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The EMBO Journal May 2024The RNA-silencing effector ARGONAUTE10 influences cell fate in plant shoot and floral meristems. ARGONAUTE10 also accumulates in the root apical meristem (RAM), yet its...
The RNA-silencing effector ARGONAUTE10 influences cell fate in plant shoot and floral meristems. ARGONAUTE10 also accumulates in the root apical meristem (RAM), yet its function(s) therein remain elusive. Here, we show that ARGONAUTE10 is expressed in the root cell initials where it controls overall RAM activity and length. ARGONAUTE10 is also expressed in the stele, where post-transcriptional regulation confines it to the root tip's pro-vascular region. There, variations in ARGONAUTE10 levels modulate metaxylem-vs-protoxylem specification. Both ARGONAUTE10 functions entail its selective, high-affinity binding to mobile miR165/166 transcribed in the neighboring endodermis. ARGONAUTE10-bound miR165/166 is degraded, likely via SMALL-RNA-DEGRADING-NUCLEASES1/2, thus reducing miR165/166 ability to silence, via ARGONAUTE1, the transcripts of cell fate-influencing transcription factors. These include PHABULOSA (PHB), which controls meristem activity in the initials and xylem differentiation in the pro-vasculature. During early germination, PHB transcription increases while dynamic, spatially-restricted transcriptional and post-transcriptional mechanisms reduce and confine ARGONAUTE10 accumulation to the provascular cells surrounding the newly-forming xylem axis. Adequate miR165/166 concentrations are thereby channeled along the ARGONAUTE10-deficient yet ARGONAUTE1-proficient axis. Consequently, inversely-correlated miR165/166 and PHB gradients form preferentially along the axis despite ubiquitous PHB transcription and widespread miR165/166 delivery inside the whole vascular cylinder.
Topics: Arabidopsis; MicroRNAs; Meristem; Arabidopsis Proteins; Argonaute Proteins; Xylem; Gene Expression Regulation, Plant; Plant Roots
PubMed: 38565948
DOI: 10.1038/s44318-024-00071-y -
The EMBO Journal May 2024A key question in plant biology is how oriented cell divisions are integrated with patterning mechanisms to generate organs with adequate cell type allocation. In the...
A key question in plant biology is how oriented cell divisions are integrated with patterning mechanisms to generate organs with adequate cell type allocation. In the root vasculature, a gradient of miRNA165/6 controls the abundance of HD-ZIP III transcription factors, which in turn control cell fate and spatially restrict vascular cell proliferation to specific cells. Here, we show that vascular development requires the presence of ARGONAUTE10, which is thought to sequester miRNA165/6 and protect HD-ZIP III transcripts from degradation. Our results suggest that the miR165/6-AGO10-HDZIP III module acts by buffering cytokinin responses and restricting xylem differentiation. Mutants of AGO10 show faster growth rates and strongly enhanced survival under severe drought conditions. However, this superior performance is offset by markedly increased variation and phenotypic plasticity in sub-optimal carbon supply conditions. Thus, AGO10 is required for the control of formative cell division and coordination of robust cell fate specification of the vasculature, while altering its expression provides a means to adjust phenotypic plasticity.
Topics: Arabidopsis; Arabidopsis Proteins; Argonaute Proteins; Cell Division; Plant Roots; Gene Expression Regulation, Plant; MicroRNAs; Cell Differentiation; Xylem
PubMed: 38565947
DOI: 10.1038/s44318-024-00072-x -
PeerJ 2024Environmental stresses diversely affect multiple processes related to the growth, development, and yield of many crops worldwide. In response, plants have developed... (Review)
Review
Environmental stresses diversely affect multiple processes related to the growth, development, and yield of many crops worldwide. In response, plants have developed numerous sophisticated defense mechanisms at the cellular and subcellular levels to react and adapt to biotic and abiotic stressors. RNA silencing, which is an innate immune mechanism, mediates sequence-specific gene expression regulation in higher eukaryotes. ARGONAUTE (AGO) proteins are essential components of the RNA-induced silencing complex (RISC). They bind to small noncoding RNAs (sRNAs) and target complementary RNAs, causing translational repression or triggering endonucleolytic cleavage pathways. In this review, we aim to illustrate the recently published molecular functions, regulatory mechanisms, and biological roles of AGO family proteins in model plants and cash crops, especially in the defense against diverse biotic and abiotic stresses, which could be helpful in crop improvement and stress tolerance in various plants.
Topics: Plant Proteins; Argonaute Proteins; Gene Expression Regulation, Plant; RNA-Induced Silencing Complex; Crops, Agricultural
PubMed: 38560454
DOI: 10.7717/peerj.17115 -
Nature Communications Mar 2024Piwi-interacting RNAs (piRNAs) are genomically encoded small RNAs that engage Piwi Argonaute proteins to direct mRNA surveillance and transposon silencing. Despite...
