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Nature Communications Feb 2024RNA interference (RNAi) is a conserved gene silencing process that exists in diverse organisms to protect genome integrity and regulate gene expression. In C. elegans,...
RNA interference (RNAi) is a conserved gene silencing process that exists in diverse organisms to protect genome integrity and regulate gene expression. In C. elegans, the majority of RNAi pathway proteins localize to perinuclear, phase-separated germ granules, which are comprised of sub-domains referred to as P granules, Mutator foci, Z granules, and SIMR foci. However, the protein components and function of the newly discovered SIMR foci are unknown. Here we demonstrate that HRDE-2 localizes to SIMR foci and interacts with the germline nuclear Argonaute HRDE-1 in its small RNA unbound state. In the absence of HRDE-2, HRDE-1 exclusively loads CSR-class 22G-RNAs rather than WAGO-class 22G-RNAs, resulting in inappropriate H3K9me3 deposition on CSR-target genes. Thus, our study demonstrates that the recruitment of unloaded HRDE-1 to germ granules, mediated by HRDE-2, is critical to ensure that the correct small RNAs are used to guide nuclear RNA silencing in the C. elegans germline.
Topics: Animals; Argonaute Proteins; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Germ Cells; RNA Interference; RNA, Small Interfering
PubMed: 38302462
DOI: 10.1038/s41467-024-45245-8 -
Vavilovskii Zhurnal Genetiki I Selektsii Dec 2023RNA interference is a gene silencing mechanism that plays an important role in genetic regulation in a number of eukaryotes. Argonaute (AGO) proteins are central to the...
The effect of salicylic and jasmonic acids on the activity of SnAGO genes in the fungus Stagonospora nodorum Berk. in in vitro culture and during infection of wheat plants.
RNA interference is a gene silencing mechanism that plays an important role in genetic regulation in a number of eukaryotes. Argonaute (AGO) proteins are central to the complex RNA interference system. However, their role in this mechanism, both in the host plant organism and in the pathogen, has not yet been fully elucidated. In this work, we identified and phylogenetically analyzed the SnAGO1, SnAGO2, SnAGO3, and SnAGO18 genes of the pathogenic fungus Stagonospora nodorum Berk., and analyzed their expression under conditions of infection of plants with varying degrees of resistance to the pathogen. The expression level against the background of plant immunization with the resistance inducers salicylic and jasmonic acids was assessed. In addition, the activity of these genes in the culture of the fungus in vitro was studied under the direct influence of resistance inducers on the mycelium of the fungus. Earlier activation of the SnAGO genes in in vitro culture under the influence of salicylic and jasmonic acids suggests their sensitivity to it. In an in vivo system, plant immunization to induce the accumulation of pathogen SnAGO transcripts was found. At the same time, the SnAGO genes of the fungus S. nodorum, when interacting with plant cells, reacted depending on the degree of host resistance: the highest level of transcripts in the resistant variety was observed. Thus, our data prove that the SnAGO genes of the fungus S. nodorum effectively interact with the host defense system in direct proportion to the degree of resistance of the latter to the pathogen. It was proposed to use the ratio of the transcriptional activity of the fungal reference gene SnTub to the host TaRLI gene as a marker of disease development in the initial period of the infectious process.
PubMed: 38239968
DOI: 10.18699/VJGB-23-115 -
G3 (Bethesda, Md.) Mar 2024Gene regulation in changing environments is critical for maintaining homeostasis. Some animals undergo a stress-resistant diapause stage to withstand harsh environmental...
