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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The recently discovered microRNAs (miRNAs) are a large family of small regulatory RNAs that have been implicated in controlling diverse pathways in a variety of organisms (1, 2). For posttranscriptional gene silencing, one strand of the miRNA is used to guide components of the RNA interference machinery, including Argonaute 2, to messenger RNAs (mRNAs) with complementary sequences (3, 4). Thus, targeted mRNAs are either cleaved by the endonuclease Argonaute 2 (5, 6), or protein synthesis is blocked by an as yet uncharacterized mechanism (7, 8). Genes encoding miRNAs are transcribed as long primary miRNAs (pri-miRNAs) that are sequentially processed by components of the nucleus and cytoplasm to yield a mature, approx 22-nucleotide (nt)-long miRNA (9). Two members of the ribonuclease (RNase) III endonuclease protein family, Drosha and Dicer, have been implicated in this two-step processing (10-13). To further our understanding of miRNA biogenesis and function it will be essential to identify the protein complexes involved. We were interested in defining the proteins required for the initial nuclear processing of pri-miRNAs to the approx 60- to 70-nt stem-loop intermediates known as precursor miRNAs (pre-miRNAs) (9, 10). This led to our identification of a protein complex we termed Microprocessor, which is necessary and sufficient for processing pri-miRNA to premiRNAs (14). The Microprocessor complex comprises Drosha and the double-stranded RNAbinding protein DiGeorge syndrome critical region 8 gene (DGCR8), which is deleted in DiGeorge syndrome (15, 16). In this chapter, we detail the methods used for the biochemical isolation and identification of the Microprocessor complex from human cells. We include a protocol for the in vitro analysis of pri-miRNA processing activity of the purified Microprocessor complex.
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PMID:MicroRNA biogenesis: isolation and characterization of the microprocessor complex. 1695 65

MicroRNAs are small noncoding 18- to 24-nt RNAs that are predicted to regulate expression of as many as 30% of protein-encoding genes. In prostate adenocarcinoma, 39 microRNAs are up-regulated, and six microRNAs are down-regulated. Production and function of microRNA requires coordinated processing by proteins of the microRNA machinery. Dicer, an RNase III endonuclease, is an essential component of the microRNA machinery. From a gene array analysis of 16 normal prostate tissue samples, 64 organ-confined, and four metastatic prostate adenocarcinomas, we identified an up-regulation of major components of the microRNA machinery, including Dicer, in metastatic prostate adenocarcinoma. Immunohistochemical studies on a tissue microarray consisting of 232 prostate specimens confirmed up-regulation of Dicer in prostatic intraepithelial neoplasia and in 81% of prostate adenocarcinoma. The increased Dicer level in prostate adenocarcinoma correlated with clinical stage, lymph node status, and Gleason score. Western blot analysis of benign and neoplastic prostate cell lines further confirmed Dicer up-regulation in prostate adenocarcinoma. Dicer up-regulation may explain an almost global increase of microRNA expression in prostate adenocarcinoma. The presence of up-regulated microRNA machinery may predict the susceptibility of prostate adenocarcinoma to RNA interference-based therapy.
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PMID:Up-regulation of dicer, a component of the MicroRNA machinery, in prostate adenocarcinoma. 1707 2

RNA interference (RNAi) is a powerful genetic tool for loss-of-function studies in mammalian cells and is also considered a potentially powerful therapeutic modality for the treatment of a variety of human diseases. During the past 3 years a number of systems for conditional RNAi have been developed that allow controlled expression of short hairpin RNA (shRNA) triggers of RNAi. The simplest strategy relies on tet-operable polymerase III-promoted shRNAs and co-expression of the tetracycline regulatory protein, TetR. In this study we have combined these features into a single lentiviral vector that upon delivery to target cells allows robust induction of shRNAs, even with low levels of doxycycline; importantly, we show minimal leakiness in the absence of inducer. We have exploited the regulatory properties of our system by targeting an essential cellular gene, the nuclear RNaseIII endonuclease Drosha. Drosha is the core catalytic component of the "microprocessor complex" and cleaves the primary microRNA (miRNA) transcripts into their pre-miRNA hairpin intermediates. We anticipate that our vector will facilitate functional studies of miRNA biogenesis.
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PMID:A facile lentiviral vector system for expression of doxycycline-inducible shRNAs: knockdown of the pre-miRNA processing enzyme Drosha. 1731 Oct 8

