak liver levels observed in all other tested organs. In vitro, no genotoxicity and no effect on human Ether-a-go-go Related Gene currents or proinflammatory cytokine production was observed, whereas in vivo, no SLN360-specific antibodies were detected in rabbit serum. In rat and nonhuman primate 29-day toxicology studies, SLN360 was well tolerated at all doses. In both species, known GalNAc-conjugated siRNA-induced microscopic changes were observed in the kidney and liver, with small increases in alanine aminotransferase and alkaline phosphatase observed in the high dose rats. Findings were in line with previously described siRNA-GalNAc platform-related effects and all observations were reversible and considered nonadverse. In cynomolgus monkeys, liver LPA messenger RNA and serum lipoprotein (a) were significantly reduced at day 30 and after an 8-week recovery period. No dose-related changes in safety assessment endpoints were noted. No SLN360-induced cytokine production, complement activation, or micronucleus formation was observed in vivo. The toxicological profile of SLN360 presented here is restricted to known GalNAc siRNA effects and no other toxicity associated with SLN360 has been noted. The preclinical profile of SLN360 confirmed suitability for entry into clinical studies., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society of Toxicology.)

Neurospora crassa protein QDE-1, a member of the two-barrel polymerase superfamily, possesses both DNA- and RNA-dependent RNA polymerase (DdRP and RdRP) activities. The dual activities are essential for the production of double-stranded RNAs (dsRNAs), the precursors of small interfering RNAs (siRNAs) in N. crassa. Here, we report five complex structures of N-terminal truncated QDE-1 (QDE-1ΔN), representing four different reaction states: DNA/RNA-templated elongation, the de novo initiation of RNA synthesis, the first step of nucleotide condensation during de novo initiation and initial NTP loading. The template strand is aligned by a bridge-helix and double-psi beta-barrels 2 (DPBB2), the RNA product is held by DPBB1 and the slab domain. The DNA template unpairs with the RNA product at position -7, but the RNA template remains paired. The NTP analog coordinates with cations and is precisely positioned at the addition site by a rigid trigger loop and a proline-containing loop in the active center. The unique C-terminal tail from the QDE-1 dimer partner inserts into the substrate-binding cleft and plays regulatory roles in RNA synthesis. Collectively, this work elucidates the conserved mechanisms for DNA/RNA-dependent dual activities by QDE-1 and other two-barrel polymerase superfamily members., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Small RNAs (sRNAs), including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are essential gene regulators for plant and animal development. The loading of sRNA duplexes into the proper ARGONAUTE (AGO) protein is a key step to forming a functional silencing complex. In Arabidopsis thaliana, the specific loading of miR166/165 into AGO10 (AtAGO10) is critical for the maintenance of the shoot apical meristem, the source of all shoot organs, but the mechanism by which AtAGO10 distinguishes miR166/165 from other cellular miRNAs is not known. Here, we show purified AtAGO10 alone lacks loading selectivity towards miR166/165 duplexes. However, phosphate and HSP chaperone systems reshape the selectivity of AtAGO10 to its physiological substrates. A loop in the AtAGO10 central cleft is essential for recognizing specific mismatches opposite the guide strand 3' region in miR166/165 duplexes. Replacing this loop with the equivalent loop from Homo sapiens AGO2 (HsAGO2) changes AtAGO10 miRNA loading behavior such that 3' region mismatches are ignored and mismatches opposite the guide 5' end instead drive loading, as in HsAGO2. Thus, this study uncovers the molecular mechanism underlying the miR166/165 selectivity of AtAGO10, essential for plant development, and provides new insights into how miRNA duplex structures are recognized for sRNA sorting., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In oocytes, mRNA decay is essential for maturation and subsequent events, such as maternal-zygotic transition, zygotic genomic activation, and embryo development. Reversible N6-methyladenosine RNA methylation directly regulates transcription, pre-mRNA splicing, mRNA export, mRNA stability, and translation. Here, we identified that downregulation of N6-methyladenosine modification by microinjecting a methyltransferase-like 3 (Mettl3)-specific small interfering RNA into mouse germinal vesicle oocytes led to defects in meiotic spindles and the first polar body extrusion during maturation in vitro. By further quantitative real-time polymerase chain reaction and Poly(A)-tail assay analysis, we found that N6-methyladenosine methylation mainly acts by reducing deadenylation of mRNAs mediated by the carbon catabolite repression 4-negative on TATA less system, thereby causing mRNA accumulation in oocytes. Meanwhile, transcriptome analysis of germinal vesicle oocytes revealed the downregulation of transcripts of several genes encoding ribosomal subunits proteins in the Mettl3 small interfering RNA-treated group, suggesting that N6-methyladenosine modification might affect translation. Together, our