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Accounts of Chemical Research Jul 2012The delivery of genes or RNA interference (RNAi) agents can increase or decrease the expression of virtually any protein in a cell, and this process opens the path for... (Review)
Review
The delivery of genes or RNA interference (RNAi) agents can increase or decrease the expression of virtually any protein in a cell, and this process opens the path for cures to most diseases that afflict humans. However, the high molecular weight, anionic nature, and instability of nucleic acids in the presence of enzymes pose major obstacles to their delivery and frustrates their use as human therapies. This Account describes current ideas about the mechanisms in nonviral nucleic acid delivery and how lipidic and polymeric carriers can overcome some of the critical barriers to delivery. Over the last 20 years, researchers have developed a multitude of polymeric and lipidic vectors, but only a small fraction of these have progressed into clinical trials. None of these vectors has received FDA approval, which indicates that the current vectors do not yet have suitable properties for effective in vivo nucleic acid delivery. Nucleic acid delivery is a multistep process and inefficiencies at any stage result in a dramatic decrease in gene delivery or gene silencing. However, the majority of studies investigating synthetic vectors focus solely on optimization of endosomal escape. A small number of studies address how to improve uptake via targeted delivery, and an even smaller fraction examine the intracellular fate of the delivery systems and nucleic acid cargo. The internalization of genes into the cell nucleus remains an inefficient and mysterious process. In the case of DNA delivery, strategies are needed to increase and accelerate the migration of DNA through the cytoplasm and transport it through the nuclear membrane. siRNA delivery involves fewer barriers. siRNA is more readily released from the carrier and more resistant to enzymatic degradation, and its target is in the cytoplasm; hence, siRNA delivery systems are becoming a clinical reality. With regard to siRNA therapy, the exact cytoplasmic location of RNA-induced silencing complex (RISC) formation and activity is unknown, which makes specific targeting of the RISC for more efficient delivery difficult. Furthermore, we would like to identify the factors that favor the binding of siRNA to Ago-2. If we could understand how the half-life of siRNA and Ago-2/siRNA complex in the cytoplasm can be modulated without interfering with RISC functions that are essential for normal cell activity, we could increase siRNA delivery efficiency. In this Account, we review the current synthetic vectors and propose alternative strategies in a few cases. We also suggest how certain cellular mechanisms might be exploited to improve gene transfection and silencing. Finally, we discuss whether some carriers that deliver the siRNA to cells could also repackage the siRNA into exosomes. The exosomes would then transport the siRNA into a subsequent population of cells that manifest the siRNA effect. This piggy-back mechanism may be responsible for reported deep tissue siRNA effects using certain carriers.
Topics: Argonaute Proteins; Endosomes; Gene Transfer Techniques; Humans; Nucleic Acids; RNA Interference; RNA, Small Interfering
PubMed: 22428908
DOI: 10.1021/ar3000162 -
Cell Cycle (Georgetown, Tex.) 2014
Topics: Animals; Argonaute Proteins; Humans; Insulin-Secreting Cells
PubMed: 24621504
DOI: 10.4161/cc.28513 -
Journal of Extracellular Vesicles Nov 2023Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein...
Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination.
Topics: Extracellular Vesicles; MicroRNAs; Argonaute Proteins
PubMed: 37885043
DOI: 10.1002/jev2.12366 -
Biomolecules Sep 2022Argonaute proteins exist widely in eukaryotes and prokaryotes, and they are of great potential for molecular cloning, nucleic acid detection, DNA assembly, and gene...
Argonaute proteins exist widely in eukaryotes and prokaryotes, and they are of great potential for molecular cloning, nucleic acid detection, DNA assembly, and gene editing. However, their overall properties are not satisfactory and hinder their broad applications. Herein, we investigated a prokaryotic Argonaute nuclease from a mesophilic bacterium (CdAgo) and explored its overall properties, especially with 5'-hydroxylated (5'-OH) guides. We found that CdAgo can exclusively use single-stranded DNA (ssDNA) as guide to cleave ssDNA and plasmid targets. Further, we found the length of the efficient guide is narrower for the 5'-OH guide (17-20 nt) than for the 5'-phosphorylated guide (5'-P, 14-21 nt). Furthermore, we discovered that the 5'-OH guides can generally offer stronger mismatch discrimination than the 5'-P ones. The 5'-OH guides offer the narrower length range, higher mismatch discrimination and more accurate cleavage than the 5'-P guides. Therefore, 5'-OH-guide-directed CdAgo has great potential in biological and biomedical applications.