Gene regulation in changing environments is critical for maintaining homeostasis. Some animals undergo a stress-resistant diapause stage to withstand harsh environmental conditions encountered during development. MicroRNAs are one mechanism for regulating gene expression during and after diapause. MicroRNAs downregulate target genes posttranscriptionally through the activity of the microRNA-induced silencing complex. Argonaute is the core microRNA-induced silencing complex protein that binds to both the microRNA and to other microRNA-induced silencing complex proteins. The 2 major microRNA Argonautes in the Caenorhabditis elegans soma are ALG-1 and ALG-2, which function partially redundantly. Loss of alg-1 [alg-1(0)] causes penetrant developmental phenotypes including vulval defects and the reiteration of larval cell programs in hypodermal cells. However, these phenotypes are essentially absent if alg-1(0) animals undergo a diapause stage called dauer. Levels of the relevant microRNAs are not higher during or after dauer, suggesting that activity of the microRNA-induced silencing complex may be enhanced in this context. To identify genes that are required for alg-1(0) mutants to develop without vulval defects after dauer, we performed an RNAi screen of genes encoding conserved kinases. We focused on kinases because of their known role in modulating microRNA-induced silencing complex activity. We found RNAi knockdown of 4 kinase-encoding genes, air-2, bub-1, chk-1, and nekl-3, caused vulval defects and reiterative phenotypes in alg-1(0) mutants after dauer, and that these defects were more penetrant in an alg-1(0) background than in wild type. Our results implicate these kinases as potential regulators of microRNA-induced silencing complex activity during postdauer development in C. elegans.
Topics: Animals; Caenorhabditis elegans; MicroRNAs; RNA Interference; Caenorhabditis elegans Proteins; Argonaute Proteins; Gene Expression Regulation, Developmental; Mutation; RNA-Binding Proteins
PubMed: 38226857
DOI: 10.1093/g3journal/jkae007 -
Nucleic Acids Research Feb 2024MicroRNAs (miRNAs) guide Argonaute (AGO) proteins to bind mRNA targets. Although most targets are destabilized by miRNA-AGO binding, some targets induce degradation of... (Review)
Review
MicroRNAs (miRNAs) guide Argonaute (AGO) proteins to bind mRNA targets. Although most targets are destabilized by miRNA-AGO binding, some targets induce degradation of the miRNA instead. These special targets are also referred to as trigger RNAs. All triggers identified thus far have binding sites with greater complementarity to the miRNA than typical target sites. Target-directed miRNA degradation (TDMD) occurs when trigger RNAs bind the miRNA-AGO complex and recruit the ZSWIM8 E3 ubiquitin ligase, leading to AGO ubiquitination and proteolysis and subsequent miRNA destruction. More than 100 different miRNAs are regulated by ZSWIM8 in bilaterian animals, and hundreds of trigger RNAs have been predicted computationally. Disruption of individual trigger RNAs or ZSWIM8 has uncovered important developmental and physiologic roles for TDMD across a variety of model organisms and cell types. In this review, we highlight recent progress in understanding the mechanistic basis and functions of TDMD, describe common features of trigger RNAs, outline best practices for validating trigger RNAs, and discuss outstanding questions in the field.
Topics: Animals; Argonaute Proteins; Binding Sites; MicroRNAs; Proteolysis; Ubiquitination; RNA Stability
PubMed: 38224449
DOI: 10.1093/nar/gkae003 -
PloS One 2024Sinonasal tumours are heterogeneous malignancies, presenting different histological features and clinical behaviour. Many studies emphasize the role of specific miRNA in...
Sinonasal tumours are heterogeneous malignancies, presenting different histological features and clinical behaviour. Many studies emphasize the role of specific miRNA in the development and progression of cancer, and their expression profiles could be used as prognostic biomarkers to predict the survival. Recently, using the next-generation sequencing (NGS)-based miRNome analysis the miR-34/miR-449 cluster was identified as miRNA superfamily involved in the pathogenesis of sinonasal cancers (SNCs). In the present study, we established an Argonaute-2 (AGO2): mRNA immunoprecipitation followed by high-throughput sequencing to analyse the regulatory role of miR-34/miR-449 in SNCs. Using this approach, we identified direct target genes (targetome), which were involved in regulation of RNA-DNA metabolic, transcript and epigenetic processes. In particular, the STK3, C9orf78 and STRN3 genes were the direct targets of both miR-34c and miR-449a, and their regulation are predictive of tumour progression. This study provides the first evidence that miR-34/miR-449 and their targets are deregulated in SNCs and could be proposed as valuable prognostic biomarkers.