results indicate that RNA methylation accelerates mRNA decay, confirming the critical role of RNA clearance in oocyte maturation., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Previously we demonstrated that in patients with preeclampsia elevated levels of endogenous Na/K-ATPase inhibitor, marinobufagenin, cause inhibition of Friend leukemia virus integration 1 (Fli1), a negative regulator of collagen-1 synthesis. We hypothesized that in vitro silencing of Fli1 in healthy human umbilical arteries would be associated with an increase in collagen-1 output, similar to the effect of preeclampsia in rat and human tissues., Methods: The isolated segments of healthy human umbilical arteries were tested for sensitivity to MBG and Fli1 silencing with Fli1 siRNA or control siRNA., Results: Following 24-hour incubation of arteries with nanomolar concentrations of marinobufagenin, Fli1 expression was inhibited 5-fold (P < 0.001), and synthesis of collagen-1 increased 3 times (P < 0.01). Twenty-four-hour incubation of umbilical artery fragments with Fli1 siRNA caused a dramatic decrease of Fli1 (7-fold; P < 0.001) and cytoplasmic PKC δ (4-fold; P < 0.001) expression in comparison to control siRNA or untreated control, followed by elevation in procollagen (3-fold; P < 0.001) and collagen-1 (3-fold; P < 0.001) levels in vascular tissue., Conclusions: Our results show that after silencing the Fli1 gene in healthy human umbilical arteries a new phenotype emerges which is typical for preeclampsia and is associated with vascular fibrosis., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Journal of Hypertension, Ltd. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The combination of genome-editing and epitope tagging provides a powerful strategy to study proteins with high affinity and specificity while preserving their physiological expression patterns. However, stably modifying endogenous genes in cells that do not allow for clonal selection has been challenging. Here, we present a simple and fast strategy to generate stable, endogenously tagged alleles in a non-transformed cell culture model. At the example of piwi in Drosophila ovarian somatic sheath cells, we show that this strategy enables the generation of an N-terminally tagged protein that emulates the expression level and subcellular localization of the wild type protein and forms functional Piwi-piRNA complexes. We further present a concise workflow to establish endogenously N-terminally and C-terminally tagged proteins, and knockout alleles through rapid selection of cell pools in fly and human models., (Published by Oxford University Press on behalf of Nucleic Acids Research 2022.)

PIWI proteins and PIWI-interacting RNAs (piRNAs) are specifically expressed in animal germlines and play essential roles during gametogenesis in animals. The primary function of PIWI/piRNAs is known to silence transposable elements for protecting genome integrity in animal germlines, while their roles beyond silencing transposons are also documented by us and others. In particular, we show that mouse PIWIL1 (MIWI)/piRNAs play a dual role in regulating protein-coding genes in mouse spermatids through interacting with different protein factors in a developmental stage-dependent manner, including translationally activating a subset of AU-rich element-containing mRNAs in round spermatids and inducing massive mRNA degradation in late spermatids. We further show that MIWI is eliminated through the ubiquitin-26S proteasome pathway during late spermiogenesis. By exploring the biological function of MIWI ubiquitination by APC/C, we identified ubiquitination-deficient mutations in human PIWIL1 of infertile men and further established their causative role in male infertility in mouse model, supporting PIWIL1 as a human male infertility-relevant gene. Additionally, we reported that PIWIL1, aberrantly induced in human tumors, functions as an oncoprotein in a piRNA-independent manner in cancer cells. In the current review, we summarize our latest findings regarding the roles and mechanisms of PIWIL1 and piRNAs in mouse spermatids and human diseases, and discuss the related works in the field., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The histone variant H3.3 is encoded by two distinct genes, H3f3a and H3f3b, exhibiting identical amino-acid sequence. H3.3 is required for spermatogenesis, but the molecular mechanism of its spermatogenic function remains obscure. Here, we have studied the role of each one of H3.3A and H3.3B proteins in spermatogenesis. We have generated transgenic conditional knock-out/knock-in (cKO/KI) epitope-tagged FLAG-FLAG-HA-H3.3B (H3.3BHA) and FLAG-FLAG-HA-H3.3A (H3.3AHA) mouse lines. We show that H3.3B, but not H3.3A, is required for spermatogenesis and male fertility. Analysis of the molecular mechanism unveils that the absence of H3.3B led to alterations in the meiotic/post-meiotic transition. Genome-wide RNA-seq reveals that the depletion of H3.3B in meiotic cells is associated with increased expression of the whole sex X and Y chromosomes as well as of both RLTR10B and RLTR10B2 retrotransposons. In contrast, the absence of H3.3B resulted in down-regulation of the expression of piRNA clusters. ChIP-seq experiments uncover that RLTR10B and RLTR10B2 retrotransposons, the whole sex chromosomes and the piRNA