Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Argonaute proteins associate with small RNAs that guide mRNA degradation, translational repression, or a combination of both. The human Argonaute family has eight members, four of which (Ago1 through Ago4) are closely related and coexpressed in many cell types. To understand the biological function of the different Ago proteins, we set out to determine if Ago1 through Ago4 are associated with miRNAs as well as RISC activity in human cell lines. Our results suggest that miRNAs are incorporated indiscriminately of their sequence into Ago1 through Ago4 containing microRNPs (miRNPs). Purification of the FLAG/HA-epitope-tagged Ago containing complexes from different human cell lines revealed that endonuclease activity is exclusively associated with Ago2. Exogenously introduced siRNAs also associate with Ago2 for guiding target RNA cleavage. The specific role of Ago2 in guiding target RNA cleavage was confirmed independently by siRNA-based depletion of individual Ago members in combination with a sensitive positive-readout reporter assay.
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PMID:Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. 1526 Sep 70

In recent years a new mechanism of posttranscriptional gene silencing has been discovered and named RNA interference. The interference is based on mRNA degradation mediated by small double-stranded RNA molecules approximately 21 nucleotides in length, the so-called short interfering or siRNAs. These molecules are produced from long dsRNAs by Dicer, a dsRNA-specific endonuclease, and cause specific degradation of their mRNA-targets by Watson-Crick base-pairing within a 300 kD multi-enzyme complex named RISC. RNAi is highly conserved between plants and animals of various phyla including mammals. The high sequence-specificity of RNAi makes it a new, promising tool in gene-function analysis as well as in potential therapeutics. In this review the discovery and molecular background of RNAi are summarized and possible fields of application pointed out.
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PMID:Silencing of disease-related genes by small interfering RNAs. 1526 22

RNA silencing is a conserved phenomenon of regulation of gene expression by small RNAs derived from cleavage of double-stranded RNA (dsRNA). The present review deals with three overlapping modes of small RNA-mediated silencing particularly in plants. In case of post-transcriptional gene silencing (PTGS), Dicer, an endonuclease, cleaves dsRNA to produce approximately 21nt-long small interfering RNAs (siRNAs), which guide RISC, another nuclease complex, to destroy specific target mRNAs based on sequence complementarity with the siRNA. Another class of siRNAs of 25nt-long is also produced from dsRNA by Dicer, different from that generates 21nt-long siRNA. These longer siRNAs are probably involved in systemic silencing during PTGS and guide methylation of both DNA and histone, and induce heterochromatinization and consequent transcriptional repression of the targeted gene. Both siRNA-mediated PTGS and epigenetic modification of the genome are considered as defense mechanisms to protect against invading viruses, transposons or aberrantly expressing transgenes. Regulation of expression of endogenous genes is mediated by another class of 21nt-long small RNAs called microRNAs (miRNA). Genes encoding the miRNAs are present either in the intergenic regions, introns or coding regions of the plant genome. Cleavage of a stem-loop precursor transcript called pre-miRNA, by another class of Dicer generates miRNAs, which in association with nuclease complex similar to RISC, if not identical, either degrade target mRNA or cause translational repression. The applications of RNA silencing in functional genomics and crop improvement are discussed.
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PMID:Small but mighty RNA-mediated interference in plants. 1569 Oct 61

Neoplasms arising from glial cells make up the most common group of primary brain tumors. The clinical outcome, especially the survival rates of the patients with brain tumours depend on tumour grade expressing its malignancy. A prognosis for glioblastomas (WHO IV) is very poor, but for astrocytomas (WHO I and II) it is relatively favourable. For oligodendrogliomas a longer survival time than for glioblastomas is observed. There is evidence that oxidative stress and reactive oxygen species (ROS) are crucial in the etiology and progression of a number of human diseases, including neoplasms. An oxidative damage of DNA, lipids and proteins is caused mainly with hydroxyl radical (*OH), the most reactive ROS species and may be seriously deleterious. In addition to all four basic nucleotides: adenosine (A), guanosine (G), tymidine (T) and cytosine (C), 5-methylcytosine (m5C) is a rare but normal component of cellular DNA and occurs mainly within a sequence of a structural gene or in regulatory regions. In the reaction with hydroxyl radical all DNA components can be modified, but m5C is relatively easily deaminated to thymine, which, in turn, pairs with adenine and after a round of replication, CG to TA transition occurs. Because thymine is a normal DNA base, therefore the product of spontaneous deamination of m5C is not so easily detected by cell's DNA repair system. Thus, 5-methylcytosine residue constitutes a mutational hotspot and DNA methylation pattern in patients might be useful as a primary diagnostic tool or as a marker for early detection of relapse of the disease. In recent years a new mechanism of posttranscriptional gene silencing has been discovered and named RNA interference (RNAi). This phenomenon is based on mRNA degradation mediated by small double-stranded RNA molecules, approximately 19-28 nucleotides in length, called short interfering RNAs (siRNAs). These molecules are produced from long dsRNAs by a dsRNA-specific endonuclease (DICER) and form 300 kD multi-enzyme complex (RISC) which by Watson-Crick base-pairing of noncoding strand with their mRNA-targets induce specific degradation. The high sequence-specificity of RNAi makes it a new, promising tool in a gene-function analysis as well as in potential therapeutics development.
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PMID:The diagnosis and therapy of brain tumours. 1624 17