Topics: Argonaute Proteins; Biomarkers; MicroRNAs; Neoplasms; Paranasal Sinuses; Humans
PubMed: 38215077
DOI: 10.1371/journal.pone.0295997 -
Nature Communications Jan 2024Argonaute (Ago) proteins are ubiquitous across all kingdoms of life. Eukaryotic Agos (eAgos) use small RNAs to recognize transcripts for RNA silencing in eukaryotes. In...
Argonaute (Ago) proteins are ubiquitous across all kingdoms of life. Eukaryotic Agos (eAgos) use small RNAs to recognize transcripts for RNA silencing in eukaryotes. In contrast, the functions of prokaryotic counterparts (pAgo) are less well known. Recently, short pAgos in conjunction with the associated TIR or Sir2 (SPARTA or SPARSA) were found to serve as antiviral systems to combat phage infections. Herein, we present the cryo-EM structures of nicotinamide adenine dinucleotide (NAD)-bound SPARSA with and without nucleic acids at resolutions of 3.1 Å and 3.6 Å, respectively. Our results reveal that the APAZ (Analogue of PAZ) domain and the short pAgo form a featured architecture similar to the long pAgo to accommodate nucleic acids. We further identified the key residues for NAD binding and elucidated the structural basis for guide RNA and target DNA recognition. Using structural comparisons, molecular dynamics simulations, and biochemical experiments, we proposed a putative mechanism for NAD hydrolysis in which an H186 loop mediates nucleophilic attack by catalytic water molecules. Overall, our study provides mechanistic insight into the antiphage role of the SPARSA system.
Topics: NAD; RNA, Guide, CRISPR-Cas Systems; Argonaute Proteins; Bacteriophages; Nucleic Acids
PubMed: 38200015
DOI: 10.1038/s41467-023-44660-7 -
Nucleic Acids Research Mar 2024Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind...
Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid targets and are responsible for gene expression regulation, mobile genome element silencing, and defence against viruses or plasmids. According to their domain organization, Agos are divided into long and short Agos. Long Agos found in prokaryotes (long-A and long-B pAgos) and eukaryotes (eAgos) comprise four major functional domains (N, PAZ, MID and PIWI) and two structural linker domains L1 and L2. The majority (∼60%) of pAgos are short pAgos, containing only the MID and inactive PIWI domains. Here we focus on the prokaryotic Argonaute AfAgo from Archaeoglobus fulgidus DSM4304. Although phylogenetically classified as a long-B pAgo, AfAgo contains only MID and catalytically inactive PIWI domains, akin to short pAgos. We show that AfAgo forms a heterodimeric complex with a protein encoded upstream in the same operon, which is a structural equivalent of the N-L1-L2 domains of long pAgos. This complex, structurally equivalent to a long PAZ-less pAgo, outperforms standalone AfAgo in guide RNA-mediated target DNA binding. Our findings provide a missing piece to one of the first and the most studied pAgos.
Topics: Archaeoglobus fulgidus; Argonaute Proteins; Bacteria; Eukaryota; Prokaryotic Cells; Protein Domains; RNA, Guide, CRISPR-Cas Systems; Archaeal Proteins
PubMed: 38197228
DOI: 10.1093/nar/gkad1241 -
Ecotoxicology and Environmental Safety Jan 2024Phthalates (PEs) are widely used plasticizers in polymer products, and humans are increasingly exposed to them. This study was designed to investigate the alleviative...
