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Science (New York, N.Y.) Dec 2019MicroRNAs (miRNAs) act within Argonaute proteins to guide repression of messenger RNA targets. Although various approaches have provided insight into target recognition,...
MicroRNAs (miRNAs) act within Argonaute proteins to guide repression of messenger RNA targets. Although various approaches have provided insight into target recognition, the sparsity of miRNA-target affinity measurements has limited understanding and prediction of targeting efficacy. Here, we adapted RNA bind-n-seq to enable measurement of relative binding affinities between Argonaute-miRNA complexes and all sequences ≤12 nucleotides in length. This approach revealed noncanonical target sites specific to each miRNA, miRNA-specific differences in canonical target-site affinities, and a 100-fold impact of dinucleotides flanking each site. These data enabled construction of a biochemical model of miRNA-mediated repression, which was extended to all miRNA sequences using a convolutional neural network. This model substantially improved prediction of cellular repression, thereby providing a biochemical basis for quantitatively integrating miRNAs into gene-regulatory networks.
Topics: Argonaute Proteins; Base Sequence; Gene Expression Regulation; HEK293 Cells; Humans; MicroRNAs; Protein Binding; Sequence Analysis, RNA
PubMed: 31806698
DOI: 10.1126/science.aav1741 -
Nature Reviews. Molecular Cell Biology Mar 2022Since the discovery of eukaryotic small RNAs as the main effectors of RNA interference in the late 1990s, diverse types of endogenous small RNAs have been characterized,... (Review)
Review
Since the discovery of eukaryotic small RNAs as the main effectors of RNA interference in the late 1990s, diverse types of endogenous small RNAs have been characterized, most notably microRNAs, small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). These small RNAs associate with Argonaute proteins and, through sequence-specific gene regulation, affect almost every major biological process. Intriguing features of small RNAs, such as their mechanisms of amplification, rapid evolution and non-cell-autonomous function, bestow upon them the capacity to function as agents of intercellular communications in development, reproduction and immunity, and even in transgenerational inheritance. Although there are many types of extracellular small RNAs, and despite decades of research, the capacity of these molecules to transmit signals between cells and between organisms is still highly controversial. In this Review, we discuss evidence from different plants and animals that small RNAs can act in a non-cell-autonomous manner and even exchange information between species. We also discuss mechanistic insights into small RNA communications, such as the nature of the mobile agents, small RNA signal amplification during transit, signal perception and small RNA activity at the destination.
Topics: Animals; Argonaute Proteins; MicroRNAs; Plants; RNA Interference; RNA, Small Interfering
PubMed: 34707241
DOI: 10.1038/s41580-021-00425-y -
Cell Apr 2022Argonaute proteins use single-stranded RNA or DNA guides to target complementary nucleic acids. This allows eukaryotic Argonaute proteins to mediate RNA interference and...
Argonaute proteins use single-stranded RNA or DNA guides to target complementary nucleic acids. This allows eukaryotic Argonaute proteins to mediate RNA interference and long prokaryotic Argonaute proteins to interfere with invading nucleic acids. The function and mechanisms of the phylogenetically distinct short prokaryotic Argonaute proteins remain poorly understood. We demonstrate that short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins form heterodimeric complexes. Upon guide RNA-mediated target DNA binding, four SPARTA heterodimers form oligomers in which TIR domain-mediated NAD(P)ase activity is unleashed. When expressed in Escherichia coli, SPARTA is activated in the presence of highly transcribed multicopy plasmid DNA, which causes cell death through NAD(P) depletion. This results in the removal of plasmid-invaded cells from bacterial cultures. Furthermore, we show that SPARTA can be repurposed for the programmable detection of DNA sequences. In conclusion, our work identifies SPARTA as a prokaryotic immune system that reduces cell viability upon RNA-guided detection of invading DNA.
Topics: Argonaute Proteins; DNA; Prokaryotic Cells; RNA, Guide, CRISPR-Cas Systems
PubMed: 35381200
DOI: 10.1016/j.cell.2022.03.012 -
Methods (San Diego, Calif.) Sep 2022Polymerase Chain Reaction (PCR) is the reigning gold standard for molecular diagnostics. However, the SARS-CoV-2 pandemic reveals an urgent need for new diagnostics that...
