Guide RNA-directed uridine insertion RNA editing in vitro.

Byrne, Elaine; Connell, Gregory J.; Simpson, Larry

doi:10.1002/j.1460-2075.1996.tb01065.x

Cited by 71 publications

(96 citation statements)

References 30 publications

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“…The same extracts, however, consistently supported accurate editing of a ND7 mRNA at quantifiable levels (data not shown, and Ref. 6), suggesting that the editing system itself was not fundamentally flawed.…”

Section: Grna-directed Editing Of a Cytochrome B Transcript-asupporting

confidence: 54%

“…However, a complete editing cycle of cytochrome b mRNA has not yet been demonstrated with these extracts, even though products are formed in the reaction that are consistent with the expected editing intermediates (5). Leishmania tarentolae mitochondrial extracts support gRNA-directed U-insertions into the first editing site of ND7 mRNA, but the guiding of insertions into a cytochrome b transcript could likewise not be demonstrated with this system (6). This is true, even though the cytochrome b mRNA isolated from the L. tarentolae mitochondria used to prepare the editing extracts is mostly edited (7).…”

mentioning

confidence: 78%

“…Editing Assays-The gRNA-directed reaction conditions are a modification of a previous procedure (6). A mRNA transcript (1 pmol) and 5 pmol of a gRNA in 25 mM Hepes, pH 7.9, and 0.2 mM EDTA, pH 8.0, were heated in a 10 l volume at 65°C for 3 min.…”

Section: Methodsmentioning

confidence: 99%

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Activation of Guide RNA-directed Editing of a Cytochromeb mRNA

Oppegard¹,

Kabb

Connell

2000

Journal of Biological Chemistry

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The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created only after the addition or deletion of specific uridines. Although in vitro systems have been valuable in characterizing the editing mechanism, only a limited number of mRNAs are accurately edited in vitro. We demonstrate here that in vitro editing of cytochrome b mRNA is inhibited by an A-U sequence present on both the 5-untranslated sequence and on a cytochrome b guide RNA. Mutation of the sequence on the guide RNA stimulates directed editing and results in the loss of binding to at least one component within the editing extract. Mutation of the sequence on the mRNA increases the accuracy of the editing. Evidence is provided that suggests the A-U sequence interacts with the editing machinery both in vitro and in vivo.

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Section: Grna-directed Editing Of a Cytochrome B Transcript-asupporting

confidence: 54%

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confidence: 78%

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Activation of Guide RNA-directed Editing of a Cytochromeb mRNA

Oppegard¹,

Kabb

Connell

2000

Journal of Biological Chemistry

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“…We previously proposed a model for the mechanism of this editing process involving a cleavage, addition or deletion of U's and ligation progressing in a 3Ј to 5Ј polarity (4), and this model has been since experimentally validated in almost every detail (5)(6)(7)(8)(9)(10).…”

mentioning

confidence: 99%

Structure of the core editing complex (L-complex) involved in uridine insertion/deletion RNA editing in trypanosomatid mitochondria

Li¹,

Hui

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Uridine insertion/deletion RNA editing is a unique form of posttranscriptional RNA processing that occurs in mitochondria of kinetoplastid protists. We have carried out 3D structural analyses of the core editing complex or ''L (ligase)-complex'' from Leishmania tarentolae mitochondria isolated by the tandem affinity purification procedure (TAP). The purified material, sedimented at 20 -25S, migrated in a blue native gel at 1 MDa and exhibited both precleaved and full-cycle gRNA-mediated U-insertion and U-deletion in vitro activities. The purified L-complex was analyzed by electron tomography to determine the extent of heterogeneity. Three-dimensional structural comparisons of individual particles in the tomograms revealed that a majority of the complexes have a similar shape of a slender triangle. An independent single-particle reconstruction, using a featureless Gaussian ball as the initial model, converged to a similar triangular structure. Another singleparticle reconstruction, using the averaged tomography structure as the initial model, yielded a similar structure. The REL1 ligase was localized on the model to the base of the apex by decoration with REL1-specific IgG. This structure should prove useful for a detailed analysis of the editing reaction.editing ͉ electron microscopy ͉ kinetoplast ͉ Leishmania ͉ trypanosome U ridine (U) insertion/deletion RNA editing is a posttranscriptional process in mitochondria of kinetoplastid protists that involves the modification of mRNA transcripts of mitochondrial cryptogenes by precise insertion and deletion of U residues to create translatable sequences (1-3). We previously proposed a model for the mechanism of this editing process involving a cleavage, addition or deletion of U's and ligation progressing in a 3Ј to 5Ј polarity (4), and this model has been since experimentally validated in almost every detail (5-10).Editing involves multiple multiprotein complexes associated by RNA linkers. The core editing complex, known as the L-complex, ''20S complex,'' ''editosome,'' or ''ϳ20S editosome,'' was detected by following the sedimentation and gel filtration of 2 adenylatable RNA ligases using Leishmania tarentolae and Trypanosoma brucei mitochondrial extracts (11,12). The activity sedimented around 20 -25S and migrated as a single band in a native gel (13). A ϳ20S multiprotein complex that showed both U-insertion and U-deletion in vitro activities was also purified from T. brucei mitochondria by column chromatography. An improved isolation of the L-complex from L. tarentolae was achieved by the tandem affinity purification procedure (TA P) (14); TA P-tagged plasmidexpressed REL1 became incorporated into the L-complex in vivo, allowing isolation of tagged complexes.Fourteen proteins were initially identified by mass spectrometry of the L. tarentolae REL1-TAP pull-down (14). All of the L. tarentolae proteins had homologs in T. brucei, and several additional proteins found in the T. brucei L-complex (15) were later identified in L. tarentolae (Tables S1 and S2).Two 3-prot...