Phthalates (PEs) are widely used plasticizers in polymer products, and humans are increasingly exposed to them. This study was designed to investigate the alleviative effect of phytochemicals quercetin (Que) against male reproductive toxicity caused by the mixture of three commonly used PEs (MPEs), and further to explore the underlying mechanism. Forty-eight male SD rats were randomly and evenly divided into control group, Que group, MPEs group and MPEs+Que group (n = 12); The oral exposure doses of MPEs and Que were 450 mg/kg/d and 50 mg/kg/d, respectively. After 91 days of continuous intervention, compared with control group, the testes weight, epididymis weight, serum sex hormones, and anogenital distance were significantly decreased in MPEs group (P < 0.05); Testicular histopathological observation showed that all seminiferous tubules were atrophy, leydig cells were hyperplasia, spermatogenic cells growth were arrested in MPEs group. Ultrastructural observation of testicular germ cells showed that the edges of the nuclear membranes were indistinct, and the mitochondria were severely damaged with the cristae disrupted, decreased or even disappeared in MPEs group. Immunohistochemistry and Western blot analysis showed that testicular CYP11A1, CYP17A1 and 17β-HSD were up-regulated, while StAR, PIWIL1 and PIWIL2 were down-regulated in MPEs group (P < 0.05); However, the alterations of these parameters were restored in MPEs+Que group. The results indicated MPEs disturbed steroid hormone metabolism, and caused male reproductive injuries; whereas, Que could inhibit MPEs' male reproductive toxicity, which might relate to the restored regulation of steroid hormone metabolism.
Topics: Humans; Rats; Male; Animals; Quercetin; Rats, Sprague-Dawley; Testis; Gonadal Steroid Hormones; Steroids; Testosterone; Argonaute Proteins; Phthalic Acids
PubMed: 38171105
DOI: 10.1016/j.ecoenv.2023.115920 -
The Chinese Journal of Physiology 2023Proven by publications, long non-coding RNAs (lncRNAs) play critical roles in the development of clear cell renal cell carcinoma (ccRCC). Although lncRNA LINC00565 has...
Proven by publications, long non-coding RNAs (lncRNAs) play critical roles in the development of clear cell renal cell carcinoma (ccRCC). Although lncRNA LINC00565 has been implicated in the progression of various cancers, its biological effects on ccRCC remain unknown. This study aimed to investigate the biological functions of LINC00565, as well as its potential mechanism in ccRCC. Here, the expression data of mature microRNAs (miRNAs) (normal: 71, tumor: 545), messenger RNAs (mRNAs), and lncRNAs (normal: 72, tumor: 539) of ccRCC were acquired from The Cancer Genome Atlas (TCGA) database and subjected to differential expression analysis. Quantitative reverse transcriptase polymerase chain reaction analyzed the expression levels of LINC00565, miR-532-3p, and ADAM19 mRNA. TCGA database, dual-luciferase report detection, and Argonaute 2 RNA immunoprecipitation were utilized to confirm the relationships between LINC00565 and miR-532-3p and between miR-532-3p and ADAM19, respectively. The progression of ccRCC cells was determined via CCK-8, colony formation, scratch healing, and transwell assays. Western blot was applied to detect the protein levels of epithelial-mesenchymal transition markers and ADAM19. We herein suggested that LINC00565 was prominently upregulated in ccRCC tissues and cells. Knockdown of LINC00565 repressed cell progression. We further predicted and validated miR-532-3p as a target of LINC00565, and miR-532-3p could target ADAM19. Knockdown of LINC00565 resulted in ADAM19 level downregulation in ccRCC cells and suppressed miR-532-3p could restore ADAM19 level. Thus, the three RNAs constructed a ceRNA network. Overexpressed ADAM19 could eliminate the anticancer effects caused by knocking down LINC00565 on ccRCC cells. In conclusion, LINC00565 upregulated ADAM19 via absorbing miR-532-3p, thereby facilitating the progression of ccRCC cells.
Topics: Humans; Carcinoma, Renal Cell; RNA, Long Noncoding; Cell Line, Tumor; Cell Proliferation; MicroRNAs; Kidney Neoplasms; Cell Movement; Gene Expression Regulation, Neoplastic; ADAM Proteins
PubMed: 38149560
DOI: 10.4103/cjop.CJOP-D-23-00078 -
Nucleic Acids Research Mar 2024Much 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...
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.
Topics: Argonaute Proteins; DNA Transposable Elements; Helminth Proteins; Piwi-Interacting RNA; Platyhelminths; Proteins; RNA Interference; RNA, Small Interfering; Stem Cells; Animals
PubMed: 38142432
DOI: 10.1093/nar/gkad1212