Topics: Argonaute Proteins; Deoxyribonucleases; DNA, Single-Stranded; DNA; Plasmids; Deoxyribonuclease I
PubMed: 36291549
DOI: 10.3390/biom12101340 -
The Journal of Neuroscience : the... Apr 2022The first evidence of local translation in the CNS appeared nearly 40 years ago, when electron microscopic studies showed polyribosomes localized to the base of... (Review)
Review
The first evidence of local translation in the CNS appeared nearly 40 years ago, when electron microscopic studies showed polyribosomes localized to the base of dendritic spines. Since then, local translation has been established as an important regulatory mechanism for gene expression in polarized or functionally compartmentalized cells. While much attention has been placed on characterizing the local transcriptome and regulatory "grammar" directing mRNA localization in neurons and glia, less is understood about how these cells subsequently de-repress mRNA translation in their peripheral processes to produce a rapid translational response to stimuli. MicroRNA-mediated translation regulation offers a possible solution to this question. Not only do miRNAs provide the specificity needed for targeted gene regulation, but association and dynamic interactions between Argonaute (AGO) with sequence-specific RNA-binding proteins may provide a molecular switch to allow for de-repression of target mRNAs. Here, we review the expression and activity of different AGO proteins in miRNA-induced silencing complexes in neurons and glia and discuss known pathways of miRNA-mediated regulation, including activity-dependent pre-miRNA maturation in dendrites. We further detail work on AGO and RNA-binding protein interactions that allow for the reversal of miRNA-mediated translational silencing, and we propose a model for how intercellular communication may play a role in the regulation of local translation.
Topics: Argonaute Proteins; MicroRNAs; Neuroglia; Neurons; RNA, Messenger; RNA-Binding Proteins
PubMed: 35444007
DOI: 10.1523/JNEUROSCI.2391-21.2022 -
Molecular Cell Sep 2019MicroRNAs (miRNAs) broadly regulate gene expression through association with Argonaute (Ago), which also protects miRNAs from degradation. However, miRNA stability is...
MicroRNAs (miRNAs) broadly regulate gene expression through association with Argonaute (Ago), which also protects miRNAs from degradation. However, miRNA stability is known to vary and is regulated by poorly understood mechanisms. A major emerging process, termed target-directed miRNA degradation (TDMD), employs specialized target RNAs to selectively bind to miRNAs and induce their decay. Here, we report structures of human Ago2 (hAgo2) bound to miRNAs and TDMD-inducing targets. miRNA and target form a bipartite duplex with an unpaired flexible linker. hAgo2 cannot physically accommodate the RNA, causing the duplex to bend at the linker and display the miRNA 3' end for enzymatic attack. Altering 3' end display by changing linker flexibility, changing 3' end complementarity, or mutationally inducing 3' end release impacts TDMD efficiency, leading to production of distinct 3'-miRNA isoforms in cells. Our results uncover the mechanism driving TDMD and reveal 3' end display as a key determinant regulating miRNA activity via 3' remodeling and/or degradation.
Topics: Animals; Argonaute Proteins; HEK293 Cells; Humans; MicroRNAs; Nucleic Acid Conformation; RNA Stability; Sf9 Cells; Spodoptera
PubMed: 31353209
DOI: 10.1016/j.molcel.2019.06.019 -
International Journal of Molecular... Oct 2022Normal testicular development plays a crucial role in male reproduction and is the precondition for spermatogenesis. PIWI-interacting RNAs (piRNAs) are novel noncoding...
Normal testicular development plays a crucial role in male reproduction and is the precondition for spermatogenesis. PIWI-interacting RNAs (piRNAs) are novel noncoding RNAs expressed in animal germ cells that form complexes with PIWI family proteins and are involved in germ cell development, differentiation, and spermatogenesis. However, changes in piRNA expression profiles during early testicular development in yak have not been investigated. In this study, we used small RNA sequencing to evaluate the differences and potential functions of piRNA expression profiles in 6-, 18-, and 30-month-old yak testis tissues. Differential expression analysis found 109, 293, and 336 differentially expressed piRNAs in M30 vs. M18, M18 vs. M6, and M30 vs. M6, respectively, and found 30 common differentially expressed piRNAs in the three groups of M6, M18, and M30. In addition, the functional enrichment analysis of differentially expressed piRNAs target genes indicated that they were related to testicular development and spermatogenesis. Finally, we detected the expression of the PIWI protein family in the yak testis at different developmental stages and found that , , , and were highly expressed in 18- and 30-month-old yak testis and almost not expressed in 6-month-old yak testis. In conclusion, this study summarizes the changes of piRNA expression patterns during the early development of yak testis and provides new clues for the regulatory role of piRNA in yak testis.
