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)

A 54 kDa protein from mustard chloroplasts was previously shown to interact specifically with a conserved U-rich sequence element in RNA derived from the 3' flanking regions of the plastid trnK and rps16 genes, which code for tRNA(Lys) and ribosomal protein CS19, respectively (Nickelsen and Link, 1991). This RNA-binding protein has now been purified by affinity chromatography on heparin Sepharose and poly(U) Sepharose. In vitro processing experiments and nuclease S1 analyses of the processing products revealed that the 54 kDa polypeptide is an endonuclease. The in vitro cleavage sites are consistent with the positions of corresponding transcript in vivo 3' ends downstream of trnK and rps16, suggesting that RNA 3' end formation takes place endonucleolytically also in vivo.
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PMID:The 54 kDa RNA-binding protein from mustard chloroplasts mediates endonucleolytic transcript 3' end formation in vitro. 822 Apr 60

Mammalian LINE-1 (L1) elements belong to the superfamily of autonomously replicating retrotransposable elements that lack the long terminal repeated (LTR) sequences typical of retroviruses and retroviral-like retrotransposons. The non-LTR superfamily is very ancient and L1-like elements are ubiquitous in nature, having been found in plants, fungi, invertebrates, and various vertebrate classes from fish to mammals. L1 elements have been replicating and evolving in mammals for at least the past 100 million years and now constitute 20% or more of some mammalian genomes. Therefore, L1 elements presumably have had a profound, perhaps defining, effect on the evolution, structure, and function of mammalian genomes. L1 elements contain regulatory signals and encode two proteins: one is an RNA-binding protein and the second one presumably functions as an integrase-replicase, because it has both endonuclease and reverse transcriptase activities. This work reviews the structure and biological properties of L1 elements, including their regulation, replication, evolution, and interaction with their mammalian hosts. Although each of these processes is incompletely understood, what is known indicates that they represent challenging and fascinating biological phenomena, the resolution of which will be essential for fully understanding the biology of mammals.
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PMID:The biological properties and evolutionary dynamics of mammalian LINE-1 retrotransposons. 1069 12

In Xenopus, estrogen induces the stabilization of vitellogenin mRNA and the destabilization of albumin mRNA. These processes correlate with increased polysomal activity of a sequence-selective mRNA endonuclease, PMR-1, and a hnRNP K homology-domain RNA-binding protein, vigilin. Vigilin binds to a region of the vitellogenin mRNA 3'-untranslated region (3'-UTR) implicated in estrogen-mediated stabilization. The vigilin-binding site in the vitellogenin B1 mRNA 3'-UTR contains two consensus PMR-1 cleavage sites. The availability of purified PMR-1 and recombinant vigilin made it possible to test the hypothesis that RNA-binding proteins interact with cis-acting elements to stabilize target mRNAs by blocking cleavage by site-specific mRNA endonucleases. Vigilin binds to the vitellogenin mRNA 3'-UTR site with at least 30-fold higher affinity than it exhibits for the albumin mRNA segment containing the mapped PMR-1 cleavage sites. This differential binding affinity correlates with differential in vitro susceptibility of the protein-RNA complexes to cleavage by PMR-1. Whereas recombinant vigilin has no detectable protective effect on PMR-1 cleavage of albumin mRNA, it retards in vitro cleavage of the vitellogenin mRNA 3'-UTR by purified PMR-1. The PMR-1 sites in the vitellogenin mRNA 3'-UTR are functional because they are readily cleaved in vitro by purified PMR-1. These results provide direct evidence for differential susceptibility to endonuclease-mediated mRNA decay resulting from the differential affinity of a RNA-binding protein for cis-acting stability determinants.
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PMID:Vigilin binding selectively inhibits cleavage of the vitellogenin mRNA 3'-untranslated region by the mRNA endonuclease polysomal ribonuclease 1. 1105 Jan 68