RNA editing in the sleeping sickness parasite Trypanosoma brucei remodels mitochondrial transcripts by the addition and deletion of uridylates as specified by guide RNAs. Editing is catalyzed by at least three distinct approximately 20S multiprotein editosomes, all of which contain KREPB4, a protein with RNase III and Pumilio motifs. RNAi repression of KREPB4 expression in procyclic forms (PFs) strongly inhibited growth and in vivo RNA editing, greatly diminished the abundance of 20S editosomes, reduced cellular levels of editosome proteins, and generated approximately 5-10S editosome subcomplexes. Editing TUTase, exoUase, and RNA ligase activities were largely shifted from approximately 20S to approximately 5-10S fractions of cellular lysates. Insertion and deletion endonuclease activities in approximately 20S fractions were strongly reduced upon KREPB4 repression but were not detected in the 5-10S subcomplex fraction. Abundance of MRP1 and RBP16 proteins, which appear to be involved in RNA processing but are not components of the 20S editosome, was unaltered upon KREPB4 repression. These data suggest that KREPB4 is important for the structural integrity of approximately 20S editosomes, editing endonuclease activity, and the viability of PF T. brucei cells.
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PMID:An essential role of KREPB4 in RNA editing and structural integrity of the editosome in Trypanosoma brucei. 1736 11

MicroRNAs are a recently discovered class of small, evolutionarily conserved, RNA molecules that negatively regulate gene expression at the post-transcriptional level. Mature microRNAs of approximately 20-22 nucleotides are formed from longer primary transcripts by two sequential processing steps mediated by a nuclear (Drosha) and a cytoplasmic (Dicer) RNAse III endonuclease. In the context of a protein complex, the RNA-induced silencing complex (RISC), microRNAs base-pair with target messenger RNA sequences causing translational repression and/or messenger RNA degradation. MicroRNAs have been implicated in the control of many fundamental cellular and physiological processes such as tissue development, cellular differentiation and proliferation, metabolic and signalling pathways, apoptosis and stem cell maintenance. Mounting evidence indicates that microRNAs also play a significant role in cellular transformation and carcinogenesis acting either as oncogenes or tumour suppressors. This review briefly introduces microRNAs in a historical perspective and focuses on the biogenesis of microRNAs, their mode of action, mammalian microRNA functions with emphasis on their involvement in disease - particularly cancer - and their potential therapeutic use.
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PMID:The role of microRNAs in cancer: no small matter. 1753 69

Chloroplast genomes in land plants harbor approximately 20 group II introns. Genetic approaches have identified proteins involved in the splicing of many of these introns, but the proteins identified to date cannot account for the large size of intron ribonucleoprotein complexes and are not sufficient to reconstitute splicing in vitro. Here, we describe an additional protein that promotes chloroplast group II intron splicing in vivo. This protein, RNC1, was identified by mass spectrometry analysis of maize (Zea mays) proteins that coimmunoprecipitate with two previously identified chloroplast splicing factors, CAF1 and CAF2. RNC1 is a plant-specific protein that contains two ribonuclease III (RNase III) domains, the domain that harbors the active site of RNase III and Dicer enzymes. However, several amino acids that are essential for catalysis by RNase III and Dicer are missing from the RNase III domains in RNC1. RNC1 is found in complexes with a subset of chloroplast group II introns that includes but is not limited to CAF1- and CAF2-dependent introns. The splicing of many of the introns with which it associates is disrupted in maize rnc1 insertion mutants, indicating that RNC1 facilitates splicing in vivo. Recombinant RNC1 binds both single-stranded and double-stranded RNA with no discernible sequence specificity and lacks endonuclease activity. These results suggest that RNC1 is recruited to specific introns via protein-protein interactions and that its role in splicing involves RNA binding but not RNA cleavage activity.
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PMID:A ribonuclease III domain protein functions in group II intron splicing in maize chloroplasts. 1769 27