Topics: Animals; Male; Cattle; RNA, Small Interfering; Spermatogenesis; Testis; Germ Cells; Sequence Analysis, RNA; Argonaute Proteins
PubMed: 36293174
DOI: 10.3390/ijms232012320 -
Nucleic Acids Research Jul 2022Mature microRNAs are bound by a member of the Argonaute (Ago1-4) protein family, forming the core of the RNA-induced silencing complex (RISC). Association of RISC with...
Mature microRNAs are bound by a member of the Argonaute (Ago1-4) protein family, forming the core of the RNA-induced silencing complex (RISC). Association of RISC with target mRNAs results in ribonucleoprotein (RNP) assembly involved in translational silencing or RNA degradation. Yet, the dynamics of RNP assembly and its underlying functional implications are unknown. Here, we have characterized the role of the RNA-binding protein Staufen2, a candidate Ago interactor, in RNP assembly. Staufen2 depletion resulted in the upregulation of Ago1/2 and the RISC effector proteins Ddx6 and Dcp1a. This upregulation was accompanied by the displacement of Ago1/2 from processing bodies, large RNPs implicated in RNA storage, and subsequent association of Ago2 with polysomes. In parallel, Staufen2 deficiency decreased global translation and increased dendritic branching. As the observed phenotypes can be rescued by Ago1/2 knockdown, we propose a working model in which both Staufen2 and Ago proteins depend on each other and contribute to neuronal homeostasis.
Topics: Argonaute Proteins; RNA-Binding Proteins; RNA-Induced Silencing Complex; Neurons
PubMed: 35687120
DOI: 10.1093/nar/gkac487 -
Nano Letters Apr 2020Super-resolution imaging allows for the visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA point accumulation in nanoscale topology) is a...
Super-resolution imaging allows for the visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA point accumulation in nanoscale topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), which allows for faster binding. Ago preorders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude, while maintaining the high spatial resolution. We envision this tool to be useful for super-resolution imaging and other techniques that rely on nucleic acid interactions.
Topics: Argonaute Proteins; Bacterial Proteins; Clostridium butyricum; DNA; Fluorescence Resonance Energy Transfer; Microscopy, Fluorescence; Nanostructures; Optical Imaging
PubMed: 32168456
DOI: 10.1021/acs.nanolett.9b04277 -
BMC Biology Apr 2023Argonaute proteins play a central role in RNA silencing by forming protein-small RNA complexes responsible for the silencing process. While most Argonaute proteins have...
BACKGROUND
Argonaute proteins play a central role in RNA silencing by forming protein-small RNA complexes responsible for the silencing process. While most Argonaute proteins have a short N-terminal region, Argonaute2 in Drosophila melanogaster (DmAgo2) harbors a long and unique N-terminal region. Previous in vitro biochemical studies have shown that the loss of this region does not impair the RNA silencing activity of the complex. However, an N-terminal mutant of Drosophila melanogaster has demonstrated abnormal RNA silencing activity. To explore the causes of this discrepancy between in vitro and in vivo studies, we investigated the biophysical properties of the region. The N-terminal region is highly rich in glutamine and glycine residues, which is a well-known property for prion-like domains, a subclass of amyloid-forming peptides. Therefore, the possibility of the N-terminal region functioning as an amyloid was tested.
RESULTS
Our in silico and biochemical assays demonstrated that the N-terminal region exhibits amyloid-specific properties. The region indeed formed aggregates that were not dissociated even in the presence of sodium dodecyl sulfate. Also, the aggregates enhanced the fluorescence intensity of thioflavin-T, an amyloid detection reagent. The kinetics of the aggregation followed that of typical amyloid formation exhibiting self-propagating activity. Furthermore, we directly visualized the aggregation process of the N-terminal region under fluorescence microscopy and found that the aggregations took fractal or fibril shapes. Together, the results indicate that the N-terminal region can form amyloid-like aggregates.
CONCLUSIONS
Many other amyloid-forming peptides have been reported to modulate the function of proteins through their aggregation. Therefore, our findings raise the possibility that aggregation of the N-terminal region regulates the RNA silencing activity of DmAgo2.
Topics: Animals; Amyloid; Argonaute Proteins; Drosophila melanogaster; Peptides; Prions; Protein Aggregates
PubMed: 37072852
DOI: 10.1186/s12915-023-01569-3