The influenza virus polymerase complex contains two associated enzymatic activities, an endoribonuclease and a RNA-dependent RNA polymerase activity. Both activities have so far been observed only with the complete polymerase complex consisting of three subunits, PB1, PB2, and PA. This chapter describes a robust and optimized procedure for the purification of active influenza virus polymerase in complex with genomic RNA and the single-stranded RNA-binding protein nucleoprotein from influenza virus particles. It also explains the synthesis of capped RNA molecules as substrates of the influenza virus endonuclease. The enzymatic properties of influenza virus-derived endoribonuclease activity have been characterized with a model RNA substrate of 20-nucleotide length, termed G20 RNA. The rate of RNA cleavage under steady state conditions appears to be limited by product dissociation. Therefore conditions have been optimized to study the chemical step of RNA cleavage under single turnover conditions. The enzyme requires divalent metal ions for activity and can use Mn(II), Co(II), and Fe(II) efficiently at pH 7, Mg(II) with intermediate efficiency, and Ni(II) and Zn(II) with lower efficiency. The reaction progress curves show slow binding of Zn(II) and Ni(II) to the protein, suggesting a conformational change of the active site as a prerequisite for endonuclease activity in the presence of these two metal ions. Low concentrations of the detergent DOC inhibit the activity and also disrupt the trimeric polymerase complex, whereas other detergents do not have a significant effect on the activity.
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PMID:Influenza virus endoribonuclease. 1158 17

The AnCOB group I intron from Aspergillus nidulans encodes a homing DNA endonuclease called I-AniI which also functions as a maturase, assisting in AnCOB intron RNA splicing. In this investigation we biochemically characterized the endonuclease activity of I-AniI in vitro and utilized competition assays to probe the relationship between the RNA- and DNA-binding sites. Despite functioning as an RNA maturase, I-AniI still retains several characteristic properties of homing endonucleases including relaxed substrate specificity, DNA cleavage product retention and instability in the reaction buffer, which suggest that the protein has not undergone dramatic structural adaptations to function as an RNA-binding protein. Nitrocellulose filter binding and kinetic burst assays showed that both nucleic acids bind I-AniI with the same 1 : 1 stoichiometry. Furthermore, in vitro competition activity assays revealed that the RNA substrate, when prebound to I-AniI, stoichiometrically inhibits DNA cleavage activity, yet in reciprocal experiments, saturating amounts of prebound DNA substrate fails to inhibit RNA splicing activity. The data suggest therefore that both nucleic acids do not bind the same single binding site, rather that I-AniI appears to contain two binding sites.
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PMID:In vitro analysis of the relationship between endonuclease and maturase activities in the bi-functional group I intron-encoded protein, I-AniI. 1265 10

LINE-1 (L1) retrotransposons are replicating repetitive elements that, by mass, are the most-abundant sequences in the human genome. Over one-third of mammalian genomes are the result, directly or indirectly, of L1 retrotransposition. L1 encodes two proteins: ORF1, an RNA-binding protein, and ORF2, an endonuclease/reverse transcriptase. Both proteins are required for L1 mobilization. Apart from the obvious function of self-replication, it is not clear what other roles, if any, L1 plays within its host. The sheer magnitude of L1 sequences in our genome has fueled speculation that over evolutionary time L1 insertions may structurally modify endogenous genes and regulate gene expression. Here we provide a review of L1 replication and its potential functional consequences.
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PMID:LINE-1 retrotransposons: modulators of quantity and quality of mammalian gene expression? 1601 95

Small RNA-mediated gene silencing (RNA silencing) has emerged as a major regulatory pathway in eukaryotes. Identification of the key factors involved in this pathway has been a subject of rigorous investigation in recent years. In humans, small RNAs are generated by Dicer and assembled into the effector complex known as RNA-induced silencing complex (RISC) by multiple factors including hAgo2, the mRNA-targeting endonuclease, and TRBP (HIV-1 TAR RNA-binding protein), a dsRNA-binding protein that interacts with both Dicer and hAgo2. Here we describe an additional dsRNA-binding protein known as PACT, which is significant in RNA silencing. PACT is associated with an approximately 500 kDa complex that contains Dicer, hAgo2, and TRBP. The interaction with Dicer involves the third dsRNA-binding domain (dsRBD) of PACT and the N-terminal region of Dicer containing the helicase motif. Like TRBP, PACT is not required for the pre-microRNA (miRNA) cleavage reaction step. However, the depletion of PACT strongly affects the accumulation of mature miRNA in vivo and moderately reduces the efficiency of small interfering RNA-induced RNA interference. Our study indicates that, unlike other RNase III type proteins, human Dicer may employ two different dsRBD-containing proteins that facilitate RISC assembly.
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PMID:The role of PACT in the RNA silencing pathway. 1642 7

