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)

RNA editing in trypanosomes has been proposed to occur through transesterification or endonuclease cleavage and RNA ligation reactions. Both models involve a chimeric intermediate in which a guide RNA (gRNA) is joined through its 3' oligo(U) tail to an editing site of the corresponding mRNA. Velocity centrifugation of Trypanosoma brucei mitochondrial extracts had been reported to completely separate the gRNA-mRNA chimera-forming activity from endonuclease activity (V. W. Pollard, M. E. Harris, and S. L. Hajduk, EMBO J. 11:4429-4438, 1992), appearing to rule out the endonuclease-RNA ligase mechanism. However, we show that an editing-domain-specific endonuclease activity does cosediment with the chimera-forming activity, as does the RNA ligase activity, but detection of the specific endonuclease requires reducing assay conditions. This report further demonstrates that the T. brucei chimera-forming activity is mimicked by mung bean nuclease and T4 RNA ligase. Using cytochrome b (CYb) preedited mRNA and a model CYb gRNA, we found that these heterologous enzymes specifically generate CYb gRNA-mRNA chimeras analogous to those formed in the mitochondrial extract. These combined results provide support for the endonuclease-RNA ligase mechanism of chimera formation.
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PMID:Trypanosoma brucei mitochondrial guide RNA-mRNA chimera-forming activity cofractionates with an editing-domain-specific endonuclease and RNA ligase and is mimicked by heterologous nuclease and RNA ligase. 753

RNA editing in kinetoplastids is the post-transcriptional insertion and deletion of uridylate residues in mitochondrial transcripts, directed by base pairing with guide RNAs. Models for editing propose transesterification or endonuclease plus RNA ligase reactions and may involve a guide RNA-mRNA chimeric intermediate. We have assessed the feasibility of the enzymatic pathway involving chimeras in vitro. Cytochrome b chimeras generated with mitochondrial extract were first found to have junctions primarily at the major endonuclease cleavage sites, supporting the role of endonuclease in chimera formation. Such cytochrome b chimeras are then specifically cleaved by extract endonuclease within the oligo(U) tract at the editing site, and the mRNA cleavage products are then joined by RNA ligase to generate partially edited mRNAs with uridylate residues transferred to an editing site. These in vitro generated partially edited mRNAs mimic partially edited mRNAs generated in vivo. Specific endonuclease cleavage in the editing region of the partially edited RNA demonstrates the potential for further in vitro editing. Finally, sensitivity to various ATP analogs suggests that all editing-like activities reported thus far utilize a mechanism involving RNA ligase.
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PMID:Trypanosoma brucei RNA editing. A full round of uridylate insertional editing in vitro mediated by endonuclease and RNA ligase. 861 22

An RNA editing-like internal uridine (U) incorporation activity (G. C. Frech, N. Bakalara, L Simpson, and A. M. Simpson, EMBO J. 14:178-187, 1995) and a 3'-terminal U addition activity (N. Bakalara, A. M. Simpson, and L. Simpson, J. Biol. Chem. 264:18679-18686, 1989) have been previously described by using a mitochondrial extract from Leishmania tarentolae. Chiral phosphorothioates were used to investigate the stereoconfiguration requirements and the stereochemical course of these nucleotidyl transfer reactions. The extract utilizes (SP)-alpha-S-UTP for both 3' and internal U incorporation into substrate RNA. The internal as well as the 3' incorporation of (SP)-alpha-S-UTP proceeds via inversion of the stereoconfiguration. Furthermore, internal U incorporation does not occur at sites containing thiophosphodiesters of the RP configuration. Our results are compatible with an enzyme cascade model for this in vitro U insertion activity involving sequential endonuclease and uridylyl transferase directly from UTP and RNA ligase steps and are incompatible with models involving the transfer of U residues from the 3' ends of guide RNAs.
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PMID:Uridine insertion into preedited mRNA by a mitochondrial extract from Leishmania tarentolae: stereochemical evidence for the enzyme cascade model. 875 59

