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Nature Reviews. Molecular Cell Biology Feb 2023PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs that associate with proteins of the PIWI clade of the Argonaute family. First identified in animal... (Review)
Review
PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs that associate with proteins of the PIWI clade of the Argonaute family. First identified in animal germ line cells, piRNAs have essential roles in germ line development. The first function of PIWI-piRNA complexes to be described was the silencing of transposable elements, which is crucial for maintaining the integrity of the germ line genome. Later studies provided new insights into the functions of PIWI-piRNA complexes by demonstrating that they regulate protein-coding genes. Recent studies of piRNA biology, including in new model organisms such as golden hamsters, have deepened our understanding of both piRNA biogenesis and piRNA function. In this Review, we discuss the most recent advances in our understanding of piRNA biogenesis, the molecular mechanisms of piRNA function and the emerging roles of piRNAs in germ line development mainly in flies and mice, and in infertility, cancer and neurological diseases in humans.
Topics: Animals; Mice; Argonaute Proteins; DNA Transposable Elements; Germ Cells; Piwi-Interacting RNA; RNA, Small Interfering
PubMed: 36104626
DOI: 10.1038/s41580-022-00528-0 -
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 -
Molecular Cell Jan 2022Small RNAs regulate a wide variety of biological processes by repressing the expression of target genes at the transcriptional and post-transcriptional levels. To... (Review)
Review
Small RNAs regulate a wide variety of biological processes by repressing the expression of target genes at the transcriptional and post-transcriptional levels. To achieve these functions, small RNAs form RNA-induced silencing complex (RISC) together with a member of the Argonaute (AGO) protein family. RISC is directed by its bound small RNA to target complementary RNAs and represses their expression through mRNA cleavage, degradation, and/or translational repression. Many different factors fine-tune RISC activity and stability-from guide-target RNA complementarity to the recruitment of other protein partners to post-translational modifications of RISC itself. Here, we review recent progress in understanding RISC formation, action, and degradation, and discuss new, intriguing questions in the field.
Topics: Animals; Argonaute Proteins; Humans; MicroRNAs; Protein Binding; Protein Stability; Proteolysis; RNA; RNA Stability; RNA, Messenger; RNA, Small Interfering; RNA-Induced Silencing Complex
PubMed: 34942118
DOI: 10.1016/j.molcel.2021.11.026 -
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 -
Science (New York, N.Y.) Dec 2020MicroRNAs (miRNAs) act in concert with Argonaute (AGO) proteins to repress target messenger RNAs. After AGO loading, miRNAs generally exhibit slow turnover. An important...
MicroRNAs (miRNAs) act in concert with Argonaute (AGO) proteins to repress target messenger RNAs. After AGO loading, miRNAs generally exhibit slow turnover. An important exception occurs when miRNAs encounter highly complementary targets, which can trigger a process called target-directed miRNA degradation (TDMD). During TDMD, miRNAs undergo tailing and trimming, suggesting that this is an important step in the decay mechanism. We identified a cullin-RING ubiquitin ligase (CRL), containing the substrate adaptor ZSWIM8, that mediates TDMD. The ZSWIM8 CRL interacts with AGO proteins, promotes TDMD in a tailing and trimming-independent manner, and regulates miRNA expression in multiple cell types. These findings suggest a model in which the ZSWIM8 ubiquitin ligase mediates TDMD by directing proteasomal decay of miRNA-containing complexes engaged with highly complementary targets.
Topics: Argonaute Proteins; Gene Knockout Techniques; Humans; K562 Cells; MicroRNAs; RNA Stability; RNA, Long Noncoding; Ubiquitin-Protein Ligases
PubMed: 33184234
DOI: 10.1126/science.abc9546 -
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 -
Nature Jan 2024Argonaute (Ago) proteins mediate RNA- or DNA-guided inhibition of nucleic acids. Although the mechanisms used by eukaryotic Ago proteins and long prokaryotic Ago...