clusters are markedly enriched of H3.3. Taken together, our data dissect the molecular mechanism of H3.3B functions during spermatogenesis and demonstrate that H3.3B, depending on its chromatin localization, is involved in either up-regulation or down-regulation of expression of defined large chromatin regions., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In plants, the biogenesis of small interfering RNA (siRNA) requires a family of RNA-dependent RNA polymerases that convert single-stranded RNA (ssRNA) into double-stranded RNA (dsRNA), which is subsequently cleaved into defined lengths by Dicer endonucleases. Here, we determined the structure of maize (Zea mays) RNA-DEPENDENT RNA POLYMERASE 2 (ZmRDR2) in the closed and open conformations. The core catalytic region of ZmRDR2 possesses the canonical DNA-dependent RNA polymerase (DdRP) catalytic sites, pointing to a shared RNA production mechanism between DdRPs and plant RDR-family proteins. Apo-ZmRDR2 adopts a highly compact structure, representing an inactive closed conformation. By contrast, adding RNA induced a significant conformational change in the ZmRDR2 Head domain that opened the RNA binding tunnel, suggesting this is an active elongation conformation of ZmRDR2. Overall, our structural studies trapped both the active and inactive conformations of ZmRDR2, providing insights into the molecular mechanism of dsRNA synthesis during plant siRNA production., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Extrahepatic delivery of small interfering RNAs (siRNAs) may have applications in the development of novel therapeutic approaches. However, reports on such approaches are limited, and the scarcity of reports concerning the systemically targeted delivery of siRNAs with effective gene silencing activity presents a challenge. We herein report for the first time the targeted delivery of CD206-targetable chemically modified mannose-siRNA (CMM-siRNA) conjugates to macrophages and dendritic cells (DCs). CMM-siRNA exhibited a strong binding ability to CD206 and selectively delivered contents to CD206-expressing macrophages and DCs. Furthermore, the conjugates demonstrated strong gene silencing ability with long-lasting effects and protein downregulation in CD206-expressing cells in vivo. These findings could broaden the use of siRNA technology, provide additional therapeutic opportunities, and establish a basis for further innovative approaches for the targeted delivery of siRNAs to not only macrophages and DCs but also other cell types., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Monocot DICER-LIKE3 (DCL3) and DCL5 produce distinct 24-nt small interfering RNAs (siRNAs), heterochromatic siRNAs (hc-siRNAs) and phased secondary siRNAs (phasiRNAs), respectively. The former small RNAs are linked to silencing of transposable elements and heterochromatic repeats, and the latter to reproductive processes. It is assumed that these DCLs evolved from an ancient 'eudicot-type' DCL3 ancestor, which may have produced both types of siRNAs. However, how functional differentiation was achieved after gene duplication remains elusive. Here, we find that monocot DCL3 and DCL5 exhibit biochemically distinct preferences for 5' phosphates and 3' overhangs, consistent with the structural properties of their in vivo double-stranded RNA substrates. Importantly, these distinct substrate specificities are determined by the PAZ domains of DCL3 and DCL5, which have accumulated mutations during the course of evolution. These data explain the mechanism by which these DCLs cleave their cognate substrates from a fixed end, ensuring the production of functional siRNAs. Our study also indicates how plants have diversified and optimized RNA silencing mechanisms during evolution., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In mammalian cells, small non-coding RNAs (sncRNAs) negatively regulate gene expression in a pathway known as RNA interference (RNAi). RNAi can be categorized into post-transcriptional gene silencing (PTGS), which involves the cleavage of target messenger RNA (mRNA) or inhibition of translation in the cytoplasm, and transcriptional gene silencing (TGS), which is mediated by the establishment of repressive epigenetic marks at target loci. Transfer RNAs (tRNAs), which are essential for translation, can be processed into small ncRNAs, termed tRNA-derived small RNAs (tsRNAs). The biogenesis of tsRNAs and their role in gene expression regulation has not yet been fully understood. Here, we show that Dicer dependent tsRNAs promote gene silencing through a mechanism distinct from PTGS and TGS. tsRNAs can lead to downregulation of target genes by targeting introns via nascent RNA silencing (NRS) in nuclei. Furthermore, we show that Ago2 slicer activity is required for this mechanism. Synthetic tsRNAs can significantly reduce expression of a target gene at both RNA and protein levels. Target genes regulated by NRS are associated with various diseases, which further underpins its biological significance. Finally, we show that NRS is evolutionarily conserved and has the potential to be explored as a novel synthetic sRNA based therapeutic., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Small noncoding RNAs such as piRNAs are guides for Argonaute proteins, enabling sequence-specific, post-transcriptional regulation