LINE-1 or L1 is an autonomous non-LTR retrotransposon in mammals. Retrotransposition requires the function of the two L1-encoded polypeptides, ORF1p and ORF2p. Early recognition of regions of homology between the predicted amino acid sequence of ORF2 and known endonuclease and reverse transcriptase enzymes led to testable hypotheses regarding the function of ORF2p in retrotransposition. As predicted, ORF2p has been demonstrated to have both endonuclease and reverse transcriptase activities. In contrast, no homologs of known function have contributed to our understanding of the function of ORF1p during retrotransposition. Nevertheless, significant advances have been made such that we now know that ORF1p is a high-affinity RNA-binding protein that forms a ribonucleoprotein particle together with L1 RNA. Furthermore, ORF1p is a nucleic acid chaperone and this nucleic acid chaperone activity is required for L1 retrotransposition.
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PMID:The ORF1 protein encoded by LINE-1: structure and function during L1 retrotransposition. 1687 16

LINE-1 retrotransposons constitute one-fifth of human DNA and have helped shape our genome. A full-length L1 encodes a 40-kDa RNA-binding protein (ORF1p) and a 150-kDa protein (ORF2p) with endonuclease and reverse transcriptase activities. ORF1p is distinctive in forming large cytoplasmic foci, which we identified as cytoplasmic stress granules. A phylogenetically conserved central region of the protein is critical for wild-type localization and retrotransposition. Yeast two-hybrid screens revealed several RNA-binding proteins that coimmunoprecipitate with ORF1p and colocalize with ORF1p in foci. Two of these proteins, YB-1 and hnRNPA1, were previously reported in stress granules. We identified additional proteins associated with stress granules, including DNA-binding protein A, 9G8, and plasminogen activator inhibitor RNA-binding protein 1 (PAI-RBP1). PAI-RBP1 is a homolog of VIG, a part of the Drosophila melanogaster RNA-induced silencing complex (RISC). Other RISC components, including Ago2 and FMRP, also colocalize with PAI-RBP1 and ORF1p. We suggest that targeting ORF1p, and possibly the L1 RNP, to stress granules is a mechanism for controlling retrotransposition and its associated genetic and cellular damage.
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PMID:LINE-1 ORF1 protein localizes in stress granules with other RNA-binding proteins, including components of RNA interference RNA-induced silencing complex. 1756 64

The endonuclease Argonaute2 (Ago2) mediates the degradation of the target mRNA within the RNA-induced silencing complex. We determined the binding and cleavage properties of recombinant human Ago2. Human Ago2 was unable to cleave preformed RNA duplexes and exhibited weaker binding affinity for RNA duplexes compared with the single strand RNA. The enzyme exhibited greater RNase H activity in the presence of Mn2+ compared with Mg2+. Human Ago2 exhibited weaker binding affinities and reduced cleavage activities for antisense RNAs with either a 5'-terminal hydroxyl or abasic nucleotide. Binding kinetics suggest that the 5'-terminal heterocycle base nucleates the interaction between the enzyme and the antisense RNA, and the 5'-phosphate stabilizes the interaction. Mn2+ ameliorated the effects of the 5'-terminal hydroxyl or abasic nucleotide on Ago2 cleavage activity and binding affinity. Nucleotide substitutions at the 3' terminus of the antisense RNA had no effect on human Ago2 cleavage activity, whereas 2'-methoxyethyl substitutions at position 2 reduced binding and cleavage activity and 12-14 reduced the cleavage activity. RNase protection assays indicated that human Ago2 interacts with the first 14 nucleotides at the 5'-pole of the antisense RNA. Human Ago2 preloaded with the antisense RNA exhibited greater binding affinities for longer sense RNAs suggesting that the enzyme interacts with regions in the sense RNA outside the site for antisense hybridization. Finally, transiently expressed human Ago2 immunoprecipitated from HeLa cells contained the double strand RNA-binding protein human immunodeficiency virus, type 1, trans-activating response RNA-binding protein, and deletion mutants of Ago2 showed that trans-activating response RNA-binding protein interacts with the PIWI domain of the enzyme.
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PMID:Binding and cleavage specificities of human Argonaute2. 1962 55


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