RNA-mediated gene silencing is recently emerged as a fundamental mechanism of regulation of gene expression in many organisms and tissues, with special emphasis with respect to the nervous system. With the aim to study the components of RNA silencing machinery, we have investigated the expression profile and localization of dicer protein RNase III endonuclease in cultures of post-mitotic neurons. Dicer catalyzes the processing of double-stranded RNAs (dsRNAs) into approximately 21-25 nucleotide-long small interfering (si)RNAs and micro (mi)RNAs, and it represents an essential step in the biogenesis of these small non-coding RNA molecules. We show that in rat primary neurons dicer is localized in the somatodendritic compartment, at the Golgi-reticulum area network level. This peculiar distribution was altered by brefeldin A treatment. Moreover the Golgi-reticulum dicer signal was observed also in primary astroglial cells. In addiction dicer was observed to be regulated during the embryogenesis and development in several tissues. In fact its expression is developmentally regulated in cultured cerebellar granule neurons. This is the first study in which dicer is shown preferentially distributed in the Golgi-reticulum area in post-mitotic terminally differentiated neuronal and glial cells and that its profile is modulated during maturation and development of in vitro cultured cerebellar granule neurons.
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PMID:Dicer expression and localization in post-mitotic neurons. 1788 88

microRNAs (miRNAs) regulate gene expression post-transcriptionally by targeting mRNAs for degradation or by inhibiting translation. Dicer is an RNase III endonuclease which processes miRNA precursors into functional 21-23 nucleotide RNAs that are subsequently incorporated into the RNA-induced silencing complex. miRNA-mediated gene regulation is important for organogenesis of a variety of tissues including limb, lung and skin. To gain insight into the roles of Dicer and miRNAs in mammalian skeletal muscle development, we eliminated Dicer activity specifically in the myogenic compartment during embryogenesis. Dicer activity is essential for normal muscle development during embryogenesis and Dicer muscle mutants have reduced muscle miRNAs, die perinatally and display decreased skeletal muscle mass accompanied by abnormal myofiber morphology. Dicer mutant muscles also show increased apoptosis and Cre-mediated loss of Dicer in Myod-converted myoblasts results in enhanced cell death. These observations demonstrate key roles for Dicer in skeletal muscle and implicate miRNAs as critical components required for embryonic myogenesis.
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PMID:Essential role for Dicer during skeletal muscle development. 1793 65

Trypanosoma brucei has three distinct approximately 20S editosomes that catalyze RNA editing by the insertion and deletion of uridylates. Editosomes with the KREN1 or KREN2 RNase III type endonucleases specifically cleave deletion and insertion editing site substrates, respectively. We report here that editosomes with KREPB2, which also has an RNase III motif, specifically cleave cytochrome oxidase II (COII) pre-mRNA insertion editing site substrates in vitro. Conditional repression and mutation studies also show that KREPB2 is an editing endonuclease specifically required for COII mRNA editing in vivo. Furthermore, KREPB2 expression is essential for the growth and survival of bloodstream forms. Thus, editing in T. brucei requires at least three compositionally and functionally distinct approximately 20S editosomes, two of which distinguish between different insertion editing sites. This unexpected finding reveals an additional level of complexity in the RNA editing process and suggests a mechanism for how the selection of sites for editing in vivo is controlled.
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PMID:RNA editing in Trypanosoma brucei requires three different editosomes. 1795 57

Sweet potato chlorotic stunt virus (genus Crinivirus) belongs to the family Closteroviridae, members of which have a conserved overall genomic organization but are variable in gene content. In the bipartite criniviruses, heterogeneity is pronounced in the 3'-proximal region of RNA1, which in sweet potato chlorotic stuat virus (SPCSV) encodes two novel proteins, RNase3 (RNase III endonuclease) and p22 (RNA silencing suppressor). This study showed that two Ugandan SPCSV isolates contained the p22 gene, in contrast to three isolates of the East African strain from Tanzania and Peru and an isolate of the West African strain from Israel, which were missing a 767 nt fragment of RNA1 that included the p22 gene. Regardless of the presence of p22, all tested SPCSV isolates acted synergistically with potyvirus sweet potato feathery mottle virus (SPFMV; genus Potyvirus, family Potyviridae) in co-infected sweetpotato plants (Ipomoea batatas), which greatly enhanced SPFMV titres and caused severe sweetpotato virus disease (SPVD). Therefore, the results indicate that any efforts to engineer pathogen-derived RNA silencing-based resistance to SPCSV and SPVD in sweetpotato should not rely on p22 as the transgene. The data from this study demonstrate that isolates of this virus species can vary in the genes encoding RNA silencing suppressor proteins. This study also provides the first example of intraspecific variability in gene content of the family Closteroviridae and may be a new example of the recombination-mediated gene gain that is characteristic of virus evolution in this virus family.
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PMID:Analysis of gene content in sweet potato chlorotic stunt virus RNA1 reveals the presence of the p22 RNA silencing suppressor in only a few isolates: implications for viral evolution and synergism. 1819 89


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