Activation of Guide RNA-directed Editing of a Cytochromeb mRNA

Oppegard, Lisa M.; Kabb, Aaron L.; Connell, Gregory J.

doi:10.1074/jbc.m003002200

Cited by 9 publications

(16 citation statements)

References 22 publications

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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: 92%

“…The A + U sequence is sufficient to induce in vitro U insertions when placed into a normally nonedited transcript, but unlike gRNAs, the A + U element is unable to stimulate editing when added in trans. Although the biological significance of the A + U element still is not clear, cytochrome b mRNA isolated from L. tarentolae mitochondria is also modified at sites immediately flanking the A + U element, suggesting that a similar reaction may occur in vivo (Oppegard et al 2000).…”

Section: Introductionmentioning

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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Section: Pai Et Almentioning

confidence: 92%

Section: Introductionmentioning

confidence: 99%

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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“…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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“…A 25 L culture of the Leishmania tarentolae UC strain was grown to a density of z1.5 3 10 8 cells/mL, and the cells were lysed as previously described (Oppegard et al 2000). A mitochondrial The edited and (C,D) pre-edited forms of RNA B were probed with DMS and CMCT under native and denaturing conditions.…”

Section: Preparation Of the Editing Extract And The In Vitro Editing mentioning

confidence: 99%

An electrochemiluminescent aptamer switch for a high-throughput assay of an RNA editing reaction

Liang

Connell²

2009

RNA

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An RNA editing reaction that is both essential and specific to the trypanosomatid parasites is an attractive target for new drug development. Although high-throughput screening of chemical libraries is a powerful strategy often used to identify new drugs, the available in vitro editing assays do not have the necessary sensitivity and format for this approach to be feasible. A ruthenium labeled reporter RNA is described here that overcomes these limitations as it can both detect edited product in the low femtomole range and is ideal for high-throughput format. The reporter RNA consists of an RNA editing substrate linked to a streptavidin-binding aptamer that is initially held within an inactive conformation. An in vitro selection strategy optimized the linkage so that the streptavidin-binding aptamer is only activated by an editing-induced conformational change. An electrochemiluminescent signal results from the ruthenium label when the reporter is bound to the bottom of a streptavidin-coated microtiter plate where it can be stimulated by a carbon electrode. Chemical probing, mutagenesis, and binding affinity measurements were used to characterize the reporter. The highly sensitive assay could be adapted to a broad range of RNA processing reactions.

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Activation of Guide RNA-directed Editing of a Cytochromeb mRNA

Cited by 9 publications

References 22 publications

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

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

Identification of specific inhibitors for a trypanosomatid RNA editing reaction

An electrochemiluminescent aptamer switch for a high-throughput assay of an RNA editing reaction

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