Piwi-interacting RNAs (piRNAs) are genomically encoded small RNAs that engage Piwi Argonaute proteins to direct mRNA surveillance and transposon silencing. Despite advances in understanding piRNA pathways and functions, how the production of piRNA is regulated remains elusive. Here, using a genetic screen, we identify casein kinase II (CK2) as a factor required for piRNA pathway function. We show that CK2 is required for the localization of PRG-1 and for the proper localization of several factors that comprise the 'upstream sequence transcription complex' (USTC), which is required for piRNA transcription. Loss of CK2 impairs piRNA levels suggesting that CK2 promotes USTC function. We identify the USTC component twenty-one-U fouled-up 4 (TOFU-4) as a direct substrate for CK2. Our findings suggest that phosphorylation of TOFU-4 by CK2 promotes the assembly of USTC and piRNA transcription. Notably, during the aging process, CK2 activity declines, resulting in the disassembly of USTC, decreased piRNA production, and defects in piRNA-mediated gene silencing, including transposons silencing. These findings highlight the significance of posttranslational modification in regulating piRNA biogenesis and its implications for the aging process. Overall, our study provides compelling evidence for the involvement of a posttranslational modification mechanism in the regulation of piRNA biogenesis.
Topics: Animals; Piwi-Interacting RNA; RNA, Small Interfering; Casein Kinase II; Phosphorylation; Soy Foods; Argonaute Proteins; Drosophila Proteins; Drosophila melanogaster
PubMed: 38548791
DOI: 10.1038/s41467-024-46882-9 -
Viruses Feb 2024Monkeypox virus (MPXV), the pathogen responsible for the infectious disease monkeypox, causes lesions on the skin, lymphadenopathy, and fever. It has posed a global...
Monkeypox virus (MPXV), the pathogen responsible for the infectious disease monkeypox, causes lesions on the skin, lymphadenopathy, and fever. It has posed a global public health threat since May 2022. Highly sensitive and specific detection of MPXV is crucial for preventing the spread of the disease. Argonaute (Ago) is an artificial DNA-guided restriction cleavage enzyme programmable with 5'-phosphorylated ssDNA sequences, which can be developed to specifically detect nucleic acids of pathogens. Here, a Ago-based system was established for the detection of MPXV-specific DNA targeting the F3L gene. A short amplicon of 79 bp could be obtained through a fast PCR procedure, which was completed within 45 min. Two 5'-phosphorylation guide DNAs were designed to guide Ago to cleave the amplicon to obtain an 18 bp 5'-phosphorylation sequence specific to MPXV, not to other orthopoxviruses (cowpox, variola, and vaccinia viruses). The 18 bp sequence guided Ago to cleave a designed probe specific to MPXV to emit fluorescence. With optimized conditions for the Ago-MPXV system, it could be completed in 60 min for the detection of the extracted MPXV DNA with the limit of detection (LOD) of 1.1 copies/reaction and did not depend on expensive instruments. Successful application of the Ago-MPXV system in sensitively detecting MPXV in simulated throat swabs, skin swabs, sera, and wastewater demonstrated the system's good performance. The Ago platform, with high sensitivity and specificity established here, has the potential to prevent the spread of MPXV.
Topics: Humans; Mpox (monkeypox); Pyrococcus furiosus; Monkeypox virus; DNA; Argonaute Proteins
PubMed: 38543748
DOI: 10.3390/v16030382 -
Nucleic Acids Research Jun 2024N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance...
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.
Topics: Animals; Male; Mice; Arginine; Argonaute Proteins; DNA Transposable Elements; Pachytene Stage; Piwi-Interacting RNA; RNA, Small Interfering; RNA-Binding Proteins; Spermatogenesis; Tudor Domain
PubMed: 38520410
DOI: 10.1093/nar/gkae193 -
Nature Communications Mar 2024The intermitochondrial cement (IMC) and chromatoid body (CB) are posited as central sites for piRNA activity in mice, with MIWI initially assembling in the IMC for piRNA...
The intermitochondrial cement (IMC) and chromatoid body (CB) are posited as central sites for piRNA activity in mice, with MIWI initially assembling in the IMC for piRNA processing before translocating to the CB for functional deployment. The regulatory mechanism underpinning MIWI translocation, however, has remained elusive. We unveil that piRNA loading is the trigger for MIWI translocation from the IMC to CB. Mechanistically, piRNA loading facilitates MIWI release from the IMC by weakening its ties with the mitochondria-anchored TDRKH. This, in turn, enables arginine methylation of MIWI, augmenting its binding affinity for TDRD6 and ensuring its integration within the CB. Notably, loss of piRNA-loading ability causes MIWI entrapment in the IMC and its destabilization in male germ cells, leading to defective spermatogenesis and male infertility in mice. Collectively, our findings establish the critical role of piRNA loading in MIWI translocation during spermatogenesis, offering new insights into piRNA biology in mammals.
Topics: Animals; Male; Mice; Argonaute Proteins; Germ Cell Ribonucleoprotein Granules; Germ Cells; Mammals; Mitochondria; Piwi-Interacting RNA; RNA, Small Interfering; Spermatogenesis; Testis
PubMed: 38491008
DOI: 10.1038/s41467-024-46664-3 -
Plant Cell Reports Mar 2024Barley AGO4 proteins complement expressional changes of epigenetically regulated genes in Arabidopsis ago4-3 mutant and show a distinct affinity for the 5' terminal...
