Gregory J. Connell

Guide RNA-directed uridine insertion RNA editing in vitro

Guide RNAs (gRNAs) have been proposed to mediate uridine (U) addition/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa. The Us are proposed to be derived either from UTP by two successive cleavage‐ligations or transesterifications, or from the 3′ end of the gRNA by the same mechanisms. We have demonstrated gRNA‐dependent U insertions into a specific editing site of a pre‐edited mRNA which was incubated in a mitochondrial extract from Leishmania tarentolae. The predominant number of U insertions was determined by the number of guiding nucleotides in the added gRNA, and the formation of a gRNA‐mRNA anchor duplex was necessary for activity. UTP and alpha‐beta bond hydrolysis of ATP were required, and the activity was inhibited above 50–100 mM KCl. A gRNA‐independent insertion of up to approximately 13 Us occurred in the absence of the added cognate gRNA; the extent of this activity was affected by sequences upstream and downstream of the edited region. Heparin inhibited the gRNA‐independent U insertion activity and had no effect on the gRNA‐dependent activity. Blocking the 3′ OH of the gRNA had little effect on the gRNA‐dependent U insertion activity. The data are consistent with a cleavage‐ligation model in which the Us are derived directly from UTP.

Guide RNA-independent and Guide RNA-dependent Uridine Insertion into Cytochrome b mRNA in a Mitochondrial Lysate from Leishmania tarentolae ROLE OF RNA SECONDARY STRUCTURE

A primer extension assay was used for the detection of uridine insertions occurring in vitro in synthetic pre-edited cytochrome b mRNA during incubation with a Leishmania tarentolae mitochondrial extract. Two different activities were detected that inserted uridines within the first two editing sites: one that is dependent on the secondary structure of the mRNA but is independent of both exogenous and endogenous guide RNA, and a second that does not put the same structural constraints on the mRNA, but is dependent on the presence of a cognate guide RNA.

A trypanosomatid protein specifically interacts with a mammalian iron-responsive element

Abstract Intracellular iron homeostasis of vertebrates and invertebrates is mediated through the interaction of iron-regulatory proteins (IRPs) with mRNAs containing a bulged hairpin-loop structure termed the iron-responsive element (IRE). We detected a protein within extracts prepared from Leishmania tarentolae that specifically interacts with a mammalian IRE; mutations to the IRE that inhibit the interaction with the mammalian protein have a corresponding effect on the interaction with the L. tarentolae protein. The disassociation constant noted for the interaction of the mammalian IRE with the L. tarentolae protein was 0.7 ± 0.3 μM, whereas that recorded for the interaction with the mammalian IRP under these conditions was 5 ± 2 nM. The interacting L. tarentolae protein potentially places the RNA-binding site of the IRP near the root of the eukaryotic evolutionary tree. However, unlike that of the mammalian IRPs, the L. tarentolae IRE-binding activity was not induced by growth in iron-depleted media.

A Cis-acting A-U Sequence Element Induces Kinetoplastid U-insertions

A 34-nucleotide A-U sequence located immediately upstream of the editing sites of the Leishmania tarentolaecytochrome b mRNA induces a mitochondrial extract to insert U nucleotides independent of guide RNA. Insertions are localized to positions immediately 5′ and 3′ of the A-U sequence. When placed within an unedited mammalian transcript, the A-U sequence is sufficient to induce U-insertions. The sequence has a high degree of similarity with the templating nucleotides of a cytochrome b guide RNA and with a sequence adjacent to the editing sites in ND7 mRNA, the other characterized kinetoplastid mRNA supporting guide RNA-independent U-insertions. At least one protein specifically interacts with the A-U sequence. The reaction is consistent with a mechanism proposed for guide RNA-directed editing.

Identification of specific inhibitors for a trypanosomatid RNA editing reaction.

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 μM. 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.

Activation of guide RNA-directed editing of a cytochrome b mRNA.

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 editedin vitro. We demonstrate here that in vitroediting 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.

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

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.

Utilization of cofactors expands metabolism in a new RNA world

RNA has been hypothesized to have preceded proteins as the major catalysts of the biosphere, yet there are only a very limited number of chemical reactions that are known to be catalyzed by modern RNA. Cofactors are used by the majority of protein enzymes to supply additional functional groups to the active site. RNA should also be able to utilize some of these same cofactors to extend its own catalytic potential. We describe here how it could be possible to use selection — amplification from a population of random RNA to obtain a coenzyme A mediated RNA transacylase. Exploitation of some of the sulphur chemistry mediated by coenzyme A could have significantly expanded a prebiotic RNA directed metabolism.

Cis-acting Elements Stimulating Kinetoplastid Guide RNA-directed Editing

The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created through the insertion and deletion of specific uridylates. The editing reactions are required to be highly specific in order to ensure that functional open reading frames are created in edited mRNAs and that potentially deleterious modification of normally nonedited sequence does not occur. Selection-amplification and mutagenesis were previously used to identify the optimal sequence requirements for in vitro editing. There is, however, a minority of natural editing sites with suboptimal sequence. Several cis-acting elements, obtained from an in vitro selection, are described here that are able to compensate for a suboptimal editing site. An A + U sequence element within the 5′-untranslated region of cytochrome b mRNA from Leishmania tarentolae is also demonstrated to function as a cis-acting guide RNA and is postulated to compensate for a suboptimal editing site in vivo. Two proteins within an enriched editing extract are UV-cross-linked to two different in vitro selected editing substrates more efficiently than poorly edited RNAs. The results suggest that these proteins contribute to the specificity of the editing reaction.

Role of RNA Structure in RNA Editing

The coding sequences of some mRNAs and the structures of some tRNAs and rRNAs in a variety of organisms are altered by posttranscriptional reactions termed editing; this can involve the modification or insertion/deletion of nucleosides. In this review, we focus on the role of RNA structure in three types of editing reactions that are known to alter the coding sequences of mRNAs: a cytosine deamination required for apoB-48 synthesis, an adenosine deamination required in some of the mammalian glutamate receptor subunits and also during the life cycle of the hepatitis delta virus, and the uridine insertion and deletion reactions needed to create functional open reading frames in many of the mitochondrial transcripts in the trypanosomatids. Although the contribution of RNA structure to these various types of RNA editing is just beginning to be understood, we attempt to summarize some of the recent advances in this field. C TO U SUBSTITUTION EDITING IN THE APOLIPOPROTEIN B mRNA Background Apolipoprotein B (apoB) is a major protein of plasma lipoproteins and exists in two forms, apoB-100 and apoB-48 (Kane et al. 1980). In mammals, apoB-100 (512 kD) is synthesized in the liver. It is the ligand for the low-density-lipoprotein receptor and is also a component of very low density lipoproteins and intermediate-density lipoproteins (for review, see Chen et al. 1990). In contrast, apoB-48 (241 kD) is synthesized in the small intestine and is present in chylomicron and chylomicron remnants; apoB-48 is also produced by the liver in rodents (mice and rats) (for review,...