Argonaute (Ago) proteins mediate RNA- or DNA-guided inhibition of nucleic acids. Although the mechanisms used by eukaryotic Ago proteins and long prokaryotic Ago proteins (pAgos) are known, that used by short pAgos remains elusive. Here we determined the cryo-electron microscopy structures of a short pAgo and the associated TIR-APAZ proteins (SPARTA) from Crenotalea thermophila (Crt): a free-state Crt-SPARTA; a guide RNA-target DNA-loaded Crt-SPARTA; two Crt-SPARTA dimers with distinct TIR organization; and a Crt-SPARTA tetramer. These structures reveal that Crt-SPARTA is composed of a bilobal-fold Ago lobe that connects with a TIR lobe. Whereas the Crt-Ago contains a MID and a PIWI domain, Crt-TIR-APAZ has a TIR domain, an N-like domain, a linker domain and a trigger domain. The bound RNA-DNA duplex adopts a B-form conformation that is recognized by base-specific contacts. Nucleic acid binding causes conformational changes because the trigger domain acts as a 'roadblock' that prevents the guide RNA 5' ends and the target DNA 3' ends from reaching their canonical pockets; this disorders the MID domain and promotes Crt-SPARTA dimerization. Two RNA-DNA-loaded Crt-SPARTA dimers form a tetramer through their TIR domains. Four Crt-TIR domains assemble into two parallel head-to-tail-organized TIR dimers, indicating an NADase-active conformation, which is supported by our mutagenesis study. Our results reveal the structural basis of short-pAgo-mediated defence against invading nucleic acids, and provide insights for optimizing the detection of SPARTA-based programmable DNA sequences.
Topics: Argonaute Proteins; Cryoelectron Microscopy; DNA; Enzyme Activation; NAD+ Nucleosidase; Nucleic Acid Conformation; Nucleic Acids; Protein Conformation; RNA, Guide, CRISPR-Cas Systems; Mutagenesis
PubMed: 37783228
DOI: 10.1038/s41586-023-06665-6 -
Molecular Biology Reports May 2021Argonaute proteins are highly conserved and widely expressed in almost all organisms. They not only play a critical role in the biogenesis of small RNAs but also defend... (Review)
Review
Argonaute proteins are highly conserved and widely expressed in almost all organisms. They not only play a critical role in the biogenesis of small RNAs but also defend against invading nucleic acids via small RNA or DNA-mediated gene silencing pathways. One functional mechanism of Argonaute proteins is acting as a nucleic-acid-guided endonuclease, which can cleave targets complementary to DNA or RNA guides. The cleavage then leads to translational silencing directly or indirectly by recruiting additional silencing proteins. Here, we summarized the latest research progress in structural and biological studies of Argonaute proteins and pointed out their potential applications in the field of gene editing.
Topics: Argonaute Proteins; Endonucleases; Gene Editing; Gene Silencing; RNA, Small Interfering; RNA, Guide, CRISPR-Cas Systems
PubMed: 34117606
DOI: 10.1007/s11033-021-06476-w -
Methods in Molecular Biology (Clifton,... 2022MicroRNAs are RNAs of about 18-24 nucleotides in lengths, which are found in the small noncoding RNA class and have a crucial role in the posttranscriptional regulation...
MicroRNAs are RNAs of about 18-24 nucleotides in lengths, which are found in the small noncoding RNA class and have a crucial role in the posttranscriptional regulation of gene expression, cellular metabolic pathways, and developmental events. These small but essential molecules are first processed by Drosha and DGCR8 in the nucleus and then released into the cytoplasm, where they cleaved by Dicer to form the miRNA duplex. These duplexes are bound by the Argonaute (AGO) protein to form the RNA-induced silencing complex (RISC) in a process called RISC loading. Transcription of miRNAs, processing with Drosha and DGCR8 in the nucleus, cleavage by Dicer, binding to AGO proteins and forming RISC are the most critical steps in miRNA biogenesis. Additional molecules involved in biogenesis at these stages can enhance or inhibit these processes, which can radically change the fate of the cell. Biogenesis is regulated by many checkpoints at every step, primarily at the transcriptional level, in the nucleus, cytoplasm, with RNA regulation, RISC loading, miRNA strand selection, RNA methylation/uridylation, and turnover rate. Moreover, in recent years, different regulation mechanisms have been discovered in noncanonical Drosha or Dicer-independent pathways. This chapter seeks answers to how miRNA biogenesis and function are regulated through both canonical and non-canonical pathways.
Topics: Argonaute Proteins; Gene Expression Regulation; MicroRNAs; RNA-Binding Proteins; RNA-Induced Silencing Complex
PubMed: 34432271
DOI: 10.1007/978-1-0716-1170-8_1