Polymerase Chain Reaction (PCR) is the reigning gold standard for molecular diagnostics. However, the SARS-CoV-2 pandemic reveals an urgent need for new diagnostics that provide users with immediate results without complex procedures or sophisticated equipment. These new demands have stimulated a tsunami of innovations that improve turnaround times without compromising the specificity and sensitivity that has established PCR as the paragon of diagnostics. Here we briefly introduce the origins of PCR and isothermal amplification, before turning to the emergence of CRISPR-Cas and Argonaute proteins, which are being coupled to fluorimeters, spectrometers, microfluidic devices, field-effect transistors, and amperometric biosensors, for a new generation of nucleic acid-based diagnostics.
Topics: Argonaute Proteins; CRISPR-Cas Systems; Humans; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction
PubMed: 35690249
DOI: 10.1016/j.ymeth.2022.06.002 -
Nature Sep 2021PIWI proteins use PIWI-interacting RNAs (piRNAs) to identify and silence transposable elements and thereby maintain genome integrity between metazoan generations. The...
PIWI proteins use PIWI-interacting RNAs (piRNAs) to identify and silence transposable elements and thereby maintain genome integrity between metazoan generations. The targeting of transposable elements by PIWI has been compared to mRNA target recognition by Argonaute proteins, which use microRNA (miRNA) guides, but the extent to which piRNAs resemble miRNAs is not known. Here we present cryo-electron microscopy structures of a PIWI-piRNA complex from the sponge Ephydatia fluviatilis with and without target RNAs, and a biochemical analysis of target recognition. Mirroring Argonaute, PIWI identifies targets using the piRNA seed region. However, PIWI creates a much weaker seed so that stable target association requires further piRNA-target pairing, making piRNAs less promiscuous than miRNAs. Beyond the seed, the structure of PIWI facilitates piRNA-target pairing in a manner that is tolerant of mismatches, leading to long-lived PIWI-piRNA-target interactions that may accumulate on transposable-element transcripts. PIWI ensures targeting fidelity by physically blocking the propagation of piRNA-target interactions in the absence of faithful seed pairing, and by requiring an extended piRNA-target duplex to reach an endonucleolytically active conformation. PIWI proteins thereby minimize off-targeting cellular mRNAs while defending against evolving genomic threats.
Topics: Animals; Argonaute Proteins; Cryoelectron Microscopy; Models, Molecular; Nucleic Acid Conformation; Porifera; RNA, Small Interfering; Substrate Specificity
PubMed: 34471284
DOI: 10.1038/s41586-021-03856-x -
Cell Jun 2018Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However,...
Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals. VIDEO ABSTRACT.
Topics: Animals; Argonaute Proteins; Cell Cycle; Gene Expression Regulation; Helminth Proteins; Planarians; Pluripotent Stem Cells; Principal Component Analysis; RNA Interference; RNA, Double-Stranded; RNA, Helminth; Regeneration; Sequence Analysis, RNA; Single-Cell Analysis; Tetraspanins; Whole-Body Irradiation
PubMed: 29906446
DOI: 10.1016/j.cell.2018.05.006 -
Nucleic Acids Research Jul 2022MicroRNAs (miRNAs) bind to complementary target RNAs and regulate their gene expression post-transcriptionally. These non-coding regulatory RNAs become functional after... (Review)
Review
MicroRNAs (miRNAs) bind to complementary target RNAs and regulate their gene expression post-transcriptionally. These non-coding regulatory RNAs become functional after loading into Argonaute (AGO) proteins to form the effector complexes. Humans have four AGO proteins, AGO1, AGO2, AGO3 and AGO4, which share a high sequence identity. Since most miRNAs are found across the four AGOs, it has been thought that they work redundantly, and AGO2 has been heavily studied as the exemplified human paralog. Nevertheless, an increasing number of studies have found that the other paralogs play unique roles in various biological processes and diseases. In the last decade, the structural study of the four AGOs has provided the field with solid structural bases. This review exploits the completed structural catalog to describe common features and differences in target specificity across the four AGOs.