Barley AGO4 proteins complement expressional changes of epigenetically regulated genes in Arabidopsis ago4-3 mutant and show a distinct affinity for the 5' terminal nucleotide of small RNAs, demonstrating functional conservation and divergence. The function of Argonaute 4 (AGO4) in Arabidopsis thaliana has been extensively characterized; however, its role in monocots, which have large genomes abundantly supplemented with transposable elements (TEs), remains elusive. The study of barley AGO4 proteins can provide insights into the conserved aspects of RNA-directed DNA methylation (RdDM) and could also have further applications in the field of epigenetics or crop improvement. Bioinformatic analysis of RNA sequencing data identified two active AGO4 genes in barley, HvAGO4a and HvAGO4b. These genes function similar to AtAGO4 in an Arabidopsis heterologous complementation system, primarily binding to 24-nucleotide long small RNAs (sRNAs) and triggering methylation at specific target loci. Like AtAGO4, HvAGO4B exhibits a preference for binding sRNAs with 5' adenine residue, while also accepting 5' guanine, uracil, and cytosine residues. In contrast, HvAGO4A selectively binds only sRNAs with a 5' adenine residue. The diverse binding capacity of barley AGO4 proteins is reflected in TE-derived sRNAs and in their varying abundance. Both barley AGO4 proteins effectively restore the levels of extrachromosomal DNA and transcript abundancy of the heat-activated ONSEN retrotransposon to those observed in wild-type Arabidopsis plants. Our study provides insight into the distinct binding specificities and involvement in TE regulation of barley AGO4 proteins in Arabidopsis by heterologous complementation.
Topics: Arabidopsis; Arabidopsis Proteins; Hordeum; RNA, Small Interfering; Nucleotides; Adenine; DNA Methylation; RNA, Plant
PubMed: 38480545
DOI: 10.1007/s00299-024-03177-z -
Nucleic Acids Research May 2024microRNAs regulate gene expression through interaction with an Argonaute protein. While some members of this protein family retain an enzymatic activity capable of...
microRNAs regulate gene expression through interaction with an Argonaute protein. While some members of this protein family retain an enzymatic activity capable of cleaving RNA molecules complementary to Argonaute-bound small RNAs, the role of the slicer residues in the canonical microRNA pathway is still unclear in animals. To address this, we created Caenorhabditis elegans strains with mutated slicer residues in the endogenous ALG-1 and ALG-2, the only two slicing Argonautes essential for the miRNA pathway in this animal model. We observe that the mutation in ALG-1 and ALG-2 catalytic residues affects overall animal fitness and causes phenotypes reminiscent of miRNA defects only when grown and maintained at restrictive temperature. Furthermore, the analysis of global miRNA expression shows that the slicer residues of ALG-1 and ALG-2 contribute differentially to regulate the level of specific subsets of miRNAs in young adults. We also demonstrate that altering the catalytic tetrad of those miRNA-specific Argonautes does not result in any defect in the production of canonical miRNAs. Together, these data support that the slicer residues of miRNA-specific Argonautes contribute to maintaining levels of a set of miRNAs for optimal viability and fitness in animals particularly exposed to specific growing conditions.
Topics: Animals; MicroRNAs; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Argonaute Proteins; Mutation; RNA-Binding Proteins
PubMed: 38477356
DOI: 10.1093/nar/gkae173 -
Nucleic Acids Research May 2024Many microRNA (miRNA)-guided Argonaute proteins can cleave RNA ('slicing'), even though miRNA-mediated target repression is generally cleavage-independent. Here we use...
Many microRNA (miRNA)-guided Argonaute proteins can cleave RNA ('slicing'), even though miRNA-mediated target repression is generally cleavage-independent. Here we use Caenorhabditis elegans to examine the role of catalytic residues of miRNA Argonautes in organismal development. In contrast to previous work, mutations in presumed catalytic residues did not interfere with development when introduced by CRISPR. We find that unwinding and decay of miRNA star strands is weakly defective in the catalytic residue mutants, with the largest effect observed in embryos. Argonaute-Like Gene 2 (ALG-2) is more dependent on catalytic residues for unwinding than ALG-1. The miRNAs that displayed the greatest (albeit minor) dependence on catalytic residues for unwinding tend to form stable duplexes with their star strand, and in some cases, lowering duplex stability alleviates dependence on catalytic residues. While a few miRNA guide strands are reduced in the mutant background, the basis of this is unclear since changes were not dependent on EBAX-1, an effector of Target-Directed miRNA Degradation (TDMD). Overall, this work defines a role for the catalytic residues of miRNA Argonautes in star strand decay; future work should examine whether this role contributes to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny.
Topics: Animals; Caenorhabditis elegans; MicroRNAs; Caenorhabditis elegans Proteins; Argonaute Proteins; RNA Stability; Mutation; Catalytic Domain; CRISPR-Cas Systems; RNA-Binding Proteins
PubMed: 38471816
DOI: 10.1093/nar/gkae170