We have studied the mechanism of accurate in vitro RNA editing of Trypanosoma brucei ATPase 6 mRNA, using four mRNA-guide RNA (gRNA) pairs that specify deletion of 2, 3, or 4 U residues at editing site 1 and mitochondrial extract. This extract not only catalyzes deletion of the specified number of U residues but also exhibits a novel endonuclease activity that cleaves the input pre-mRNA in a gRNA-directed manner, precisely at the phosphodiester bond predicted in a simple enzymatic model of RNA editing. This cleavage site is inconsistent with a chimera-based editing mechanism. The U residues to be deleted, present at the 3' end of the upstream cleavage product, are then removed evidently by a 3' U-specific exonuclease and not by a reverse reaction of terminal U transferase. RNA ligase can then join the mRNA halves through their newly formed 5' P and 3' OH termini, generating mRNA faithfully edited at the first editing site. This resultant, partially edited mRNA can then undergo accurate, gRNA-directed cleavage at editing site 2, again precisely as predicted by the enzymatic editing model. All of these enzymatic activities cofractionate with the U-deletion activity and may reside in a single complex. The data imply that each round of editing is a four-step process, involving (i) gRNA-directed cleavage of the pre-mRNA at the bond immediately 5' of the region base paired to the gRNA, (ii) U deletion from or U addition to the 3' OH of the upstream mRNA half, (iii) ligation of the mRNA halves, and (iv) formation of additional base pairing between the correctly edited site and the gRNA that directs subsequent nuclease cleavage at the next editing site.
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PMID:Trypanosome U-deletional RNA editing involves guide RNA-directed endonuclease cleavage, terminal U exonuclease, and RNA ligase activities. 879 25

Kinetoplastid mitochondrial RNA editing, the insertion and deletion of U residues, is catalyzed by sequential cleavage, U addition or removal, and ligation reactions and is directed by complementary guide RNAs. We have purified a approximately 20S enzymatic complex from Trypanosoma brucei mitochondria that catalyzes a complete editing reaction in vitro. This complex possesses all four activities predicted to catalyze RNA editing: gRNA-directed endonuclease, terminal uridylyl transferase, 3' U-specific exonuclease, and RNA ligase. However, it does not contain other putative editing complex components: gRNA-independent endonuclease, RNA helicase, endogenous gRNAs or pre-mRNAs, or a 25 kDa gRNA-binding protein. The complex is composed of eight major polypeptides, three of which represent RNA ligase. These findings identify polypeptides representing catalytic editing factors, reveal the nature of this approximately 20S editing complex, and suggest a new model of editosome assembly.
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PMID:Purification of a functional enzymatic editing complex from Trypanosoma brucei mitochondria. 923 16

RNA editing in trypanosome mitochondria entails the posttranscriptional internal addition and occasional deletion of uridines from precursor mRNAs. Ample evidence exists to show that the information specifying the site and number of uridines added or deleted comes from small, mitochondrially encoded guide RNAs (gRNAs). More recent work indicates that the process involves an enzymatic cascade, initiating with an endonucleolytic cleavage of the pre-mRNA at an editing site. The cleaved editing site can undergo uridine (U) addition to or deletion from the 3' end of the 5' fragment via a mitochondrial terminal uridylyl transferase (TUTase) or terminal uridylyl exonuclease, respectively. Mitochondrial RNA ligase subsequently rejoins the mRNA. Activities to carry out these processes have been found in trypanosome mitochondria, including an editing-site-specific endonuclease activity which cleaves preedited but not edited mRNAs. We have found that this enzymatic activity cosediments with the same 19S ribonucleoprotein particle previously shown to contain TUTase, RNA ligase, and gRNAs and remains stable after salt treatment. Depletion of endogenous cytochrome b gRNAs by the addition of complementary oligonucleotides in vitro completely inhibits editing-site cleavage of synthetic preedited cytochrome b mRNA. The addition of synthetic cognate gRNA for cytochrome b but not unrelated small RNA restores editing-site cleavage. These studies show that in addition to specifying the site and number of uridines added or deleted, gRNAs provide the necessary information for cleavage by the editing-site-specific endonuclease.
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PMID:Guide RNA requirement for editing-site-specific endonucleolytic cleavage of preedited mRNA by mitochondrial ribonucleoprotein particles in Trypanosoma brucei. 927 15