of gene expression. The piRNAs of Caenorhabditis elegans have been observed to bind targets with high mismatch tolerance and appear to lack specific transposon targets, unlike piRNAs in Drosophila melanogaster and other organisms. These observations support a model in which C. elegans piRNAs provide a broad, indiscriminate net of silencing, competing with siRNAs associated with the CSR-1 Argonaute that specifically protect self-genes from silencing. However, the breadth of piRNA targeting has not been subject to in-depth quantitative analysis, nor has it been explained how piRNAs are distributed across sequence space to achieve complete coverage. Through a bioinformatic analysis of piRNA sequences, incorporating an original data-based metric of piRNA-target distance, we demonstrate that C. elegans piRNAs are functionally random, in that their coverage of sequence space is comparable to that of random sequences. By possessing a sufficient number of distinct, essentially random piRNAs, C. elegans is able to target arbitrary nonself sequences with high probability. We extend this approach to a selection of other nematodes, finding results which elucidate the mechanism by which nonself mRNAs are silenced, and have implications for piRNA evolution and biogenesis., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A critical challenge for the successful development of RNA interference-based therapeutics therapeutics has been the enhancement of their in vivo metabolic stability. In therapeutically relevant, fully chemically modified small interfering RNAs (siRNAs), modification of the two terminal phosphodiester linkages in each strand of the siRNA duplex with phosphorothioate (PS) is generally sufficient to protect against exonuclease degradation in vivo. Since PS linkages are chiral, we systematically studied the properties of siRNAs containing single chiral PS linkages at each strand terminus. We report an efficient and simple method to introduce chiral PS linkages and demonstrate that Rp diastereomers at the 5' end and Sp diastereomers at the 3' end of the antisense siRNA strand improved pharmacokinetic and pharmacodynamic properties in a mouse model. In silico modeling studies provide mechanistic insights into how the Rp isomer at the 5' end and Sp isomer at the 3' end of the antisense siRNA enhance Argonaute 2 (Ago2) loading and metabolic stability of siRNAs in a concerted manner., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A promising approach to tackle the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. So far it is unclear, which viral replication steps can be efficiently inhibited with siRNAs. Here, we report that siRNAs can target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, and thereby terminate replication before start of transcription and prevent virus-induced cell death. Coronaviruses replicate via negative sense RNA intermediates using a unique discontinuous transcription process. As a result, each viral RNA contains identical sequences at the 5' and 3' end. Surprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting common sequences shared by all viral transcripts allowed simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. The most effective suppression of viral replication and spread, however, was achieved by siRNAs that targeted open reading frame 1 (ORF1) which only exists in gRNA. In contrast, siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs leading to an impaired antiviral efficacy. Verifying the translational relevance of these findings, we show that a chemically modified siRNA that targets a highly conserved region of ORF1, inhibited SARS-CoV-2 replication ex vivo in explants of the human lung. Our work encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with specifically targeting gRNA, might be key for high antiviral efficacy., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

PIWI-interacting RNAs (piRNAs) and their partnering PIWI proteins defend the animal germline against transposable elements and play a crucial role in fertility. Numerous studies in the past have uncovered many additional functions of the piRNA pathway, including gene regulation, anti-viral defense, and somatic transposon repression. Further, comparative analyses across phylogenetic groups showed that the PIWI/piRNA system evolves rapidly and exhibits great evolutionary plasticity. However, the presence of so-called piRNA clusters as the major source of piRNAs is common to nearly all metazoan species. These genomic piRNA-producing loci are highly divergent across taxa and critically influence piRNA populations in different evolutionary lineages. We launched the initial version of the piRNA cluster database to facilitate research on regulation and evolution of piRNA-producing loci across tissues und species. In recent years the amount of small RNA sequencing data that was generated and the abundance of species that were studied has grown rapidly. To keep up with this recent progress, we have released a major update for the piRNA cluster database (https://www.smallrnagroup.uni-mainz.de/piRNAclusterDB), expanding it from 12 to a total of 51 species with hundreds of new datasets, and revised its overall structure to enable easy navigation through this large amount of data., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)