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“…Then, in the case of U-deletion editing, a proposed U-specific 3Ј-5Ј-exonuclease removes non-base-paired Us from the 3Ј-end of the 5Ј-cleavage fragment, which is followed by ligation of the two mRNA cleavage fragments. Evidence from analysis of in vitro editing systems from the trypanosomatids Trypanosoma brucei (11)(12)(13) and Leishmania tarentolae (14,15) has provided strong evidence for this model. All of the proposed enzymatic activities have been detected in mitochondrial extracts, including a U-specific 3Ј-5Ј-exoribonuclease activity (16).…”

mentioning

confidence: 99%

Isolation and Characterization of a U-specific 3′-5′-Exonuclease from Mitochondria of Leishmania tarentolae

Aphasizhev

Simpson

2001

Journal of Biological Chemistry

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We have purified a 3-5-exoribonuclease from mitochondrial extract of Leishmania tarentolae over 4000-fold through six column fractionations. This enzyme digested RNA in a distributive manner, showed a high level of specificity for 3-terminal Us, and was blocked by a terminal dU; there was slight exonucleolytic activity on a 3-terminal A or C but no activity on a 3-terminal G residue. The enzyme preferred single-stranded 3-oligo(U) overhangs and did not digest duplex RNA. Two other 3-5-exoribonuclease activities were also detected in the mitochondrial extract, one of which was stimulated by a 3-phosphate and the other of which degraded RNAs with a 3-OH to mononucleotides in a processive manner. The properties of the distributive U-specific 3-5-exoribonuclease suggest an involvement in the U-deletion RNA editing reaction that occurs in the mitochondrion of these cells.3Ј-5Ј-Exoribonucleases have been shown to play important roles in the maturation and degradation of RNAs, including the 3Ј-end maturation of the 5.8 S rRNA (1) and pre-tRNAs (2) and both deadenylation-dependent (3) and deadenylation-independent mRNA decay in eukaryotes (4). An exonuclease activity specific for removal of the post-transcriptionally added (5) 3Ј-oligo(U) tail of U6 small nuclear RNA has been partially purified and characterized (6). A number of 3Ј-5Ј-exoribonucleases have also been described in bacteria and shown to be involved in RNA processing. For example, RNase E from Escherichia coli is a site-specific endoribonuclease that also has a 3Ј-5Ј-exonuclease activity on poly(A) and poly(U) tails (7). There is conservation of some exoribonucleases between bacteria and eukaryotes. In yeast, four of the five components of the "exosome" complex required for the 3Ј-end processing of the 5.8 S rRNA are homologous to bacterial 3Ј-5Ј-exoribonucleases that have distributive, processive, and phosphorolytic activities (8). In addition, a conserved 3Ј-5Ј-exonuclease domain that is similar to the 3Ј-5Ј-proofreading domain of DNA polymerases has been identified by hidden Markov model and phylogenetic analysis (9).The presence of a U-specific 3Ј-5Ј-exoribonuclease in mitochondria of kinetoplastid protozoa was predicted by the enzyme cascade model of U-insertion/deletion RNA editing (10). This model involves an initial base-pairing interaction of specific guide RNAs with the pre-edited mRNAs just downstream of the first editing site, and a cleavage occurs at the first mismatched base of the mRNA. Then, in the case of U-deletion editing, a proposed U-specific 3Ј-5Ј-exonuclease removes non-base-paired Us from the 3Ј-end of the 5Ј-cleavage fragment, which is followed by ligation of the two mRNA cleavage fragments. Evidence from analysis of in vitro editing systems from the trypanosomatids Trypanosoma brucei (11-13) and Leishmania tarentolae (14, 15) has provided strong evidence for this model. All of the proposed enzymatic activities have been detected in mitochondrial extracts, including a U-specific 3Ј-5Ј-exoribonuclease activity (16). This activity has...

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Guide RNA-directed uridine insertion RNA editing in vitro.

Cited by 71 publications

References 30 publications

Activation of Guide RNA-directed Editing of a Cytochromeb mRNA

Activation of Guide RNA-directed Editing of a Cytochromeb mRNA

Structure of the core editing complex (L-complex) involved in uridine insertion/deletion RNA editing in trypanosomatid mitochondria

Isolation and Characterization of a U-specific 3′-5′-Exonuclease from Mitochondria of Leishmania tarentolae

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