The mitochondrial mRNAs of trypanosomes are often post-transcriptionally modified by an RNA processing event, termed RNA editing, which results in the insertion or deletion of uridylate (U) residues in mRNAs. RNA editing is necessary for the formation of complete coding sequences for several essential mitochondrial proteins. The number and site of U addition and deletion is directed by small guide RNAs (gRNAs). Recent studies indicate that the mechanism of RNA editing in trypanosomes involves a series of enzymatic steps. We show that the initial step in this enzymatic cascade requires the formation of a binary RNA complex between the gRNA and its cognate pre-mRNA. Depletion of specific gRNAs inhibits cleavage of the pre-mRNA by an editing site specific endoribonuclease. Addition of synthetic gRNAs reverses this inhibition. All of the activities needed for RNA editing in vitro are present within a 19S ribonucleo-protein complex (RNP) composed of gRNAs, the editing site specific endonuclease, an RNA ligase, a terminal uridylate transferase (TUTase) and approximately 15 other unidentified proteins. We have recently identified and cloned the gene for a 45kDa protein, the RNA Editing Associated Protein-1 (REAP-1), which is a component of trypanosome editing complexes. REAP-1 co-purifies with RNA ligase and TUTase activities and is part of a > 700 kDa RNP containing gRNAs. Antibodies against REAP-1 inhibit in vitro RNA editing reactions confirming its role in RNA editing.
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PMID:Insertional and deletional RNA editing in trypanosome mitochondria. 947 93

RNA editing in kinetoplastids is a type of post-transcriptional processing that changes mitochondrial mRNA sequences by the addition or deletion of uridines. Multiple enzymatic activities, such as endoribonuclease and RNA ligase, are associated with this process and exist in a multienzyme complex. Endonuclease activities from Trypanosoma brucei mitochondrial extracts were fractionated by sequential ion exchange and gel filtration chromatography. The RNA editing specific endonuclease activity co-fractionated with in vitro editing while another endonuclease activity with a different substrate specificity, and the majority of mtRNase P activity fractionated away from the editing activity. The pH, salt, temperature, and Mg(2+) optima of all three endonucleases were determined. All three activities are sensitive to high temperature and protease digestion. In addition, treatment with micrococcal nuclease resulted in partial disruption of the editing complex and decreased pre-cleaved in vitro insertion editing activity, suggesting that both RNA(s) and protein(s) are necessary in the intact functional complex.
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PMID:Endoribonuclease activities of Trypanosoma brucei mitochondria. 1184 2

Pre-tRNA splicing is an essential process in all eukaryotes. It requires the concerted action of an endonuclease to remove the intron and a ligase for joining the resulting tRNA halves as studied best in the yeast Saccharomyces cerevisiae. Here, we report the first characterization of an RNA ligase protein and its gene from a higher eukaryotic organism that is an essential component of the pre-tRNA splicing process. Purification of tRNA ligase from wheat germ by successive column chromatographic steps has identified a protein of 125 kDa by its potentiality to covalently bind AMP, and by its ability to catalyse the ligation of tRNA halves and the circularization of linear introns. Peptide sequences obtained from the purified protein led to the elucidation of the corresponding proteins and their genes in Arabidopsis and Oryza databases. The plant tRNA ligases exhibit no overall sequence homologies to any known RNA ligases, however, they harbour a number of conserved motifs that indicate the presence of three intrinsic enzyme activities: an adenylyltransferase/ligase domain in the N-terminal region, a polynucleotide kinase in the centre and a cyclic phosphodiesterase domain at the C-terminal end. In vitro expression of the recombinant Arabidopsis tRNA ligase and functional analyses revealed all expected individual activities. Plant RNA ligases are active on a variety of substrates in vitro and are capable of inter- and intramolecular RNA joining. Hence, we conclude that their role in vivo might comprise yet unknown essential functions besides their involvement in pre-tRNA splicing.
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PMID:Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins. 1565 39

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


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