Direct visualisation of RNA editing within a Leishmania tarentolae mitochondrial extract

Oppegard, Lisa M.; Connell, Gregory J.

doi:10.1016/s0020-7519(02)00015-2

Cited by 4 publications

(10 citation statements)

References 25 publications

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“…The peak Q2 fractions (13-15) contain z1/6000 of the original crude mitochondrial extract protein and exhibit a simpler protein pattern than the parental D and Q1 fractions. This extent of purification is consistent with others reported using similar protocols (Rusche et al 1997;Panigrahi et al 2001a;Oppegard and Connell 2002). There is at least an z10-fold further purification compared to the whole-cell protein content; however, the specific activity of editing complexes could not be calculated since the in vitro editing assay is not linear with protein added, particularly in cruder fractions (Rusche et al 1997;Panigrahi et al 2001a;Oppegard and Connell 2002;data FIGURE 2. p40, p50, p60, and p100 copurify with editing complexes after extensive ion-exchange chromatography.…”

Section: Resultssupporting

confidence: 89%

RNA editing complex interactions with a site for full-round U deletion in Trypanosoma brucei

Sacharidou

Cifuentes-Rojas

Halbig

et al. 2006

RNA

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Trypanosome U insertion and U deletion RNA editing of mitochondrial pre-mRNAs is catalyzed by multisubunit editing complexes as directed by partially complementary guide RNAs. The basic enzymatic activities and protein composition of these high-molecular mass complexes have been under intense study, but their specific protein interactions with functional pre-mRNA/gRNA substrates remains unknown. We show that editing complexes purified through extensive ion-exchange chromatography and immunoprecipitation make specific cross-linking interactions with A6 pre-mRNA containing a single 32 P and photoreactive 4-thioU at the scissile bond of a functional site for full-round U deletion. At least four direct protein-RNA contacts are detected at this site by cross-linking. All four interactions are stimulated by unpaired residues just 59 of the premRNA/gRNA anchor duplex, but strongly inhibited by pairing of the editing site region. Furthermore, competition analysis with homologous and heterologous transcripts suggests preferential contacts of the editing complex with the mRNA/gRNA duplex substrate. This apparent structural selectivity suggests that the RNA-protein interactions we observe may be involved in recognition of editing sites and/or catalysis in assembled complexes.

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Section: Resultssupporting

confidence: 89%

RNA editing complex interactions with a site for full-round U deletion in Trypanosoma brucei

Sacharidou

Cifuentes-Rojas

Halbig

et al. 2006

RNA

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“…Likewise, the addition of a third potential guiding nucleotide (A-42) resulted in three U insertions (lanes 5,6). The guiding is significantly more precise than any other previously described gRNA-directed reaction that is catalyzed by mitochondrial extracts (Oppegard et al 2000;Kabb et al 2001;Oppegard and Connell 2002) or lysates (Byrne et al 1996;Kapushoc and Simpson 1999) prepared from L. tarentolae. Linear molecules, some of which are consistent with the size expected of editing reaction intermediates, are formed during the reaction, although other unrelated processes could also be producing these products.…”

Section: Pai Et Almentioning

confidence: 96%

“…It has been difficult to obtain an editing assay that is linear with the quantity of added extract in both the direct L. tarentolae (Oppegard and Connell 2002) and T. brucei (Rusche et al 1997;Panigrahi et al 2001a) in vitro assays. As illustrated in Figure 7, the editing assay using the in vitro selected A-1 RNA is linear with extract concentration over the range from 3% to 40% correctly edited product.…”

Section: In Vitro Editing Activity Increases With Increasing Amounts mentioning

confidence: 99%

“…The activity cofractionates with the gRNA-directed activity (Oppegard and Connell 2002;Oppegard et al 2000) and was shown to be dependent upon an A + U element that is present within the 5Ј untranslated sequence of the mRNA encoding cytochrome b (Brown et al 1999). The U insertions are induced within the sequence both 5Ј and 3Ј of the element.…”

Section: Introductionmentioning

confidence: 99%

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Sequence and structural requirements for optimal guide RNA-directed insertional editing within Leishmania tarentolae

Pai

Oppegard

Connell³

2003

RNA

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The coding sequence of several mitochondrial mRNAs of the trypanosomatid family of protozoa is created by the guide RNA-directed insertion and deletion of uridylates (Us). Selection-amplification was used to explore the sequence and structure of the guide RNA and mRNA required for efficient insertional editing within a mitochondrial extract prepared from Leishmania tarentolae. This study identifies several novel features of the editing reaction in addition to several that are consistent with the previous mutagenesis and phylogenetic analysis of the reaction in Trypanosoma brucei, a distantly related trypanosomatid. Specifically, there is a strong bias against cytidines 5 of the editing sites and guanosines immediately 3 of guiding nucleotides. U insertions are directed both 5 and 3 of a genomically encoded U, which was previously assumed not to occur. Base pairing immediately flanking an editing site can significantly stimulate the editing reaction and affect the reaction fidelity but is not essential. Likewise, single-stranded RNA in the region upstream of the editing site, not necessarily immediately adjacent, can facilitate editing but is also not essential. The editing of an RNA containing many of the optimal features is linear with increasing quantities of extract permitting specific activity measurements to be made that are not possible with previously described T. brucei and L. tarentolae assays. The reaction catalyzed by the L. tarentolae extract can be highly accurate, which does not support a proposed model for editing that was based largely on the inaccuracy of an earlier in vitro reaction.

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“…This secondary screen exploited a radiolabeled reporter that is based on the cytochrome b mRNA (Oppegard et al 2000;Oppegard and Connell 2002). The gRNA for the cytochrome b mRNA acts in trans, like the majority of natural gRNAs.…”

Section: Secondary Screens To Identify False Positivesmentioning

confidence: 99%

Identification of specific inhibitors for a trypanosomatid RNA editing reaction

Liang¹,

Connell²

2010

RNA

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Several mitochondrial mRNAs of the trypanosomatid protozoa are edited through the post-transcriptional insertion and deletion of uridylates. The reaction has provided insights into basic cellular biology and is also important as a potential therapeutic target for the diseases caused by trypanosomatid pathogens. Despite this importance, the field has been hindered by the lack of specific inhibitors that could be used as probes of the reaction mechanism or developed into novel therapeutics. In this study, an electrochemiluminescent aptamer-switch was utilized in a high-throughput screen for inhibitors of a trypanosomatid RNA editing reaction. The screen identified GW5074, mitoxantrone, NF 023, protoporphyrin IX, and D-sphingosine as inhibitors of insertion editing, with IC 50 values ranging from 1 to 3 mM. GW5074 and protoporphyrin IX are demonstrated to inhibit at or before the endonuclease cleavage that initiates editing and will be valuable biochemical probes for the early events of the in vitro reaction. Since protoporphyrin IX and sphingosine are both naturally present within the trypanosomatids, their effectiveness as in vitro inhibitors is also suggestive of the potential for in vivo modulatory roles.

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Direct visualisation of RNA editing within a Leishmania tarentolae mitochondrial extract

Cited by 4 publications

References 25 publications

RNA editing complex interactions with a site for full-round U deletion in Trypanosoma brucei

RNA editing complex interactions with a site for full-round U deletion in Trypanosoma brucei

Sequence and structural requirements for optimal guide RNA-directed insertional editing within Leishmania tarentolae

Identification of specific inhibitors for a trypanosomatid RNA editing reaction

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