Short interfering RNA (siRNA) duplexes are currently being evaluated as antisense agents for gene silencing. Chemical modification of siRNAs is widely expected to be required for therapeutic applications in order to improve delivery, biostability and pharmacokinetic properties. Beyond potential improvements in the efficacy of oligoribonucleotides, chemical modification may also provide insight into the mechanism of mRNA downregulation mediated by the RNA-protein effector complexes (RNA-induced silencing complex or RISC). We have studied the in vitro activity in HeLa cells of siRNA duplexes against firefly luciferase with substitutions in the guide strand of U for the apolar ribo-2,4-difluorotoluyl nucleotide (rF) [Xia, J. et al. (2006) ACS Chem. Biol., 1, 176-183] as well as of C for rF. Whereas an internal rF:A pair adjacent to the Ago2 ('slicer' enzyme) cleavage site did not affect silencing relative to the native siRNA duplex, the rF:G pair and other mismatches such as A:G or A:A were not tolerated. The crystal structure at atomic resolution determined for an RNA dodecamer duplex with rF opposite G manifests only minor deviations between the geometries of rF:G and the native U:G wobble pair. This is in contrast to the previously found, significant deviations between the geometries of rF:A and U:A pairs. Comparison between the structures of the RNA duplex containing rF:G and a new structure of an RNA with A:G mismatches with the structures of standard Watson-Crick pairs in canonical duplex RNA leads to the conclusion that local widening of the duplex formed by the siRNA guide strand and the targeted region of mRNA is the most likely reason for the intolerance of human Ago2 (hAgo2), the RISC endonuclease, toward internal mismatch pairs involving native or chemically modified RNA. Contrary to the influence of shape, the thermodynamic stabilities of siRNA duplexes with single rF:A, A:A, G:A or C:A (instead of U:A) or rF:G pairs (instead of C:G) show no obvious correlation with their activities. However, incorporation of three rF:A pairs into an siRNA duplex leads to loss of activity. Our structural and stability data also shed light on the role of organic fluorine as a hydrogen bond acceptor. Accordingly, UV melting (T(M)) data, osmotic stress measurements, X-ray crystallography at atomic resolution and the results of semi-empirical calculations are all consistent with the existence of weak hydrogen bonds between fluorine and the H-N1(G) amino group in rF:G pairs of the investigated RNA dodecamers.
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PMID:Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme. 1788 74

RNAi is widely applied to inhibit expression of specific genes, but it is limited by variable efficiency and specificity of empirically designed siRNA or shRNA constructs. This complicates studies targeting individual genes and significantly impairs large-scale screens using genome-wide knockdown libraries. Here, we show that ectopic expression of the RISC slicer Argonaute-2 (Ago2, eIF2C2) dramatically enhances RNAi specifically for mRNA targets with perfectly matched binding sites. This effect depends on its endonuclease activity and is uncoupled from its regulation of microRNA expression. To model the application of Ago2 coexpression with shRNA knockdown, we targeted the EGF receptor (EGFR) in lung cancer cells exhibiting oncogene addiction to EGFR. Whereas multiple empirically designed shRNA constructs exhibited highly divergent efficiencies in mediating EGFR knockdown and cell killing, coexpression of Ago2 resulted in uniform and highly specific target gene suppression and apoptosis in EGFR-dependent cells. Codelivery of Ago2 with shRNA constructs or siRNA duplexes thus provides a strategy to enhance the efficacy and the specificity of RNAi in experimental and potentially therapeutic settings.
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PMID:Coexpression of Argonaute-2 enhances RNA interference toward perfect match binding sites. 1859 65

Endonuclease cleavage was one of the first identified mechanisms of mRNA decay but until recently it was thought to play a minor role to the better-known processes of deadenylation, decapping, and exonuclease-catalyzed decay. Most of the early examples of endonuclease decay came from studies of a particular mRNA whose turnover changed in response to hormone, cytokine, developmental, or nutritional stimuli. Only a few of these examples of endonuclease-mediated mRNA decay progressed to the point where the enzyme responsible for the initiating event was identified and studied in detail. The discovery of microRNAs and RISC-catalyzed endonuclease cleavage followed by the identification of PIN (pilT N-terminal) domains that impart endonuclease activity to a number of the proteins involved in mRNA decay has led to a resurgence of interest in endonuclease-mediated mRNA decay. PIN domains show no substrate selectivity and their involvement in a number of decay pathways highlights a recurring theme that the context in which an endonuclease function is a primary factor in determining whether any given mRNA will be targeted for decay by this or the default exonuclease-mediated decay processes.
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PMID:Mechanisms of endonuclease-mediated mRNA decay. 2195 46

miRNAs are 20-22 nucleotide long noncoding RNAs that act as post-transcriptional regulators of gene expression controlling more than half of protein coding genes in humans. Being the critical modulators of the mRNA translation process, biogenesis, function, and turnover of these small RNAs are tightly regulated in cells. We have reported that target mRNAs induce increased biogenesis of cognate miRNAs from pre-miRNAs by increased activity of Ago-associated Dicer endonuclease that processes precursor miRNAs to their mature form. In the current chapter, we discuss how target mRNA-driven RISC loading can be monitored in vitro using affinity-purified miRISC or recombinant AGO2 and DICER1 proteins and scoring the processivity of AGO2-associated DICER1 in vitro.
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PMID:Target mRNA-Driven Biogenesis of Cognate MicroRNAs In Vitro. 2943 20