Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.26.3 (RNase III)
1,015 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNA interference (RNAi) is the mechanism through which double-stranded RNAs silence cognate genes. In plants, this can occur at both the transcriptional and the post-transcriptional levels; however, in animals, only post-transcriptional RNAi has been reported to date. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC, to destroy specific messenger RNAs. Here we identify an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.
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PMID:Role for a bidentate ribonuclease in the initiation step of RNA interference. 1120 23

Double-stranded (ds) RNA causes the specific degradation of homologous RNAs in a process called "RNA interference (RNAi)"[1-4]; this process is called "posttranscriptional gene silencing (PTGS)" in plants [5-7]. Both classes of gene silencing have been reviewed extensively [8-13]. The duplex RNA becomes processed by Dicer [14] or another RNase III-like enzyme to short dsRNA fragments of about 21-23 nucleotides (nt) [15], which are incorporated in the RNA-induced silencing complex (RISC)[16] that directs target-specific RNA degradation [17, 18]. Here, we show that different synthetic dsRNA cassettes, consisting of two 5'-phosphorylated RNA strands of 22 nt each, can initiate RNAi in Drosophila embryos. The cassettes were active at similar quantities required to initiate RNAi by conventional dsRNA. Their sequence specificity was confirmed using synthetic dsRNA cassettes for two different genes, Notch and hedgehog; each time, only the relevant embryonic phenotype was observed. Introduction of point mutations had only a moderate effect on the silencing potential, indicating that the silencing machinery does not require perfect sequence identity. 5'-phosphorylated synthetic RNA was more active than its hydroxylated form. Substitution of either RNA strand by DNA strongly reduced activity. Synthetic cassettes of siRNA will provide a new tool to induce mutant phenotypes of genes with unknown function.
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PMID:Short 5'-phosphorylated double-stranded RNAs induce RNA interference in Drosophila. 1171 20

Members of the ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoribonucleases participate in diverse cellular RNA maturation and degradation pathways. A recently identified eukaryotic RNase III family member, named "Dicer", functions in the RNA interference (RNAi) pathway by producing 21--23 bp dsRNAs which target the selective destruction of homologous RNAs. RNAi is operative in animals, plants, and fungi, where it is proposed to inhibit viral reproduction and retroposon movement, as well as to participate in developmental pathways. RNAi functions in mammalian cells, including mouse oocytes and embryos. This article reports the cDNA sequence characterization and expression analysis of the mouse Dicer ortholog. On the basis of the cDNA sequence, the Dicer polypeptide is 1906 amino acids and has a predicted molecular mass of 215 kDa. Mouse Dicer contains a DExH/DEAH helicase motif; a PAZ domain; a tandem repeat of RNase III catalytic domain sequences; and a dsRNA-binding motif. The Dicer gene maps to a single locus on the distal portion of mouse Chromosome (Chr) 12. The Dicer transcript is expressed from the embryonic through adult stages of development. The Dicer transcript is also present in a wide variety of adult mouse organs. The highly conserved set of functional domains and the occurrence of a single-copy gene strongly indicate that the encoded protein is the RNase III ortholog responsible for dsRNA processing in the RNAi pathway.
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PMID:Molecular characterization of a mouse cDNA encoding Dicer, a ribonuclease III ortholog involved in RNA interference. 1188 53

MicroRNAs (miRNAs) are an extensive class of ~22-nucleotide noncoding RNAs thought to regulate gene expression in metazoans. We find that miRNAs are also present in plants, indicating that this class of noncoding RNA arose early in eukaryotic evolution. In this paper 16 Arabidopsis miRNAs are described, many of which have differential expression patterns in development. Eight are absolutely conserved in the rice genome. The plant miRNA loci potentially encode stem-loop precursors similar to those processed by Dicer (a ribonuclease III) in animals. Mutation of an Arabidopsis Dicer homolog, CARPEL FACTORY, prevents the accumulation of miRNAs, showing that similar mechanisms direct miRNA processing in plants and animals. The previously described roles of CARPEL FACTORY in the development of Arabidopsis embryos, leaves, and floral meristems suggest that the miRNAs could play regulatory roles in the development of plants as well as animals.
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PMID:MicroRNAs in plants. 1210 Nov 21

Dicer is a multi-domain RNase III-related endonuclease responsible for processing double-stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the purification and properties of a recombinant human Dicer. The protein cleaves dsRNAs into approximately 22 nucleotide siRNAs. Accumulation of processing intermediates of discrete sizes, and experiments performed with substrates containing modified ends, indicate that Dicer preferentially cleaves dsRNAs at their termini. Binding of the enzyme to the substrate can be uncoupled from the cleavage step by omitting Mg(2+) or performing the reaction at 4 degrees C. Activity of the recombinant Dicer, and of the endogenous protein present in mammalian cell extracts, is stimulated by limited proteolysis, and the proteolysed enzyme becomes active at 4 degrees C. Cleavage of dsRNA by purifed Dicer and the endogenous enzyme is ATP independent. Additional experiments suggest that if ATP participates in the Dicer reaction in mammalian cells, it might be involved in product release needed for the multiple turnover of the enzyme.
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PMID:Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. 1241 5

