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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 -
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 -
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 -
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 Plants Mar 2021Plants use extracellular vesicles (EVs) to transport small RNAs (sRNAs) into their fungal pathogens and silence fungal virulence-related genes through a phenomenon...
Plants use extracellular vesicles (EVs) to transport small RNAs (sRNAs) into their fungal pathogens and silence fungal virulence-related genes through a phenomenon called 'cross-kingdom RNAi'. It remains unknown, however, how sRNAs are selectively loaded into EVs. Here, we identified several RNA-binding proteins in Arabidopsis, including Argonaute 1 (AGO1), RNA helicases (RHs) and annexins (ANNs), which are secreted by exosome-like EVs. AGO1, RH11 and RH37 selectively bind to EV-enriched sRNAs but not to non-EV-associated sRNAs, suggesting that they contribute to the selective loading of sRNAs into EVs. Conversely, ANN1 and ANN2 bind to sRNAs non-specifically. The ago1, rh11 rh37 and ann1 ann2 mutants showed reduced secretion of sRNAs in EVs, demonstrating that these RNA-binding proteins play an important role in sRNA loading and/or stabilization in EVs. Furthermore, rh11 rh37 and ann1 ann2 showed increased susceptibility to Botrytis cinerea, suggesting that RH11, RH37, ANN1 and ANN2 positively regulate plant immunity against B. cinerea.
Topics: Annexins; Arabidopsis; Arabidopsis Proteins; Argonaute Proteins; Botrytis; DEAD-box RNA Helicases; Extracellular Vesicles; Plant Diseases; Proteome; RNA, Plant; RNA, Small Interfering; RNA-Binding Proteins; Tetraspanins
PubMed: 33633358
DOI: 10.1038/s41477-021-00863-8 -
Cell Research Feb 2021RNA interference (RNAi) has been thought to be a gene-silencing pathway present in most eukaryotic cells to safeguard the genome against retrotransposition. Small...
RNA interference (RNAi) has been thought to be a gene-silencing pathway present in most eukaryotic cells to safeguard the genome against retrotransposition. Small interfering RNAs (siRNAs) have also become a powerful tool for studying gene functions. Given the endosymbiotic hypothesis that mitochondria originated from prokaryotes, mitochondria have been generally assumed to lack active RNAi; however, certain bacteria have Argonaute homologs and various reports suggest the presence of specific microRNAs and nuclear genome (nDNA)-encoded Ago2 in the mitochondria. Here we report that transfected siRNAs are not only able to enter the matrix of mitochondria, but also function there to specifically silence targeted mitochondrial transcripts. The mitoRNAi effect is readily detectable at the mRNA level, but only recordable on relatively unstable proteins, such as the mtDNA-encoded complex IV subunits. We also apply mitoRNAi to directly determine the postulated crosstalk between individual respiratory chain complexes, and our result suggests that the controversial observations previously made in patient-derived cells might result from differential adaptation in different cell lines. Our findings bring a new tool to study mitochondrial biology.
Topics: Animals; Argonaute Proteins; DNA, Mitochondrial; Fibroblasts; HEK293 Cells; HeLa Cells; Humans; Membrane Potential, Mitochondrial; Mice; Mitochondria; Myocytes, Cardiac; Oxygen; RNA Interference; RNA, Messenger; RNA, Small Interfering; Transfection
PubMed: 32807841
DOI: 10.1038/s41422-020-00394-5 -
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 -
Cell Host & Microbe Sep 2021RNA interference (RNAi) is an across-kingdom gene regulatory and defense mechanism. However, little is known about how organisms sense initial cues to mobilize RNAi....
RNA interference (RNAi) is an across-kingdom gene regulatory and defense mechanism. However, little is known about how organisms sense initial cues to mobilize RNAi. Here, we show that wounding to Nicotiana benthamiana cells during virus intrusion activates RNAi-related gene expression through calcium signaling. A rapid wound-induced elevation in calcium fluxes triggers calmodulin-dependent activation of calmodulin-binding transcription activator-3 (CAMTA3), which activates RNA-dependent RNA polymerase-6 and Bifunctional nuclease-2 (BN2) transcription. BN2 stabilizes mRNAs encoding key components of RNAi machinery, notably AGONAUTE1/2 and DICER-LIKE1, by degrading their cognate microRNAs. Consequently, multiple RNAi genes are primed for combating virus invasion. Calmodulin-, CAMTA3-, or BN2-knockdown/knockout plants show increased susceptibility to geminivirus, cucumovirus, and potyvirus. Notably, Geminivirus V2 protein can disrupt the calmodulin-CAMTA3 interaction to counteract RNAi defense. These findings link Ca signaling to RNAi and reveal versatility of host antiviral defense and viral counter-defense.
Topics: Argonaute Proteins; Calcium; Calcium Signaling; Calmodulin; Cucumovirus; Endonucleases; Geminiviridae; MicroRNAs; Plant Diseases; Plants; Potyviridae; RNA Interference; RNA, Small Interfering; RNA-Dependent RNA Polymerase; Ribonuclease III; Nicotiana; Transcription Factors
PubMed: 34352216
DOI: 10.1016/j.chom.2021.07.003