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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 -
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 -
The Plant Cell Feb 2016Argonaute (AGO) family proteins are effectors of RNAi in eukaryotes. AGOs bind small RNAs and use them as guides to silence target genes or transposable elements at the... (Review)
Review
Argonaute (AGO) family proteins are effectors of RNAi in eukaryotes. AGOs bind small RNAs and use them as guides to silence target genes or transposable elements at the transcriptional or posttranscriptional level. Eukaryotic AGO proteins share common structural and biochemical properties and function through conserved core mechanisms in RNAi pathways, yet plant AGOs have evolved specialized and diversified functions. This Review covers the general features of AGO proteins and highlights recent progress toward our understanding of the mechanisms and functions of plant AGOs.
Topics: Argonaute Proteins; Models, Biological; Phylogeny; Plant Proteins; Plants; RNA Interference; RNA, Plant; RNA, Small Interfering
PubMed: 26869699
DOI: 10.1105/tpc.15.00920 -
International Journal of Molecular... Feb 2016Small RNAs are essential for a variety of cellular functions. Argonaute (AGO) proteins are associated with all of the different classes of small RNAs, and are... (Review)
Review
Small RNAs are essential for a variety of cellular functions. Argonaute (AGO) proteins are associated with all of the different classes of small RNAs, and are indispensable in small RNA-mediated regulatory pathways. AGO proteins have been identified in various types of stem cells in diverse species from plants and animals. This review article highlights recent progress on how AGO proteins and AGO-bound small RNAs regulate the self-renewal and differentiation of distinct stem cell types, including pluripotent, germline, somatic, and cancer stem cells.
Topics: Animals; Argonaute Proteins; Carrier Proteins; Gene Expression Regulation; Humans; Multigene Family; Protein Binding; Protein Interaction Domains and Motifs; RNA, Small Untranslated; Stem Cells
PubMed: 26861290
DOI: 10.3390/ijms17020208 -
Nature Communications Dec 2018Recognition and repression of RNA targets by Argonaute proteins guided by small RNAs is the essence of RNA interference in eukaryotes. Argonaute proteins with diverse... (Review)
Review
Recognition and repression of RNA targets by Argonaute proteins guided by small RNAs is the essence of RNA interference in eukaryotes. Argonaute proteins with diverse structures are also found in many bacterial and archaeal genomes. Recent studies revealed that, similarly to their eukaryotic counterparts, prokaryotic Argonautes (pAgos) may function in cell defense against foreign genetic elements but, in contrast, preferably act on DNA targets. Many crucial details of the pAgo action, and the roles of a plethora of pAgos with non-conventional architecture remain unknown. Here, we review available structural and biochemical data on pAgos and discuss their possible functions in host defense and other genetic processes in prokaryotic cells.
Topics: Argonaute Proteins; Catalysis; Catalytic Domain; DNA; Eukaryota; Eukaryotic Cells; Genetic Engineering; Immune System; Prokaryotic Cells; Protein Binding; Protein Domains; Protein Interaction Domains and Motifs; RNA; RNA Interference; RNA, Small Interfering
PubMed: 30514832
DOI: 10.1038/s41467-018-07449-7 -
Journal of Molecular Biology Aug 2017Nearly every cell in the human body contains a set of programmable gene-silencing proteins named Argonaute. Argonaute proteins mediate gene regulation by small RNAs and... (Review)
Review
Nearly every cell in the human body contains a set of programmable gene-silencing proteins named Argonaute. Argonaute proteins mediate gene regulation by small RNAs and thereby contribute to cellular homeostasis during diverse physiological process, such as stem cell maintenance, fertilization, and heart development. Over the last decade, remarkable progress has been made toward understanding Argonaute proteins, small RNAs, and their roles in eukaryotic biology. Here, we review current understanding of Argonaute proteins from a structural prospective and discuss unanswered questions surrounding this fascinating class of enzymes.
Topics: Argonaute Proteins; Eukaryota; Eukaryotic Cells; RNA Interference
PubMed: 28757069
DOI: 10.1016/j.jmb.2017.07.018