RNA interference (RNAi) as a protecting mechanism against invasion by foreign genes was first described in C. elegans and has subsequently been demonstrated in diverse eukaryotes such as insects, plants, fungi and vertebrates. RNAi is the mechanism of sequence-specific, post-transcriptional gene silencing initiated by double-stranded RNAs (dsRNA) homologous to the gene being suppressed. dsRNAs are processed by Dicer, a cellular ribonuclease III, to generate duplexes of about 21 nt with 3'-overhangs (small interfering RNA, siRNA) which mediate sequence-specific mRNA degradation. In mammalian cells siRNA molecules are capable of specifically silencing gene expression without induction of the unspecific interferon response pathway. Thus, siRNAs have become a new and powerful alternative to other genetic tools such as antisense oligonucleotides and ribozymes to analyze loss-of-function phenotypes. Application of siRNA duplexes to interfere with the expression of a specific gene requires knowledge of target accessibility, highly effective delivery of siRNAs into target cells and for some applications long-term siRNA expression. Effective strategies to deliver siRNAs to target cells in cell culture include transduction by physical or chemical transfection. An alternative strategy uses the endogenous expression of siRNAs by various Pol III promoter expression cassettes that allow transcription of functional siRNAs or their precursors. This review summarizes some genetic and biochemical aspects of RNAi, the delivery and application of siRNAs to target cells, the kinetics of RNAi and the utility of siRNAs as analytical and potential therapeutic tools.
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PMID:Gene silencing mediated by small interfering RNAs in mammalian cells. 1257 Jul 11

RNA interference (RNAi) is a powerful method for specifically silencing gene expression in diverse cell types. RNAi is mediated by approximately 21-nucleotide small interfering RNAs (siRNAs), which are produced from larger double-stranded RNAs (dsRNAs) in vivo through the action of Dicer, an RNase III-family enzyme. Transfecting cells with siRNAs rather than larger dsRNAs avoids the nonspecific gene silencing of the interferon response, underscoring the importance of developing efficient methods for producing reliable siRNAs. Here we show that pools of 20- to 21-base pair (bp) siRNAs can be produced enzymatically in vitro using active recombinant Dicer. Yields of < or = 70% are obtained, and the siRNAs can be easily separated from any residual large dsRNA by a series of spin columns or gel purification. Dicer-generated siRNAs (d-siRNAs) are effective in silencing transiently transfected reporter genes and endogenous genes, making in vitro dicing a useful, practical alternative for the production of siRNAs.
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PMID:Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing. 1259 10

Hundreds of small RNAs of approximately 22 nucleotides, collectively named microRNAs (miRNAs), have been discovered recently in animals and plants. Although their functions are being unravelled, their mechanism of biogenesis remains poorly understood. miRNAs are transcribed as long primary transcripts (pri-miRNAs) whose maturation occurs through sequential processing events: the nuclear processing of the pri-miRNAs into stem-loop precursors of approximately 70 nucleotides (pre-miRNAs), and the cytoplasmic processing of pre-miRNAs into mature miRNAs. Dicer, a member of the RNase III superfamily of bidentate nucleases, mediates the latter step, whereas the processing enzyme for the former step is unknown. Here we identify another RNase III, human Drosha, as the core nuclease that executes the initiation step of miRNA processing in the nucleus. Immunopurified Drosha cleaved pri-miRNA to release pre-miRNA in vitro. Furthermore, RNA interference of Drosha resulted in the strong accumulation of pri-miRNA and the reduction of pre-miRNA and mature miRNA in vivo. Thus, the two RNase III proteins, Drosha and Dicer, may collaborate in the stepwise processing of miRNAs, and have key roles in miRNA-mediated gene regulation in processes such as development and differentiation.
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PMID:The nuclear RNase III Drosha initiates microRNA processing. 1450 93

The completion of the human genome project has left researchers searching for an efficient method to study gene function in mammalian cells. RNA interference (RNAi) is an evolutionarily conserved post-transcriptional gene silencing (PTGS) mechanism mediated by double-stranded RNA (dsRNA). The dsRNA is processed into small duplex RNA molecules of approximately 21-22 nucleotides (nts) termed small interfering RNAs (siRNAs) by a RNase III enzyme called Dicer. Interaction of siRNAs with a multi-protein complex, termed the RNA-induced silencing complex (RISC), results in sequence specific association of the activated RISC complex with the cognate RNA transcript. This interaction leads to sequence-specific cleavage of the target transcript. Originally discovered in Caenorhabditis elegans, the study of RNAi in mammalian cells has blossomed in the last couple of years with the discovery that introduction of siRNA molecules directly into somatic mammalian cells circumvents the non-specific response vertebrate cells have against larger dsRNA molecules. Emerging as a powerful tool for reverse genetic analysis, RNAi is rapidly being applied to study the function of many genes associated with human disease, in particular those associated with oncogenesis and infectious disease. This review summarizes the mechanism of RNAi and provides an overview of its current applications in medicine.
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PMID:RNA interference and human disease. 1456 61

The bidentate RNase III Dicer cleaves microRNA precursors to generate the 21-23 nt long mature RNAs. These precursors are 60-80 nt long, they fold into a characteristic stem-loop structure and they are generated by an unknown mechanism. To gain insights into the biogenesis of microRNAs, we have characterized the precise 5' and 3' ends of the let-7 precursors in human cells. We show that they harbor a 5'-phosphate and a 3'-OH and that, remarkably, they contain a 1-4 nt 3' overhang. These features are characteristic of RNase III cleavage products. Since these precursors are present in both the nucleus and the cytoplasm of human cells, our results suggest that they are generated in the nucleus by the nuclear RNase III. Additionally, these precursors fit the minihelix export motif and are thus likely exported by this pathway.
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PMID:Human let-7 stem-loop precursors harbor features of RNase III cleavage products. 1460 19


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