Topics: Humans; Argonaute Proteins
PubMed: 35736234
DOI: 10.1093/nar/gkac519 -
Trends in Cell Biology Mar 2023Ribosome-associated protein quality control (RQC) is a protein surveillance mechanism that eliminates defective nascent polypeptides. The E3 ubiquitin ligase, Ltn1, is a... (Review)
Review
Ribosome-associated protein quality control (RQC) is a protein surveillance mechanism that eliminates defective nascent polypeptides. The E3 ubiquitin ligase, Ltn1, is a key regulator of RQC that targets substrates for ubiquitination. Argonaute proteins (AGOs) are central players in miRNA-mediated gene silencing and have recently been shown to also regulate RQC by facilitating Ltn1. Therefore, AGOs directly coordinate post-transcriptional gene silencing and RQC, ensuring efficient gene silencing. We summarize the principles of RQC and the functions of AGOs in miRNA-mediated gene silencing, and discuss how AGOs associate with the endoplasmic reticulum (ER) to assist Ltn1 in controlling RQC. We highlight that RQC not only eliminates defective nascent polypeptides but also removes unwanted protein products when AGOs participate.
Topics: Humans; Argonaute Proteins; Ribosomes; Ubiquitination; Ubiquitin-Protein Ligases; Saccharomyces cerevisiae Proteins; Peptides; MicroRNAs; Protein Biosynthesis
PubMed: 35981909
DOI: 10.1016/j.tcb.2022.07.007 -
The Plant Journal : For Cell and... Mar 2022Argonaute (AGO) proteins are central players in RNA interference in eukaryotes. They associate with small RNAs (sRNA) and lead to transcriptional or posttranscriptional...
Argonaute (AGO) proteins are central players in RNA interference in eukaryotes. They associate with small RNAs (sRNA) and lead to transcriptional or posttranscriptional silencing of targets, thereby regulating diverse biological processes. The molecular and biological functions of AGO proteins have been extensively characterized, particularly in a few angiosperm species, leading to the recognition that the AGO family has expanded to accommodate diverse sRNAs thereby performing diverse biological functions. However, understanding of the expansion of AGO proteins in plants is still limited, due to a dearth of knowledge of AGO proteins in green algal groups. Here, we identified more than 2900 AGO proteins from 244 plant species, including green algae, and performed a large-scale phylogenetic analysis. The phylogeny shows that the plant AGO family gave rise to four clades after the emergence of hydrobiontic algae and prior to the emergence of land plants. Subsequent parallel expansion in ferns and angiosperms resulted in eight main clades in angiosperms: AGO2, AGO7, AGO6, AGO4, AGO1, AGO10a, AGO10b and AGO5. On the basis of this phylogeny, we identified two novel AGO4 orthologs that Arabidopsis does not have, and redefined AGO10, which is composed of AGO10a and AGO10b. Finally, we propose a hypothetical evolutionary model of AGO proteins in plants. Our studies provide a deeper understanding of the phylogenetic relationships of AGO family members in the green lineage, which would help to further reveal their roles as RNAi effectors.
Topics: Arabidopsis; Argonaute Proteins; Magnoliopsida; Phylogeny; Plant Proteins; Plants
PubMed: 34845788
DOI: 10.1111/tpj.15615 -
Current Opinion in Microbiology Aug 2023Both eukaryotes and prokaryotes (archaea and bacteria) encode an arsenal of immune systems that protect the host against mobile genetic elements (MGEs) including... (Review)
Review
Both eukaryotes and prokaryotes (archaea and bacteria) encode an arsenal of immune systems that protect the host against mobile genetic elements (MGEs) including viruses, plasmids, and transposons. Whereas Argonaute proteins (Agos) are best known for post-transcriptional gene silencing in eukaryotes, in all domains of life, members from the highly diverse Argonaute protein family act as programmable immune systems. To this end, Agos are programmed with small single-stranded RNA or DNA guides to detect and silence complementary MGEs. Across and within the different domains of life, Agos function in distinct pathways and MGE detection can trigger various mechanisms that provide immunity. In this review, we delineate the diverse immune pathways and underlying mechanisms for both eukaryotic Argonautes (eAgos) and prokaryotic Argonautes (pAgos).
Topics: Argonaute Proteins; Prokaryotic Cells; Bacteria; Eukaryota; Archaea
PubMed: 37023508
DOI: 10.1016/j.mib.2023.102313